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INACTIVATION OF STREPTOMYCES GRISEUS BY COMMON WATER
TREATMENT DISINFECTANTS
by
Alby Aguilar
A Thesis Presented in Partial Fulfillment of the Requirements for the Degree
Master of Science
ARIZONA STATE UNIVERSITY
December 2004
INACTIVATION OF STREPTOMYCES GRISEUS BY COMMON WATER
TREATMENT DISINFECTANTS
by
Alby Aguilar
has been approved
December 2004
APPROVED:
________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________
Supervisory Committee ACCEPTED: ____________________________________ Department Chair ____________________________________
, Chair
iii
ABSTRACT
Actinomycetes are known to be odor - causing bacteria. The present project used
Streptomyces griseous subsps. Griseous ATCC ® 3343 for different inactivation
procedures and to spike them into an existing pipe – loop apparatus. Streptomycetes are
members of the Actinomycetales family. The objectives define in this project were to
obtain inactivation rates for streptomyces using different disinfectants,
compare Actinomycetes inactivation with literature values and other bacteria and finally
to determine the fate of Actinomycetes spiked into a laboratory pipe-loop PVC apparatus
with subsequent chlorination. Several studies have demonstrated this subspecies
produced very strong and foul odors. Among the metabolites produced by Streptomycetes
are Geosmin and 2-Methylisoborneol (MIB). The maximum geosmin concentration
achieved during this project was 6.63 ng/L, while the maximum MIB concentration was
125.23 ng/L. During the length of this research it was observed that stressful conditions
altered physical properties such as shape, size and color of the colonies. Odor -
production was also affected by media variations and their insertion into the polyvinyl
chloride (PVC) pipe – loop.
Actinomycetes were found on several samples sites within Arizona State University Main
Campus and on a canal wall prior to the Deer Valley Water Treatment Plant.
Nevertheless, ascertaining subspecies found on those sites goes beyond the scope of this
research. Since Actinomycetes were attached to canal walls, it could be expected that they
could also be found at the bottom of the canal. Canal deposits could provide
Actinomycetes with nutrients that could help them to reproduce and generate odorous
iv
metabolites that affect water’s aesthetic properties. Various levels of inactivation were
achieved with the different disinfectants and inactivation procedures used throughout this
project. Streptomyces spiked onto previously autoclaved phosphate buffer solution (PBS)
diminished an average of 43% of its original concentration after 5 days without any type
of inactivation. Higher removal values were obtained with chlorine, monochloramine,
ozone and ultraviolet radiation.
v
To my husband, Rafa I could not have done it without you.
To my beautiful daughters Tamara and Alejandra
And last but not least to my Family in Ecuador.
Padres mios muchas gracias por todo el apoyo
que ustedes me han brindado siempre.
vi
ACKNOWLEDGMENTS
First of all I will like to thank Dr Paul Westerhoff for his support and patience throughout
this research. He really needed lots of patience to understand my Spanish thinking mind.
When writing and thinking in Spanish, we Latin-Americans have the tendency to write
long sentences, loaded with lots of passive voices. I guess it must have been difficult to
read such writings. I really appreciate your support while I was doing my research.
Thank you very much.
I will also like to thanks Dr Jordan Peccia and Dr Morteza Abbaszadegan. Without their
help I would not have been able to finish this investigation. They both allowed me to
work in their laboratories. The results obtained in those labs are registered throughout
this project. I learned real microbiology basis within those walls.
My appreciation goes also to Mr. Terri Kitchen from the City of Phoenix Laboratory. He
introduced me to the world of Actinomycetes. He taught me how to recognize them and to
plate them. I can not forget Dr Absar, either. Thanks for your help, while I worked with
the PVC pipe-loop.I will also like to thanks Tania Paez, Thank you for reading my thesis,
I appreciate it very much. Rafa thank you very much, without your help, I would not be
here. Thanks to Mario Esparza, Luis Lesser and Maikel Mendez, I learned a lot from you
guys. Finally I will like to thanks Fundacyt (Fundación para la Ciencia y la Tecnología)
and LASPAU for their support throughout my program here in the USA
vii
TABLE OF CONTENTS
Page
LIST OF TABLES............................................................................................................. X
LIST OF FIGURES .........................................................................................................XII
CHAPTER
1 INTRODUCTION ....................................................................................................... 1
What are Actinomycetes? ....................................................................................... 1
Culturing Actinomycetes ........................................................................................ 2
Relationship with the taste and odor issue.............................................................. 3
Objectives ............................................................................................................... 4
2 LITERATURE REVIEW ............................................................................................ 5
Introduction............................................................................................................. 5
Actinomycetes and Source Water........................................................................... 5
Actinomycetes and Treated Water.......................................................................... 6
Laboratory studies................................................................................................... 7
Biofilm formation in water distribution systems .................................................... 9
Summary............................................................................................................... 10
3 MATERIALS AND METHODS............................................................................... 13
Introduction........................................................................................................... 13
Experimental Methods.......................................................................................... 13
Sources of Actinomycetes................................................................. 13
viii
CHAPTER Page
Streptomycetes Enumeration ............................................................ 17
Inactivation Studies........................................................................... 18
Pipe- Loop experiment...................................................................... 22
Analytical procedure used to determine inactivation rates................................... 24
4 RESULTS .................................................................................................................. 27
Introduction........................................................................................................... 27
Actinomycetes culturing from field sample.......................................................... 27
Inactivation Studies............................................................................................... 28
Chlorination ...................................................................................... 28
Monochloramine and Ozone inactivation of actinomycetes............ 31
Ultra violet inactivation of actinomycetes ....................................... 32
Inactivation Comparison with other Bacteria ................................... 32
Pipe- Loop experiment...................................................................... 32
5 DISCUSSION............................................................................................................ 52
Introduction........................................................................................................... 52
Actinomycetes, Streptomycetes and the media .................................................... 52
Streptomycetes Griseous................................................................... 52
Actinomycetes................................................................................... 53
Inactivation Results............................................................................................... 54
Chlorine Results................................................................................ 55
Graphs plotted for pH5, pH 7 and pH 9............................................ 57
ix
CHAPTER Page
Monochloramine, Ozone and Ultraviolet inactivation Results......... 60
Pipe – loop Experiments ....................................................................................... 60
6 CONCLUSIONS........................................................................................................ 63
Introduction........................................................................................................... 63
Actinomycetes, Streptomycetes and the media .................................................... 63
Inactivation experiments....................................................................................... 63
Pipe loop assays .................................................................................................... 65
Recommendations for future research .................................................................. 66
REFERENCES ................................................................................................................. 67
APPENDIX
A STARCH - CASEIN MEDIA................................................................................... 74
B 3 X PHOSPHATE BUFFER SALINE...................................................................... 77
C DATA OBTAINED FROM CHLORINE EXPERIMENTS .................................... 79
D DATA OBTAINED FROM MONOCHLORAMINE, OZONE AND UV
EXPERIMENTS ....................................................................................................... 82
E STREPTOMYCETES DECAY ON PBS ................................................................. 84
F PVC PIPE-LOOP: COLONY COUNTS, MIB AND GEOSMIN ............................ 86
x
LIST OF TABLES
TABLE Page
1 Physical and chemical properties of MIB and Geosmin.............................................. 4
2 Description of the set up used for ultraviolet inactivation........................................ 22
3 Sampling Ports within the PVC pipe-loop system.................................................... 24
4 Number of colony forming units of actinomycetes obtained from........................... 36
5 Data obtained from the chlorination assays for pH5 ................................................ 37
6 Data obtained from the chlorination assays for pH7 ................................................ 38
7 Data obtained from the chlorination assays for pH9 ............................................... 38
8 Values used to determine K, m and n using multiple regressions (pH5).................. 39
9 Values used to determine K, m and n using multiple regressions (pH7)................. 39
10 Values used to determine K, m and n using multiple regressions (pH9)................. 40
11 Coefficients k, m and n for obtained after the multiple regression........................... 40
12 Coefficients k, m and n for obtained using Mathematica ......................................... 40
13 Kinetic coefficient for chlorine decay (2nd order reaction) and r2 ......................... 40
14 Kinetic coefficient for chlorine decay (1st order reaction) and r2........................... 41
15 Average, Standard Deviation and Standard Error for experiment A set at pH 5...... 41
16 Average, Standard Deviation and Standard Error for experiment A set at pH 7...... 41
17 Average, Standard Deviation and Standard Error for experiment A set at pH 9...... 41
xi
TABLE Page
18 Concentration – Time values (mg/L – minutes) for pH 5, pH 7 and pH 9 .............. 45
19 Rate constant K and coefficients m, n and correlation factor R ........................... 45
20 Estimated chlorine demands for the PVC pipe-loop................................................ 45
xii
LIST OF FIGURES
FIGURE Page
1 Streptomyces griseous subsps. Griseous ATCC ® 3343.......................................... 15
2 Actinomycetes isolated from natural waters. These were isolated from samples
collected at the inlet of Deer Valley Water Treatment Plant (R-16)......................... 15
3 Scheme of the set up used for the ultraviolet inactivation procedure ....................... 22
4 Pipe – Loop set up spiked with Streptomyces griseous subsps. Griseous ATCC ®
3343. (Ghatpande, 2002) ........................................................................................... 23
5 Free Chlorine Demand for super Q water, 0.1 % peptone water and PBS. .............. 36
6 Streptomyces decay on PBS ..................................................................................... 37
7 Survival Ratio vs. Time (Set at pH 5)....................................................................... 42
8 Chlorine decay vs. Time (Set at pH 5)...................................................................... 42
9 Streptomyces inactivation curve for pH 7 ................................................................ 43
10 Chlorine decay vs. Time (Set at pH 7)...................................................................... 43
11 Streptomyces inactivation curve for pH 9 ................................................................ 44
12 Chlorine decay vs. Time (Set at pH 9)...................................................................... 44
13 Streptomyces inactivation using monochloramine. Curve for pH 8.5...................... 46
14 Streptomyces inactivation using Ozone. Curve for pH 7. ........................................ 47
15 Streptomyces inactivation using UV as disinfectant. Curve for pH 7. ..................... 47
FIGURE Page
xiii
16 Colony counts, MIB and Geosmin Concentration.................................................... 48
17 Free available chlorine within the pipe-loop. These reading were obtained before
adding chlorine to the system. ................................................................................... 48
18 Chlorine demand within the PVC Pipe-Loop System demand (1st run) .................. 49
19 Chlorine demand within the PVC Pipe-Loop System demand (2nd run)................. 49
20 Combined Chloramine, colony counts (1st run) ....................................................... 50
Figure 21 Combined Chloramines, colony counts (2nd run)............................................ 50
Figure 22 Pictures obtained from the samples taken from the pipe-loop ......................... 51
Figure 23 Watson’s plot constructed for different types of microorganisms. .................. 59
1INTRODUCTION
Taste and odor issues have influenced water treatment for a very long time. They affect
consumers’ perception of the sanitation of potable water. The presence of off-flavors and
odors affect organoleptic properties in potable water (Young W.F., 1996), and diminishes
its acceptance by the community. The first report of taste and odor issues in the United
States, was issued in 1855, however it was only until 1890 when the first attempts to
identify the earthy smell in potable water were conducted (Persson, 1995).
The main biological sources of off-flavors on water systems are certain types of bacteria
and algae. Both kinds of microorganisms produce metabolites and release them into the
environment (J. Mallavialle 1987). In many cases algae, especially cyanobacteria, are the
culprit for taste and odor problems (Izaguirre et al., 1994). Studies to remove algae and
its metabolites have been conducted before(Bruce et al., 2002). Actinomycetes are odor-
causing bacteria known to be found in raw water supplies. These bacteria have caused
taste and odor events, however, much less is known about Actinomycetes in water
distribution systems, their ability to produce off-flavors and their susceptibility to
disinfectants.
WHAT ARE ACTINOMYCETES?
Actinomycetes are a large group of branching unicellular microorganisms. They are
about 1µm in diameter. These colonies look like a mass of unicellular mycelium, with
branching filaments extensions of the original cell or cells, in addition to spores and
degradation products (Waksman, 1950). The variety of Actinomycetes is considerable;
2therefore it was difficult for researchers to classify them either as fungi or as bacteria.
Actinomycetes are generally gram positive and prokaryotes, this means that they lack of a
defined nucleus. However they should be considered bacteria (Waksman, 1950, 1962,
1967). Most of its subspecies are aerobic, although a few can live under anaerobic
conditions. They reproduce by fission or by spores, and this was one of the reasons why
Actinomycetes were originally typified as fungi. Actinomycetes form colonies of radiating
structure, which is the origin of their name (Ray Fungus)(Adams, 1929; Waksman,
1950). Actinomycetes can be found almost in any substrate, although they prefer alkaline
and neutral conditions in order to grow. The optimal pH range in which they grow is
between 7 and 8. Nevertheless, they can live under acidic conditions between pH 4.8-5;
however this is a critical condition for these bacteria. Most of the Actinomycetes growth
at temperatures between 15 and 30 degrees Celsius, however, some like the thermophiles
Actinomycetes live in very high temperature, about 60 degrees Celsius (Waksman, 1950,
1962, 1967).
In this present study the word Actinomycetes will be used to name the entire
Actinomycetes Genus, which includes Streptomycetes, Nocardia, Micromonospora, etc.,
or when it is not possible to determine which of the Actinomycetes sub-species are
identify during the study.
CULTURING ACTINOMYCETES
Actinomycetes need carbon and nitrogen in order to grow. Starch is the usual carbon
source used, while casein provides the nitrogen requirement under artificial
conditions(Waksman, 1950). Mineral traces such as NaCl, K2HPO4, MgSO2•7H2O,
3CaCO3 and FeSO4•7H2O are also necessary. In certain cases, antibiotics are added to
the culture in order to eliminate fungi and general bacteria from Petri plates. (Waksman,
1950, 1962, 1967). Previous studies used copper when plating Actinomycetes to inhibit
the growth of algae, bacteria, fungi and other aquatic species that could be present in the
samples(Silvey, 1954).
Once plated, Actinomycetes have a compact, leathery appearance. They present a dry
surface. When Actinomycetes are grown and maintained in artificial media, they lose
some morphological properties. Among these properties are the ability to form aerial
mycelium and spores, certain physiological and biochemical properties like pigmentation
and odor(Waksman, 1967; Cross, 1981).
RELATIONSHIP WITH THE TASTE AND ODOR ISSUE
In 1929 Actinomycetes were reported as the source of an earthy smell in chlorinated water
(Adams, 1929). Later, an earthy smelling substance was isolated from various
Actinomycetes(Gerber et al., 1965). Both, Geosmin and 2-methylisoborneol (MIB) are
organic metabolites that have been isolated from Actinomycetes, mostly Streptomyces sp.
(Gerber, 1979). Table 1 shows some of the physical and chemical properties of both
MIB and geosmin. Actinomycetes not only produce geosmin and MIB, they also generate
several other metabolites such as selinadienol, which smells like freshly plowed soil and
is produced by some streptomyces strains (Gerber, 1979; Thiemer, 1982). Mucidone is
another chemical compound that was isolated from Actinomycetes. This compound had a
threshold odor number (TON) of approximately 300,000,000. The threshold odor number
4is the greatest dilution of a sample with odor free water that still yields a just-detectable
odor. This odor was described as musty, and it was present in Cedar River (Dougherty et
al., 1967). Naturally occurring microbial metabolites are not considered to pose health
risk at low concentrations (Young W.F., 1996), but are a nuisance due to their odors. On
the other hand, some of the metabolites pose a threat to bodies of water and their
inhabitants (Thayssen, 1936).
Table 1 Physical and chemical properties of MIB and Geosmin MIB Geosmin (1-R-exo)-1,2,7,7- trans-1,10-demethyl-
Chemical Name tetramethyl bicyclo-[2,2,1]- trans-9-decalol heptan-2-ol
Molecular Weight (g/mole) 168 182 Boiling Point (degree C) 196.7 165.1
Aqueous Solubility (mg/l) 194.5(in methanol) 150.2(in methanol) Log of Octanol/Water
Partition Coefficient (Kow) 3.13 3.7
Henry's Law Constant (atm M3/mole)
5.70E-05 6.66E-05
Source:(Pirbazari et al., 1992)
OBJECTIVES
The goal of this research was to study Actinomycetes inactivation by chlorine,
monochloramine, ozone and ultraviolet inactivation. The specific objectives of this study
included:
To obtain inactivation rates for isolated streptomyces for different disinfectants.
To compare Actinomycetes inactivation with literature values and other bacteria.
To determine the fate of Actinomycetes spiked into a laboratory pipe-loop PVC apparatus
with subsequent chlorination.
5LITERATURE REVIEW
INTRODUCTION
Actinomycetes have been linked to taste and odor occurrences for over a century. In 1895
Cladothrix Odofer was isolated and described as producing an earthy odor(Adams,
1929). This microorganism was later referred as Cladothrix Dichotoma, and due to their
description, thought to be what nowadays are described as Actinomycetes (Romano et al.,
1963a). Ever since then, Actinomycetes have been linked to several off-flavor episodes
not only in the United States, but in several other countries as well. Nonetheless,
Actinomycetes are not the only source of earthy-musty odor problems in drinking
water(Mallavialle et al., 1987; Izaguirre et al., 1994).
ACTINOMYCETES AND SOURCE WATER
Actinomycetes have been isolated in lakes and rivers that were used as sources for
different water distribution systems (Adams, 1929; Silvey et al., 1953; Burnan, 1973;
Jensen et al., 1994). In 1951, the Oklahoma City Water Works (USA) had serious taste
and odor problems. At first, a type of Cladophora algae was thought to be the source of
the problem, since they were attached to rip-rap across a lake. Nevertheless, microscopic
studies lead to the discovery of thousands of Actinomycetes colonies living inside the
green algae, including, Streptomyces, Micromonospora and Nocardia (Silvey et al.,
1953). Cladophoras contained nutrients that Actinomycetes depended on, especially
nitrogen. Moreover, Actinomycetes were the cause of an earthy odor within several
distribution systems in Great Britain in 1973. The ranges observed in the Thames River
6were between 5000 and 20000 Streptomyces/100 ml of water and between 1000 and
2000 Micromonosporas/100 ml of water (Burnan, 1973). In 1991 Streptomycetes were
found to be the source of taste and odor problems in Edmonton, Alberta (Jensen et al.,
1994). Recent studies performed on Lake Ontario associated mussel beds and
Actinomycetes to MIB and Geosmin production within the lake(Zaitlin et al., 2003).
Previous research stated that Actinomycetes, due to hyphae formation, tend to form
clumps and settle with time (Waksman, 1950, 1962, 1967). This explains the fact that
most of the time, Actinomycetes are found on sediments, or attached to other living
organisms (Silvey et al., 1953). When these living organisms provide the right nutrients,
especially carbon and nitrogen (Silvey et al., 1953; Waksman, 1967), Actinomycetes are
not only able to produce metabolites that generate taste and odor in the water, but also
contaminate the species that live within that body of water (Thayssen, 1936).
ACTINOMYCETES AND TREATED WATER
Chlorination has generated contradictory statements regarding taste and odor issues. On
one hand it is considered for lessening taste and odor problems. On the other hand,
chlorinated water from the Nile River with a two-hour contact time, and 0.6 ppm chlorine
dose still showed taste and odor problems (Adams, 1929). Actinomycetes isolated from
the Thames River and exposed to chlorine demonstrated that chlorination had little effect
on taste removal. It was even claimed that the reaction with chlorine may have even
worsened the taste than the original substance (Burnan, 1973). The British research
stated that Actinomycetes presented as spores were more resistant to chlorination, and that
chlorine resistance also depended on the sub-specie under study as well.
7Micromonospores, were found to be more resistant to chlorine than streptomyces
(Burnan, 1973). Both microorganisms are members of the actinomyceteles family.
In 1991, Actinomycetes isolated from the Saskatchewan River were diluted in water set at
pH 8 and exposed to both chlorine and monochloramine solutions. The concentration of
the applied oxidant, multiplied by its contact time was used to obtain 99 percent
inactivation (CT99). Values obtained ranged from 3.6 to 92 min-mg/L (Jensen et al.,
1994).
LABORATORY STUDIES
Several studies were conducted on Actinomycetes and its subspecies from 1960 to
1980(Romano et al., 1963b; Gerber et al., 1965; Dougherty et al., 1967; Medsker et al.,
1969; Sipma et al., 1972; Burnan, 1973; Gerber, 1973, 1979; Bentley et al., 1981; Cross,
1981). In 1963 an odorous compound was extracted from Streptomyces Griseoluteus IM
3718. This compound was diluted with water and then chlorinated. It was found that
chlorine did not remove odor completely. On the other hand, effective removal was
achieved when various grades of activated carbon were used (Romano et al., 1963a). In
1968 geosmin was isolated from aquatic Actinomycetes (Actinomycete No. 18). This type
of bacteria produced the most geosmin in the shortest time. The culture was kept at room
temperature, ranging between 22 and 26 degrees Celsius. By day 12th geosmin
concentration was approximately 200µg per liter (Lloyd L. Medsker, 1968). In a
different study, it was stated that another Streptomycetes sub-specie, Streptomyces
8Albudoflavus, produced geosmin in water that was supplemented with sufficient
concentrations of available carbon, nitrogen and phosphorous. (Wood S, 1985).
Geosmin concentration produced by Actinomycetes under laboratory conditions reached
3.6 mg/l(Blevins et al., 1995). The Streptomyces sub-specie used was Streptomyces
Halstedii, and it was grown at 30 degrees Celsius. That same study found that no
geosmin was detected above 40 degrees Celsius, or below 10 degrees Celsius. Optimal
Streptomyces growth occurred at pH values between 6 and 7. Nitrate and ammonium
were used as a nitrogen source on the streptomyces growth. Comparing the growth
results using both nitrogen sources, the study showed that biomass and geosmin
production was greater with nitrate. However limiting concentrations of either source of
nitrogen favored geosmin synthesis (W. T. Blevins, 1995).
Most of the experiments conducted on Actinomycetes were performed on terrestrial
species. These were later diluted in water and then either the threshold odor number
(TON) was determined or its concentration was measured with a gas chromatography
(Gerber, 1979). Researchers encountered difficulties distinguishing the aquatic from the
soil species (Silvey et al., 1953). For this reason it was hard to establish if the organisms
under study were water or soil based organisms. On the other hand, one study suggested
that Actinomycetes are terrestrial species may have been drawn to the water by different
transport mechanisms(Cross, 1981).
9BIOFILM FORMATION IN WATER DISTRIBUTION SYSTEMS
A biofilm could be defined as a microbial community that adheres to a substratum or to a
surface in aqueous environment. It is attributed to sessile cells attached to a surface
embedded in a matrix of extra-cellular polymeric substance. This polymeric matrix traps
nutrients required by microbes, hampering removal methodologies at the same
time(MarK E Schirliff, 2002).
A Finnish study performed on biofilm formation established that Actinomycetes are
among the microorganisms that attach to solid surfaces such as pipe walls. That same
study determined the microbiological quality of old biofilm deposits on pipe walls and
found that Actinomycetes were found among the sample deposits (Zacheus et al., 2001).
This agrees with the British research results that showed a ratio of Actinomycetes to
bacteria as high as 105: 1 (Burnan, 1973).
An 18-month study done on biofilm formation in potable water distribution systems
related biofilm growth with the reduction of residual disinfectant concentration. It was
demonstrated that chlorinated water did not have the potential for biofilm formation. It
was also found that a free chlorine residual of as low as 0.05 mgr/L prevented the
presence of biofilm on the interior walls of the water distribution pipes. (Lund et al.,
1995). Since chlorine was used as a bacteria inhibitor in previous studies when isolating
the Actinomycetes, the chlorine residual maintained within the distribution system might
not affect them at all (Silvey, 1954). Nevertheless, chlorine has been effective on other
microorganisms that might be present in the distribution system and that might act as
10Actinomycetes nutrients as happened in the case of the taste and odor problem in
Oklahoma City in 1951 (Silvey et al., 1953).
The presence of available organic carbon within the distribution system is directly related
with the growth of microorganisms inside the system. When the presence of organic
carbon is significant, 1mg/l of free chlorine residual is not enough to maintain the system
free of microorganisms(Silvey, 1954). In order for Actinomycetes to be present in the
distribution system enough nutrients (carbon and nitrogen) must be available for them to
survive and reproduce(Burnan, 1973).
SUMMARY
The following has been noted:
Actinomycetes have been linked to taste and odor occurrences for over a century.
However, they are not the only source of earthy-musty odor problems in drinking
water(Mallavialle et al., 1987; Izaguirre et al., 1994).
The maximum measured concentration of geosmin obtained in a laboratory and
produced by Actinomycetes was 3.6 mg/l. No geosmin was detected above 40
degrees Celsius, nor below 10 degrees Celsius(Blevins et al., 1995).
Actinomycetes have been isolated from lake and river waters that were used as
source for different water distribution systems (Adams, 1929; Silvey et al., 1953;
Burnan, 1973; Jensen et al., 1994).
11
Actinomycetes tend to form clumps and settle with time (Waksman, 1950, 1962,
1967). For this reason they are mostly found on sediments, or attached to other
living organisms(Silvey et al., 1953). These living organisms provide the right
nutrients, specially carbon and nitrogen (Silvey et al., 1953; Waksman, 1967).
Water containing Actinomycetes still showed taste and odor problems after
chlorination(Adams, 1929). By-products generated by odorous substances, after
their reaction with chlorine, may have a worse taste than the original
substance(Burnan, 1973).
Actinomycetes presented as spores were more resistant to chlorination, and
chlorine resistance also depended on the actinomycete sub-specie under study as
well (Burnan, 1973).
Actinomycetes are among the microbes that attach to solid surfaces such as pipe
walls. (Zacheus et al., 2001). Enough nutrients are required in the distribution
system for them to survive and reproduce(Burnan, 1973).
The presence of available organic carbon within the distribution system is directly
related with the growth of microorganisms inside the system. When the presence
of organic carbon is significant, 1mg/l of free chlorine residual is not enough to
maintain the system free of microorganisms(Silvey, 1954).
12
Even though numerous studies have been done regarding Actinomycetes, and
specially Streptomycetes, there is very little information concerning their
resistance to other inactivation mechanisms such as ultraviolet light, or ozone.
This means that this is an area that could be studied further.
Although Actinomycetes have been found within certain distribution systems,
such as those in England, Finland and the USA, very little is known of their fate
within a distribution system and their ability to produce MIB and geosmin is
affected under set laboratory conditions. This work intends to look into the
aspects stated above. First evaluating Actinomycetes inactivation procedures and
then by determining their response under a control system such as the PVC pipe –
loop.
13MATERIALS AND METHODS
INTRODUCTION
This chapter describes the materials and methodology used to perform the present study.
It will give a brief description of the actinomycete sub-specie employed during the
experiments and a summary of the protocols adopted throughout this investigation.
EXPERIMENTAL METHODS
Sources of Actinomycetes
Inactivation experiments were conducted on streptomyces acquired from the American
Type Culture Collection ATCC (Manassas, VA). In addition, some Actinomycetes were
isolated from the sediments attached to one of the canal walls that supply water for the
metropolitan Phoenix area. Sediments were scrubbed from the wall and kept at 4 degrees
Celsius until plating. Besides canal sampling, several water samples were collected
within the Arizona State University Main Campus. These first sets of samples were
taken between noon and 1 PM. Samples were diluted and plated approximately 2 hours
after been collected.
Culture Collections
The culture used during the present study was acquired from the American Type Culture
Collection (Manassas, VA). The strain employed was the Streptomyces griseous subsps.
14griseous ATCC ® 3343, which is one of the genera of Actinomycetes(Waksman, 1950).
These Streptomycetes had been study before, specially in their relationship with taste and
odor issues, not only for MIB and geosmin production(Gerber et al., 1965), but for other
metabolites such as mucidone as well (Dougherty et al., 1967; Wood et al., 2001 Re-
printed).
Natural Water Isolates
Water samples were taken from one of canals that run across the City of Phoenix
metropolitan area. This specific canal is located at the entrance of the Deer Valley
Treatment Plant. Besides water samples, attached material was scrubbed from the canal
walls. Samples were filtered and diluted before plating. Figures 1 and 2 show
Actinomycetes plated during this research. Figure 1 displays pictures of Streptomycetes
acquired from the American Type Culture Collection. Figure 2 shows Actinomycetes
obtained from the canal. Comparing both sets of pictures it could be observed that figure
1 exhibits whiter, rounder Streptomyces, with very similar diameters. On the other hand,
on figure 2, Actinomycetes look more grayish, the shape is not as round as those on the
previous picture, and more size variation could be observed. Both results were obtained
using Starch-Casein media. Appendix A lists the ingredients and steps required to prepare
this media.
15
Figure 1 Streptomyces griseous subsps. Griseous ATCC ® 3343.
Figure 2 Actinomycetes isolated from natural waters. These were isolated from samples collected at the inlet of Deer Valley Water Treatment Plant (R-16).
Actinomycetes Stock Preparation and Maintenance
Upon receipt, Streptomyces were unfrozen following the procedure indicated by ATCC
(2001). Two types of media were used with this culture. The first medium used is based
16on a Yeast Malt Extract Agar (ISP Medium 2), This medium is recommended by
ATCC to use after the streptomyces are defrost(BD, 2001). The second medium used is
based on a Starch - Casein Agar(Kitchen, 2002). This second medium had been used on
previous studies (Romano et al., 1963b). It is a selective medium and it forms part of the
protocol to determine the presence of Actinomycetes (American Public Health
Association. et al., 1995).
Both broth and solid media were prepared using de-ionized water, and both were
autoclaved at 121 degrees Celsius and 15 psi during at least 15 minutes.
Bacterial enumeration was done using the protocol established for Actinomycetes
isolation. This protocol forms part of the Standard Methods for the Examination of Water
and Wastewater (18th Edition)(American Public Health Association. et al., 1995).
Streptomyces concentration was determined by the preparation of serial dilution plate
counts.
Actinomycetes required a double layer agar plating method. Medium for the bottom layer
agar was prepared, autoclaved and poured into disposable plastic Petri dishes (VWR).
This last part was done inside a bio-safety hood to ensure that there would not be
contamination affecting the Petri dishes. The amount poured into the dishes was between
10 and 15 mL. The top layer agar was also Starch - Casein medium and it was dispensed
into borosilicate test tubes (VWR). After the medium was poured, the tubes were
covered using plastic caps (VWR). Both test tubes and caps were previously autoclaved
under the same conditions as the media (pressure, temperature and time). When
17necessary, both bottom layer and top layer media were prepared in advanced and kept
at 4 degrees Celsius.
Before plating, the top layer agar needed to be softened. This procedure was done so that
0.5 mL of Cyclohexamide could be added to each test tube containing the top layer
agar(American Public Health Association. et al., 1995; Kitchen, 2002). Test tubes were
kept in a water-bed set at 45 degrees Celsius while the cyclohexamide was added.
Cyclohexamide is a an antibiotic which inhibits fungi growth without affecting
Actinomycetes reproduction(Waksman, 1950). This antifungal agent is produced by the
Streptomyces Griseous (Waksman, 1950), which is the genus used in this study.
Cyclohexamide helps to assure that only Actinomycetes grow on the Petri dishes. After
the cyclohexamide was added, 1 ml of water sample containing Actinomycetes was
pipetted into each test tube. After the temperature inside the water bed reached 45
degrees Celsius, the contents of each test tube were poured onto the bottom layer agar.
Once the Petri dishes were plated, they were kept upside down in an incubator set at 28
degrees Celsius during 7 days.
Streptomycetes Enumeration
Bacterial concentration was determine using the procedure established for Actinomycetes
on the Standard Methods(American Public Health Association. et al., 1995). All colony-
forming units CFU that appeared on each Petri dish were individually counted. In the
case of streptomyces and of Actinomycetes in general, counting could be very difficult.
Actinomycetes tend to attach to each other, and for this reason, in some cases CFU varied
18in size. This could be observed on figures 1 and 2. The formula used to determine
bacterial concentration from the CFU enumeration is:
C = 2 ⋅Navg ⋅DF−1 (1)
Where
Navg = The average of the three Petri dishes counting
DF = The dilution factor
Inactivation Studies
Before performing disinfection assays, broth media (2 ml) with Actinomycetes from the
stock was centrifuged and re-suspended in a buffer solution. This procedure was
repeated 3 times to separate Actinomycetes from the media they suspended in. The reason
for this procedure was to reduce the possibility of oxidant demand from the media. The
centrifuged pellet was then re-suspended in a phosphate buffer solution (PBS) at a known
pH, and diluted with PBS to 500 mL. The initial concentration of Streptomycetes was
obtained by removing 1 mL from the flask and then diluting it by 10 fold. Oxidants were
quench using sodium thiosulfate (Na2S203*5H2O).
Chlorine Solution
Chlorine was the first chemical used during this research. The initial concentration used
was set at 2 mg/L free chlorine. For each experiment a phosphoric buffer saline (PBS)
set at a specific pH was prepared. The pH values were set at 5, 7 and 9. A known
volume of autoclaved PBS was dispensed on a beaker. Then 10 mL of the same solution
containing Actinomycetes, at the same pH as the one contained in the beaker was add to
19it. While the streptomyces were added, the PBS was continuously stirred. To
determine the initial Streptomycetes concentration, 1 mL of the stirred solution was
extracted and serially diluted into sterilized test tubes that contained 9.2 mL of sterilized
PBS at the same pH as the stirred solution. Afterwards, as soon as the solution was well
mix, chlorine was added, and again 1 mL of the stirred solution was withdrawn and the
dilutions were repeated. This procedure was repeated at several intervals during one
hour. These extractions served to determine how the streptomyces concentration varied
with the increase the contact time between the microorganisms and the chlorine.
Monochloramine Inactivation
Chloramines are used as disinfectants in drinking water to control taste and odor
problems(Letterman et al., 1999). For this research a monochloramine solution (NH2Cl)
was prepared using 500 mg L-1 NH4Cl (7m M). This solution was adjusted to pH 8-9 with
NaOH. A second solution containing 500 mg L-1 Cl2 (9.35 mM) was prepared from
NaOCl. This second solution was also adjusted to pH 8-9 with HCL. Later, 50 mL of 7
mM NH4C solution were placed in a flask and mixing was started. Then, 50 mL of 9.35
mM chlorine solution were added to the flask that already contained NH4Cl. The
monochloramine solution was refrigerated for three hours before it was used(Westerhoff,
2003). The procedure that followed was exactly the same as the one already mentioned
for chlorine. Microbial concentration was determine the same way as the stated before for
the chlorine experiments. Chloramine concentration was determined with Total and Free
available chlorine readings using the procedure indicated by Hach DR 2000
manual(Hach).
20
Ozonation
Ozone was generated using model 03V-0 (Ozone research & equipment corporation,
Phoenix Arizona). De-ionized water at 4 degrees Celsius was used for the procedure. The
flask containing the cold water was placed inside a bucket on top of a stir plate. The flask
was surrounded by ice and covered by a polystyrene top that left only the top of the flask
open. In order to generate ozone, and after turning the power on the generator, the voltage
is adjusted to its maximum value and the flow rate of O2 gas adjusted until the generator
indicated to be 0.5 l/min. After approximately 45minutes the ozone (O3) stock solution
should be at saturation40 mg L-1. Ozone concentration was obtained indirectly, by
measuring UV absorbency. A 1:3 dilution of the stock solution in dionized water was
clone prior to UV measurements. This was done to ensure that absorbency readings will
be within the linear range (Pei, 2003).
Ultraviolet Irradiation
During the ultra violet (UV) inactivation process, electromagnetic energy is transfer from
a mercury arc lamp to an organism ribonucleic acid (RNA) or deoxyribonucleic acid
(DNA). UV destroys the microorganisms cell wall, and inhibits microbial ability to
reproduce(Agency, 1999). In this case, 50 mL of PBS containing streptomyces were
dispensed onto a Petri dish. Then this solution was exposed to UV radiation.
21UV lamps were located inside a bio-safety cabinet. A colorimetric column ensured that
the UV light was directed over the Petri dish. Streptomyces concentration was
determined by serial dilution, followed by plating(Peccia, 2000). Figure 3 represents a
scheme of the set up used for these set of experiments, and table 2 describes the different
components of the set-up used at that time.
Before performing the inactivation procedure, chemical actinometry was used to measure
the intensity of the electromagnetic radiation. One of the differences between UV
radiation and the other methods exposed above is that UV iridescence is maintained
constant during the experiments. The main difference, is that the is no residual effect
In order to determine the UV dose to which streptomyces were exposed. First three
actinometry cells containing a 0.6 M solution of KI with 0.1 M KIO3 in a 0.01 M borate
buffer (pH 9.25) were exposed to UV light. This actinometry cells were kept under the
UV radiation until the solution turned yellowish, which occurred when tri-iodide was
formed under UV light(Peccia, 2000). At this point a Hach 4000 DR spectrophotometer
was used to measured UV absorbency.
22
Figure 3 Scheme of the set up used for the ultraviolet inactivation procedure
Table 2 Description of the set up used for ultraviolet inactivation
Part Description 1 UV lamp covered with aluminum foil 2 Colorimetric column 3 Petri Dish containing sample 4 Stirring Magnetic plate
Pipe- Loop experiment
A polyvinyl chloride (PVC) pipe-loop has been running for nearly two years as part of a
previous AwwaRF project at the Arizona State University main campus. Figure 4 shows
the pipe-loop set up. This system contains well developed biofilm. Streptomyces were
pipetted into the existing pipe-loop to study their fate and reactions within this apparatus.
1
2
3
4
23Initial liquid samples were smelled and analyzed by GC/MS to determine if MIB or
Geosmin were present in the water.
Figure 4 Pipe – Loop set up spiked with Streptomyces griseous subsps. Griseous ATCC ® 3343. (Ghatpande, 2002)
Table 3 enumerates and describes the sampling ports designated in the system. Samples
were taken every day for seven days, and then every 3 days during 2 weeks. Before
taking the sample, 500 mL were flushed from the system at the sampling port. The first
sample was collected on a 40 mL amber bottle that contained 0.5 mL of sodium azide for
sample preservation. This first sample was used for the GC/MS runs. The second
sample was taken on a plastic 50 mL plastic centrifuge tube (VWR). The tube was left
24on a rack for 30 minutes to allow the streptomyces to settle to the bottom. Disposable
1 mL pipettes were used to transfer sample from the centrifuge tubes into Petri dishes,
which contained the bottom layer agar. Then the normal platting procedure, described on
3.2.1.3 was followed.
Table 3 Sampling Ports within the PVC pipe-loop system
Sampling Port Description 1 Gate valve, unthreaded, soldered 2 PVC labcock valve 3 Dedicated sampling tap at dead end 4 Gate valve (hose bib type) ¾ ” cooper service line 6 ” 5 Gate valve (hose bib type) ¾ ” cooper service line 18 ” 6 Dedicated sampling tap 7 Reservoir
(Ghatpande, 2002)
ANALYTICAL PROCEDURE USED TO DETERMINE INACTIVATION RATES
Since for most of the sets of experiments performed during this study, disinfectant
concentration was not constant, but decay with time, neither Chick's Law, nor Chick -
Watson model could be used to fit the data. Both models require constant disinfectant
concentration throughout the experiment.
For this case, Hom's Model was used to determine the rates for each of the chlorination
assays. This is an empirical model, a generalization of the Chick-Watson pseudo first-
order rate law, which takes into account disinfectants decay. Hom’s Model could be
expressing as follows (Hass et al., 2001):
mnTkNNLn C−=⎟
⎠⎞⎜
⎝⎛
0 (2)
Where:
25N/N0 is the survival ratio
n and m are empirical coefficients
C is the disinfectant concentration
T is contact time
At first, this expression does not consider disinfectant decay. For it to consider
disinfectant decay, C (concentration) has to be replaced by an equation that defines its
decay. The Hom model considers a first order decay equation to replace C.
This expression could be written as follows:
( ) ∫ −−−=⎟
⎠⎞⎜
⎝⎛
xmz
m
n
dzzenkkmC
NNLn
0
1
'0
0 (3)
Where C0 is the initial concentration and 'k is the disinfectant’s decay rate constant. The
integral part of this equation could be replaced by the Incomplete Gamma Function.
Since the Gamma Function is expressed as:
∫ −−=x
z dzzex0
1),( ααγ (4)
α >0 , x > 0
Then the Hom model with disinfectant decay could be expressed as follows:
( ) ( )TnkmnkkmC
NNLn m
n
','
0
0γ•−
=⎟⎠⎞⎜
⎝⎛ (5)
Haas and Hoff stated Hom Model using an Efficiency Hom Factor.
η•−=⎟⎠⎞⎜
⎝⎛ mnTkN
NLn C0
(6)
26Where η is the Hom Efficiency factor. This Factor considers disinfectant decay. η is
not as accurate as the use of the incomplete gamma function, but it is an analytic
approximation.
m
mTnk
mTnk
e
⎥⎥⎥
⎦
⎤
⎢⎢⎢
⎣
⎡−
=⎟⎠
⎞⎜⎝
⎛
⎟⎠
⎞⎜⎝
⎛ −
'
'
1η (7)
The Hom Model with constant disinfectant (equation 2) could also be expressed as
follows:
( )[ ] T) Ln( m Ln(C)n Ln(K) N/NLn- Ln 0 ++= (8)
Equation 8 is the equation used by this researcher to define the values for K, m and n.
27RESULTS
INTRODUCTION
The present chapter describes results obtained from:
Actinomycetes inactivation assays
PVC Pipe-loop assays
The inactivation study includes kinetics for both disinfectant concentration and bacteria
inactivation.
ACTINOMYCETES CULTURING FROM FIELD SAMPLE
Streptomyces griseous subsps. Griseous ATCC ® 3343 were used for all the inactivation
procedures. However, Actinomycetes were isolated from one sample site at the inlet of
Deer Valley Water Treatment Plant, and from eight sample sites within the Arizona State
University main campus. Sediments were scrub from the side of the canal wall (site 16)
and then plated following the procedure indicated in the previous chapter. When plated,
Actinomycetes obtained from this sampling site grew bigger than those obtained from
ATCC. Even thought the test tubes that contain the sample were vortex before the
plating, Actinomycetes grew larger and cells attached each other, for this reason it was
difficult to count and obtain a precise number of colonies from the Petri dish even after a
7 fold dilution.
Tap water Actinomycetes isolates were obtained after sampling at eight different locations
at the Arizona State University main campus. Samples were taken in 250 ml amber
28bottles. The bottles were cleaned and ashed before used. Sodium thiosulfate was
added to each bottle before the sample was taken to quench any chlorine residual that
might have been present at the time. Table 4 displays results obtained from these sample
sites.
INACTIVATION STUDIES
Chlorination
Chlorine Demand from water solution
The Standard Methods for the Examination of Water and Wastewater establishes that
bacterial samples could be diluted using two different solutions. The firsts one is a
peptone water solution, and the second one is a phosphoric buffer solution(American
Public Health Association. et al., 1995). Both solutions were tested for chlorine demand
using chlorine free glassware. Figure 5 shows the results for this experiment. The X axis
represents time in minutes, while the Y axis represents free chlorine concentration in mg
L-1. It is possible to observe that chlorine demand is higher for the 0.1% peptone water
solution. On the other hand, the phosphate buffer saline solution (PBS), has almost the
same trend line as just as the super Q water. All three water solutions contain
streptomyces that had been previously centrifuged and re - suspended as described in a
prior chapter. For this experiment streptomyces concentration was not determine, since
the objective was only to determine chlorine demand from the solutions used.
29For the rest of the experiments, peptone water was not used. This was decided since
after one hour contact time, chlorine had decay 48% on peptone water, while it decay
11% for super Q water and 8% for PBS.
Streptomycetes decay on PBS
During five days Streptomycetes were left on previously autoclaved PBS solution.
Before taking samples from the recipient, the closed container was slightly shaken.
Actinomycetes in general tend to settle, and shaking the container would help to obtain an
even distribution within the recipient. Figure 6 represents the decay of the Streptomycetes
on PBS in the absence of disinfectant.
Curves for pH 5, pH7 and pH 9
Figures 7, 9 and 11 summarized Streptomycetes inactivation using chlorine as
disinfectant. For all the graphs, the X - axis is time (minutes), and the Y- axis is the
survival ratio of microorganisms (N/No), expressed as percentage. No is the initial
Streptomycetes concentration, and N is its concentration at different contact times
throughout the experiments. Data obtained from the chlorine assays was used to
determine the unknown coefficients. Tables5, 6 and 7 below show data collected from the
experiments done for pH 5, 7 and 9. Data was re-arranged and expressed as indicated on
equation 8 (Chapter 3). This procedure was done for pH5, pH 7 and pH 9. Tables 8, 9
and 10 show the parameters used to determine the unknown coefficients from equation 8.
Values for k, m and n were estimated applying a multiple regression procedure to the data
30from tables 8, 9 and 10. This procedure was performed using an office excel
spreadsheet. The results obtained are listed on table 11.
A second analysis was performed using the same data using Mathematica for Students
version 4.2(Wolfram, 2002). This computer program analyzed data using nonlinear data
fitting procedures. Mathematica uses iterative procedures in order to obtain the required
parameters. Table 12 shows results obtained using Mathematica. This second set of
kinetic results have the advantage that takes into account disinfectant decay, since K’ is
one of the coefficients introduced into the equation. While, results obtained on table 11
do not take into account disinfectant decay.
Kinetic coefficients obtained from chlorine decay
The information listed in tables 5, 6 and 7 include chlorine concentration throughout the
assays. These data was used to determine a chlorine decay constants for each of the
experiments. To be congruent with was stated while defining the Hom Model, the
disinfectant decay constant is going to be represented with 'k . The values listed on table
13 and 14 show kinetic coefficients for chlorine decay. These values were obtained by
plotting the data listed on tables 5, 6 and 7. Linearized equation forms (first and second
order) were used to graphically represent the data. For all three cases (pH5, pH 7 and pH
9) highest correlation factors (r2) were obtained using second order functions. But since
the Hom Model uses 'k coefficient obtained from first order kinetic reaction, these
second values were the ones used for the present project. Table 13 shows the values for
'k using a second order reaction, and table 14 show the 'k using first order reaction.
31Number of Streptomycetes during the disinfection process using chlorine
Figures 7, 9 and 11 show Actinomycetes decay through time. Each data point represents
the average of the number of colony forming units counted from the Petri dishes multiply
by 2 and by the dilution coefficient. As it was stated before, for each contact time,
triplicate plates were set. Tables 15 to 17 show the values obtained for each of the Petri
Dishes counted for the first experiment (A) performed at pH 5, pH 7 and pH 9. Data
obtained for experiments B and C is listed on Appendix C. Values obtained as initial
concentration Streptomycetes concentration were unusually high, nevertheless, water
used during the inactivation experiments did not show any apparent turbidity.
Concentration - Time values obtained for chlorine disinfection
Since one of the main objectives of this work was to obtain concentration – time values
for pH 5, pH 7 and pH 9; values calculated using Hom’s model are display on table 18.
For each percent removal (50 %, 99 % and 99.9 %) concentration and time values were
determine using the values k, m and n listed on table 12.
Monochloramine and Ozone inactivation of actinomycetes
Since for monochloramine and ozone only one set of experiment was performed on each
case, a time - concentration curve could not be achieved. For these two cases, only decay
curves are presented. Since the disinfectants used were not constant throughout the
experiment, this researcher used Hom’s Model to obtain inactivation rates for
monochloramine and ozone experiments. Figures 13 and 14 show decay curves for both
32monochloramine and ozone assays. Table 19 display the values obtained for the decay
rate constants k, and for the empiric parameters m and n.
Ultra violet inactivation of actinomycetes
Finally, for the UV assay, and since the ultra violet light is maintain constant throughout
the experiment, the Chick model was used to determine the reaction rate.
kteNN −=0/
Where N/N0 is the survival rate, k is the rate constant and t is the contact time.
Figure 15 shows the results obtained from the inactivation assay.
Inactivation Comparison with other Bacteria
Figure 18 shows the Watson’s Plot for 99% removal using chlorine as disinfectant.
It shows several Watson’s plot for E.Coli, Polio, Coxsackie, Hepatitis, E. Histolytica, etc.
All the curves are set for 99% to 100 % microbes kill. The disinfectant use for those
graphs is measure as ppm of free available chlorine. Each line provides a correlation
between free available chlorine and contact time.
Pipe- Loop experiment
Four different runs were performed using the PVC pipe loop. Each run consisted on
seeding Streptomycetes into the system. Water samples were taken and analyzed for
Actinomycetes before introducing streptomyces into the pipe-loop. This was done using
the protocol described on chapter 3. Figure 19 shows the result for the first run. It could
33be observed that there were no streptomyces present within the pipe-loop before
adding them to the system. After the streptomyces were added, 1156 colony forming
units (CFU) were counted on a single Petri dish. This was the maximum number of CFU
achieved, and it was reached after 5 days. The maximum geosmin concentration reached
was 6.63 ng/L, while the maximum MIB concentration was 125.23 ng/L, both obtained at
day third. No chlorine was added for this first assay, nevertheless, free chlorine average
within the system was 0.02 mg/L.
The results for the second seven-day run are shown on figure 20. In this case, the initial
Streptomycetes count was not zero, since the system still had some of the bacteria that
were previously added. For this second assay, no chlorine was added either, the free
chlorine readings were the chlorine residuals within the pipe-loop. As it was stated
before, this pipe-loop system run with water treated by the City of Tempe.
Pipe-Loop Chlorination
After obtaining the first results from the pipe-loop assay, chlorine was added to the
system. The first step of this third run was to estimate chlorine demand within the PVC
pipe -loop set up. For this reason, a predetermined amount of chlorine was added to the
pipe-loop reservoir and then distributed within the experimental set up.
Total chlorine and free chlorine levels were measure every 30 minutes at each sampling
port. The results were then plotted on a excel spreadsheet and a linear equation was
obtained from sample port 4 chlorine decay values. This sample port was chosen because
it was the one the presented the most acceptable correlation parameter (r2 = 0.97)
34between the chlorine levels measured and the lineal trend originated from the data read
at the time. Chlorine demand for the PVC pipe-loop was estimated from the slope of
linear equation obtained from sample port 4. This assay was replicated to confirm the
systems demand. Table 20 shows the results for both assays. The chlorine consumption
rate obtained for the first run, was the rate used to determine the pumping rate required
to inject chlorine into the system.
Since both total and free chlorine were measure at each sampling port, the combine
chloramines formed within the system was calculating by subtracting free chlorine
values, from total chlorine values at each of the sampling ports. This result was then
plotted against the number of Streptomycetes counts at each of the port, and the results
are shown on Figures 23 and 24.
Streptomycetes within the pipe – loop system
The four pictures shown on figure 25 are from samples taken from the PVC pipe-loop
system during different stages of the process. Picture A was taken at the beginning of the
first pipe-loop run. Before adding Streptomycetes to the system, they were kept in liquid
media. Then they were centrifuged and re-suspended on water. Streptomycetes were
introduced into the system through a valve located on the top of the PVC pipe-loop set
up.
Picture B comes from the second seed of Streptomycetes. At this point it could be
observed that the bacteria displayed are showing signs of stress. Colony forming units
35are not as big and as round as the ones observed on picture A. Nevertheless, at this
point no chlorine was added to the system. The average free chlorine available within the
system at that time was 0.02 mg/L.
Pictures C and D came from the third and forth runs. These two pictures differed from
the previous ones in the fact that some other bacteria, different from the seeded
Streptomycetes appeared on the Petri dishes. At this point is necessary to recall that the
last two runs of the pipe – loop were done while chlorine was been pumped into the
system. This means that some of the bacteria that were present on the PVC pipe –loop
detached from the pipe walls and re-circulate through the system. On the other hand, the
Starch – casein media used to plate Streptomycetes is selective for Actinomycetes in
general. Hence, the bacteria that appear on pictures C and D could be Actinomycetes, but
from different subspecies than the ones seed during this project. For instance, Nocardia is
a type of Actinomycete, which is very ubiquitous within distribution systems. When
plated, Nocardia appears reddish, instead of white, which is the color associated to
Streptomycetes. On pictures C and D a reddish type of bacteria could be easily seen on
the Petri dishes.
At this point, this researcher can not confirm that the reddish bacteria present on the
plates are in fact Nocardia. That type of assessment goes beyond the scope of the present
project. Nevertheless, Nocardia was listed as one of the bacteria present within the pipe
– loop system during a previous research.
36
Table 4 Number of colony forming units of actinomycetes obtained from the samples taken at the Arizona State University main campus.
Sample Location CFU/100 mL ERC fourth floor 9.0 x 102
Laboratory Water fountain # 1 8.20 x 102 Goldwater Center sixth floor 1.24 x 103 Goldwater Center first floor 1.34 x 104
Library 8. 0 x 102 Laboratory Water fountain # 2 6.6 x 102
Tap water ECE 108 4.08 x 103 Super Q water dispenser ECE 108 8.4 x 102
0
0 . 5
1
1 . 5
2
2 . 5
0 1 0 2 0 3 0 4 0 5 0 6 0 7 0
T i m e ( m i n u t e s )
Chl
orin
e C
once
ntra
tion
(mg/
L)
C o n c e n t r a t io n o f F r e e C h lo r in e ( m g / L ) D is t i l l e d W a t e r +S t r e p t o m y c e t e s
C o n c e n t r a t io n o f F r e e C h lo r in e ( m g / L ) 0 . 1 % P e p t o n e W a t e r+ S t r e p t o m y c e t e s
C o n c e n t r a t io n o f T o t a l c h lo r i n e ( m g / L ) P B S + S t r e p t o m y c e s
Figure 5 Free Chlorine Demand for super Q water, 0.1 % peptone water and PBS.
37
Streptomyces (PBS)
10.00
100.00
1 2 3 4 5time (days)
(N/N
o) Set ASet BSet C
Figure 6 Streptomyces decay on PBS
Table 5 Data obtained from the chlorination assays for pH5
Experiment Time
(minutes) Concentration
(mg/L) N/N0
A
0
2.2
1.0000
A 5 1.87 0.0230 A 60 1.45 0.0000 B 0 2.1 1.0000 B 2 1.83 0.1000 B 5 1.72 0.0022 B 10 1.65 0.0000 B 30 1.54 0.0000 B 60 1.42 0.0000 C 0 2.1 1.0000 C 5 1.83 0.1000 C 15 1.67 0.0003 C 30 1.34 0.0000 C 60 1.3 0.0000
38Table 6 Data obtained from the chlorination assays for pH7
Experiment
Time (minutes)
Concentration (mg/L)
N/N0
A 0 2.04 1.0000 A 5 1.93 0.9216 A 15 1.76 0.0784 A 30 1.56 0.0007 A 60 1.39 0.0000 B 0 2.00 1.0000 B 5 1.97 0.9206 B 10 1.73 0.0757 B 15 1.69 0.0103 B 30 1.61 0.0009 B 60 1.53 0.0001 C 0 1.95 1.0000 C 5 1.86 0.0831 C 10 1.72 0.0064 C 15 1.67 0.0009 C 30 1.53 0.0001 C 60 1.47 0.0000
Table 7 Data obtained from the chlorination assays for pH9
Experiment
Time (minutes)
Concentration (mg/L)
N/N0
A 0 2.00 1.0000 A 5 1.58 0.8049 A 10 1.45 0.1228 A 15 1.42 0.0012 A 30 1.39 0.0002 A 60 1.23 0.0001 B 0 2.00 1.0000 B 5 1.84 0.9174 B 10 1.78 0.0092 B 15 1.56 0.0012 B 30 1.45 0.0001 B 60 1.15 0.0001 C 0 2.00 1.0000 C 5 1.67 0.8431 C 10 1.46 0.1745 C 15 1.38 0.0114 C 30 1.23 0.0009 C 60 1.12 0.0001
39Table 8
Values used to determine K, m and n using multiple regressions (pH5)
ln(-ln(N/N0)) ln(t) ln ( C )
1.328 1.609 0.626 2.370 4.094 0.372 0.834 0.693 0.604 1.812 1.609 0.542 2.360 2.303 0.501 2.520 3.401 0.432 2.557 4.094 0.351 0.834 1.609 0.604 2.115 2.708 0.513 2.382 3.401 0.293 2.609 4.094 0.262
Table 9 Values used to determine K, m and n
using multiple regressions (pH7) ln(-ln(N/N0)) ln(t) ln ( C )
-2.505 1.609 0.658 0.934 2.708 0.565 1.988 3.401 0.445 2.310 4.094 0.329 -2.492 1.609 0.678 0.948 2.303 0.548 1.521 2.708 0.525 1.943 3.401 0.476 2.292 4.094 0.425 0.912 1.609 0.621 1.620 2.303 0.542 1.946 2.708 0.513 2.229 3.401 0.425 2.453 4.094 0.385
40Table 10 Values used to determine K, m and n
using multiple regressions (pH9) ln(-ln(N/N0)) ln(t) ln ( C )
-1.528 1.609 0.457 0.741 2.303 0.372 1.911 2.708 0.351 2.148 3.401 0.329 2.231 4.094 0.207 -2.451 1.609 0.610 1.545 2.303 0.577 1.911 2.708 0.445 2.219 3.401 0.372 2.228 4.094 0.140 -1.768 1.609 0.513 0.557 2.303 0.378 1.499 2.708 0.322 1.944 3.401 0.207 2.190 4.094 0.113
Table 11 Coefficients k, m and n for obtained after the multiple regression
K M n Correlation Coefficient
pH5 4.42 -1.15 0.38 0.78
pH7 193947.66 -18.35 -0.58 0.71 pH9 4.56E-03 2.72 1.93 0.72
Table 12 Coefficients k, m and n for obtained using Mathematica Coefficients K m n
pH 5 0.0102 0.5826 5.947 pH 7 0.0589 0.7820 2.022 pH 9 0.05526 0.7968 2.118
Table 13 Kinetic coefficient for chlorine decay (2nd order reaction) and r2
'k (L/mg*minutes) r2
pH 5 0.0037 0.763 pH 7 0.003 0.8432 pH 9 0.0053 0.7927
41
Table 14 Kinetic coefficient for chlorine decay (1st order reaction) and r2
'k (1/minutes) r2
pH 5 -0.0061 0.7385 pH 7 -0.0049 0.8198 pH 9 -0.0077 0.7442
Table 15 Average, Standard Deviation and Standard Error for experiment A set at pH 5 Time
(minutes) n1
(CFU/100 mL) n2
(CFU/100 mL) n3
(CFU/100 mL) average standard deviation
Standard error %
0 1.56E+14 1.24E+14 1.78E+14 1.53E+14 2.72E+13 2.52E-01 18% 5 3.60E+12 3.58E+12 3.34E+12 3.51E+12 1.45E+11 4.19E-03 4%
60 3.56E+09 3.18E+09 3.62E+09 3.45E+09 2.39E+08 4.32E-06 7%
Table 16 Average, Standard Deviation and Standard Error for experiment A set at pH 7 Time
(minutes) n1
(CFU/100 mL) n2
(CFU/100 mL) n3
(CFU/100 mL) average standard deviation
Standard error %
0 1.92E+12 1.74E+12 1.44E+12 1.70E+12 2.42E+11 2.02E-01 14% 5 1.92E+12 1.44E+12 1.34E+12 1.57E+12 3.10E+11 2.25E-01 20%
15 1.56E+11 1.18E+11 1.26E+11 1.33E+11 2.00E+10 1.62E-02 15% 30 8.20E+08 1.52E+09 1.10E+09 1.15E+09 3.52E+08 2.28E-04 31% 60 7.20E+07 8.20E+07 6.20E+07 7.20E+07 1.00E+07 8.43E-06 14%
Table 17 Average, Standard Deviation and Standard Error for experiment A set at pH 9
Time (minutes)
n1 (CFU/100 mL)
n2 (CFU/100 mL)
n3 (CFU/100 mL) average standard
deviation Standard
error %
0 8.00E+10 7.80E+10 8.80E+10 8.20E+10 5.29E+09 9.13E-02 6% 5 6.60E+10 7.00E+10 6.20E+10 6.60E+10 4.00E+09 7.13E-02 6%
10 9.00E+09 1.14E+10 9.80E+09 1.01E+10 1.22E+09 1.69E-02 12% 15 1.58E+08 1.70E+08 1.78E+08 1.69E+08 1.01E+07 1.81E-04 6% 30 1.56E+07 1.78E+07 1.34E+07 1.56E+07 2.20E+06 2.95E-05 14% 60 6.40E+06 7.20E+06 8.60E+06 7.40E+06 1.11E+06 1.48E-05 15%
42
Figure 7 Survival Ratio vs. Time (Set at pH 5)
1.2
1.4
1.6
1.8
2
2.2
2.4
0 20 40 60
Time (minutes)
Chlo
rini
ne C
once
ntra
tion
pH
5 (m
g/L) Experiment A
Experiment BExperiment C
Figure 8 Chlorine decay vs. Time (Set at pH 5)
0.00001
0.00010
0.00100
0.01000
0.10000
1.00000
10.00000
100.00000
0 10 20 30 40 50 60 70
T (minutes)
Surv
ival
Rat
io (N
/No)
Experiment AExperiment BExperiment C
99% removal
99.9% removal
43
Figure 9 Streptomyces inactivation curve for pH 7
1.20
1.40
1.60
1.80
2.00
2.20
0 20 40 60
Time (minutes)
Chlo
rine
Conc
entra
tion
pH 7
(mg/
L) Experiment AExperiment BExperiment C
Figure 10 Chlorine decay vs. Time (Set at pH 7)
0.0001
0.0010
0.0100
0.1000
1.0000
10.0000
100.0000
0 10 20 30 40 50 60 70
T (minutes)
(N/N
o) Experiment AExperiment BExperiment C
99% removal
99.9% removal
44
Figure 11 Streptomyces inactivation curve for pH 9
1.00
1.20
1.40
1.60
1.80
2.00
2.20
0 20 40 60
Time (minutes)
Chl
orin
e C
once
ntra
tion
pH
9 (m
g/L)
Experiment AExperiment BExperiment C
Figure 12 Chlorine decay vs. Time (Set at pH 9)
0.001
0.010
0.100
1.000
10.000
100.000
0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00
T (minutes)
(N/N
o) Experiment AExperiment BExperiment C
45
Table 18 Concentration – Time values (mg/L – minutes) for pH 5, pH 7 and pH 9
Percent Removal 50% 99% 99.9%
pH 5 4.25 26.50 27.39 pH 7 12.48 28.79 25.99 pH 9 11.94 17.54 18.77
Table 19 Rate constant K and coefficients m, n and correlation factor R
Coefficients K m n
Monochloramine 0.0194 1.1382 2.4576 Ozone 0.0068 1.0084 6.2715
Ultra-Violet inactivation 0.1305 (1 / minutes, r2 =
0.92)
Table 20 Estimated chlorine demands for the PVC pipe-loop CL2 Consumption Rate
(mg/L - minutes) r2
1st Run
0.007 0.97
2nd Run 0.004 0.83
46
Figure 13 Streptomyces inactivation using monochloramine. Curve for pH 8.5
0.00100
0.01000
0.10000
1.00000
10.00000
100.00000
0.0 10.0 20.0 30.0 40.0 50.0 60.0
Time (minutes)
Surv
ival
Rat
io (N
/No)
99%
99.9%
47
Figure 14 Streptomyces inactivation using Ozone. Curve for pH 7.
Figure 15 Streptomyces inactivation using UV as disinfectant. Curve for pH 7.
0.01
0.10
1.00
10.00
100.00
0.0 5.0 10.0 15.0 20.0 25.0 30.0
T (minutes)
99% removal
0.00
0.01
0.10
1.00
10.00
100.00
0 10 20 30 40 50 60 70
Time (minutes)
99% removal
99.9% removal
48
0
200
400
600
800
1000
1200
1400
1 2 3 4 5 6 7 8 10 15 20
Days
Num
ber o
f Col
onie
s
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
90.00
100.00
110.00
120.00
130.00
MIB
-Geo
smin
(nG
/L)
Colony CountAverage
Geosmin Average
MIB Average
Figure 16 Colony counts, MIB and Geosmin Concentration
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
0 1 2 3 4 5 6 7
Days
mg/
L
0.00
500.00
1000.00
1500.00
2000.00
2500.00
colo
nies
Free Chlorine (Average)Colony Count (Average)
Figure 17 Free available chlorine within the pipe-loop. These reading were obtained before adding
chlorine to the system.
49
Free Chlorine
y = -0.0067x + 2.0868R2 = 0.9731
0.00
0.50
1.00
1.50
2.00
2.50
3.00
0 50 100 150 200 250 300 350
Time
Free
Chl
orin
e C
once
ntra
tion
(mg/
L)
abcdeLinear (b)
Figure 18 Chlorine demand within the PVC Pipe-Loop System demand (1st run)
Free Chlorine
y = -0.0042x + 1.1218R2 = 0.8365
-0.4
-0.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 50 100 150 200 250 300 350
Time
Free
Chl
orin
e C
once
ntra
tion
(mg/
L)
abcdeLinear (b)
Figure 19 Chlorine demand within the PVC Pipe-Loop System demand (2nd run)
50
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
30 60 90 120 150 180 210 240 300
Time (minutes)
Com
bine
chl
oram
ine
conc
entr
atio
n (m
g/L)
0.0
500.0
1000.0
1500.0
2000.0
2500.0
Col
ony
Cou
nts
ChloramineConcentration
CFU (average)
Figure 20 Combined Chloramine, colony counts (1st run)
Figure 21 Combined Chloramines, colony counts (2nd run)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
30 60 90 120 150 180 200 240 300
Time (minutes)
Com
bine
d C
hlor
amin
e C
once
ntra
tion
(mg/
L)
0.0
200.0
400.0
600.0
800.0
1000.0
1200.0
1400.0
1600.0
1800.0
Col
ony
Cou
nts
Chloramine Concentration
CFU (average)
51.
(a)
(b)
(c)
(d)
Figure 22 Pictures obtained from the samples taken from the pipe-loop
52DISCUSSION
INTRODUCTION
This chapter analyzes and describes the results obtained after the experiments were
completed. It will cover the differences obtained using different media. I will describe
the graphics obtained from the inactivation procedures as well as the results obtained
from the pipe-loop experiments after Streptomycetes were introduced into the system.
ACTINOMYCETES, STREPTOMYCETES AND THE MEDIA
Streptomycetes Griseous
One of the first observations that have to be done regarding this research is related to the
bacterial culture used for the project and the media utilized to maintain it. Streptomyces
griseous subsps. griseous ATCC ® 3343, used during this project, were acquired from the
American Type Culture Collection (Manassas, VA). The strain employed is a genera of
the Actinomycetes group(Waksman, 1950). This culture was chosen due to its ability to
produce off-flavors metabolites.
Two kinds of media were used for this study. The first media used is listed in the
Standard Methods, and cited in Appendix A (Starch-Casein). The main purpose of this
media is to detect the presence of Actinomycetes. When Streptomycetes were first plated
on this media, they spread and reproduced rapidly. According to the literature,
Actinomycetes should be expected to appear after a seven days incubation period. In this
case, Streptomycetes became visible after the third day. By day seven it was very difficult
53to count the amount of colony forming units present on the Petri Dishes. One thing
that was noticed at that time was the smell that came from the dishes. The Streptomycetes
plated generated a very strong and foul smell. As successive platings were done using
Starch – Casein medium, the foulness of the smell diminished.
The second thing that was noticed was the way Streptomycetes’ shape change after
several platings. First results showed white round Streptomycetes, as those seen on figure
1. This first batch of Streptomycetes was kept on Starch – Casein media, without agar.
After several platings the size of the bacteria was perceptible smaller than the first ones
and not as white either. At that point it was believed that Streptomycetes were
experimenting stressful conditions. This due to the physical changes on the
Streptomycetes characteristics and a noticeable diminish of their odor production. For
this reason a second type of media was employed. This second media based on a Yeast
Malt Extract Agar (ISP Medium 2), helped guaranteed that the bacteria kept its physical
characteristics as well as its ability for odor production.
Actinomycetes
Water samples were taken from different places around the Arizona State University
Main Campus. The first set of samples was taken between noon and 2 pm. No
Actinomycetes were present on the plated Petri dishes this first time. A second set of
samples was taken from the same campus, but for this second time, samples were taken
between 6 and 7 am. On this occasion Actinomycetes appeared in all the Petri dishes that
were plated. At this point it could be say that the bacteria that show up on the Petri
54dishes were Actinomycetes, since the media used was Starch – Casein, which is use to
detect Actinomycetes presence. Nevertheless, at the current time it can not be assert
which of the subspecies of the Actinomycetes appeared on the Petri dishes. Further
studies were necessary to determine which subspecies appeared on the plates, and that is
beyond the present project.
Samples were also taken form the canal that carries water into the Deer Valley Treatment
Plant. In this case, Actinomycetes were also present on the Petri dishes that were plated
using sampled water. These Actinomycetes grew bigger than that once plated from the
samples taken from the main campus, and than the Streptomycetes acquired from the
American Type Culture Collection. Figure 2 shows some examples of these
Actinomycetes. Their shape was irregular, their color was more grayish than those plated
from the ASU samples, and the colonies grew bigger than those purchase from the
ATCC. It was also observed that these Actinomycetes developed an aerial mycelium after
7 days. The only thing common in both cases was the foul smell that was perceived from
the samples taken at ASU as well as those obtained at the entrances of the treatment
plant.
INACTIVATION RESULTS
Various levels of inactivation were achieved with the different disinfectants and
inactivation procedures used throughout this project. The most obvious result is the one
obtained from spiking streptomyces onto previously autoclaved PBS. After 5 days
Actinomycetes concentration diminished an average of 43% of its original concentration.
55In this first case, it was the lack of nutrients that provoked concentration reduction,
since no disinfectant was used at this time.
Chlorine Results
Chlorine was the first chemical used to inactivate Streptomycetes used during this present
project. Three separate inactivation procedures were performed at pH 5, pH7 and pH9.
Since during these nine assays, chlorine was not maintain constant throughout the
process, Chick Law was not use to determine the decay constant for these assays. An
alternative procedure was explored for this case. Hom’s Model appeared to be a good
choice, but just as Chick’s law it did not contemplate disinfectant decay. Hass presented
a variation to Hom’s Model in which included disinfectant decay. The formula use to
express the results of the k, m and n included the computation of the incomplete gamma
function in order to obtain the results. One of the parameters that goes into the
incomplete gamma function is precisely the first order kinetic disinfectant
coefficient(Hass et al., 2001).
Values for k, m and n were achieved using a multivariable lineal regression and
Mathematica for students(Wolfram, 2002). These results are shown on tables 11 and 12.
By examining table 11 could be observed the values obtained for k varies several orders
of magnitude depending on the water’s pH. Results obtained on table 11 do not take into
account disinfectant decay. On the other hand, since Mathematica can determine
nonlinear parameters for a given experimental set (time, N/N0), kinetic values were
56obtained for each experiment. The only parameter that could affect the result is the
bacteria itself. Older streptomyces developed hyphae (Waksman, 1962) that might cause
clump formation, which results in a less contact between bacteria and disinfectant. Also
hyphae formation could increase the difficulty to separate bacteria from the broth media
in which streptomyces were kept until before chlorination. If media residual went into
the disinfection experimental set up, chlorine would be consumed faster and it would
diminish its bactericidal effect.
It is important to recall how pH affects chlorine. Above pH 7.5, less than 50% of
chlorine is present as hypochlorous acid (HOCl,), which is known to be a very active
disinfectant. At the same time, hypochlorite (OCl-), which is known to be the less active
than HOCl, Putting this in context with the curves obtained for pH5 (figure 7), (HOCl
predominant), could be observed that almost 2 log removal were obtained when
Streptomycetes where suspended in water at a pH level below 7.5 for less than 7 minutes..
On the other hand, figures 9 and 11, where obtained when water’s pH was set at 7 and 9
respectively, show a shoulder or lag during the first minitues after disinfection stantes.
At this point and looking back at the data obtained, it would have been better to try to
maintain a constant disinfectant concentration throughout each of the batch experiments
that were performed. If that was the case, Chick’s Law could have been applied with out
a problem to determine kinetic coefficients.
57Graphs plotted for pH5, pH 7 and pH 9
Figures 7 to 12 were plotted using the information obtained from the nine assays
performed to obtained decay rate values for chlorine disinfection. Figures 7, 9 and 11
show Streptomycetes decay through time. Figures 8, 10 and 12 show chlorine’s decay
throughout each of the experiments. Figures 9 and 11, which correspond to the graphs
obtained for Streptomycetes decay for the experiments set at pH7 and pH 9, respectively,
shown similarities between them. A shoulder could be appreciated during the first five to
ten minutes of disinfection, then a sharp decay for the next 20 to 30 minutes and finally a
tailing off. On previous researches this type of shoulder or lag has been attributed to
inadequate mixing, diffusion delay or the presence of multiple targets. For this research,
a magnetic stirring plate was used throughout each assay. The stirring plate ensured
complete mixture within the system. The lag could have been caused by the fact that
before adding Streptomycetes into the flask, they were centrifuged and reduced to a very
compact pellet. Even though the pellet was then re-suspended, it is very possible that
clumps of Streptomycetes were present, and it was more difficult for the disinfectant to
go through the clumps.
Figure 7 shows the bacteria decay for pH 5 does not show any shoulder, but is does show
and slightly tail off. Tailing has been attributed to the presence of very resistant
microbes, or to spores. For the present study it is quite obvious in these three graphs is
that the tailing starts after 30 minutes of inactivation. All three graphs appeared to show
two decay rates. First a very fast decay between 0 and 30 minutes, then a slower rate
58between 30 and 60 minutes of contact time between the Streptomycetes and the
disinfectant.
In the case of pH5, one log removal is obtained in the first five minutes of contact times.
This is because there is no shoulder seen on the graph during this period. One remark
that is need to be made is that experiment B set at pH 5, was a re-suspension of
previously chlorinated Streptomycetes. When comparing this result (figure 7), to
experiments A and C, both at the same pH, it can be observed that almost one more log
removal was attained for experiment B, than those obtained for experiments A and C. For
the case of pH 7, to achieve the same one log removal, contact time varies from 10 to 20
minutes. Finally for pH 9, one log removal was attained in the first 15 minutes of
contact time with chlorine. Nevertheless, for the experiment set at pH 9, a couple more
minutes were required to reach 2 log removals. As it was indicated before, this concurs
with the shoulder, follow by a sharp steep that is observed for the assays set at pH 7 and
9.
Concentration – time graph
The figure 23 is a summery of previous research works. Its importance lies on the fact
that it allows the comparison of different inactivation result obtained during this project,
with those obtained on previous research studies for other types of bacteria. Figure 23
shows concentration-time values for E.Coli, Polio, Coxsackie, Hepatitis, E. Histolytica,
etc. All the curves are set for 99% to 100 % kill. The disinfectant use for those graphs is
measure as ppm of free available chlorine. From this graph it is possible to infer that E.
59Coli could be inactivated with low free available chlorine doses (0.01 – 0.05 ppm) and
high contact times, up to 80 minutes, for the lowest chlorine concentration. On the other
side of the spectrum is Ento Amoeba Histolytica, which requires much higher chlorine
doses (2 – 20 ppm), and up to 100 minutes of contact time for the lower chlorine dose, in
order to achieve 99% removal. Comparing this values with the ones obtained on this
project, this researcher can state that 99% inactivation of Streptomycetes was attained
with chlorine concentrations between 0.6 and 1.75 of free chlorine, with contact times
ranging from 4 minutes for the higher chlorine concentrations , and up to 30 minutes for
the lower chlorine concentration. These ranges covered all three pH values under study
for this project. Table 18 lists C-T values for 0.3, 1 and 2 log removal (50, 99 and 99.9
percent removal) for pH5, pH 7 and pH 9. The data presented was calculated using
Hom’s coefficients.
Figure 23 Watson’s plot constructed for different types of microorganisms.
60
Monochloramine, Ozone and Ultraviolet inactivation Results
These inactivation procedures were performed only one time each one of them.
Significant decay rates were obtained using monochloramine, ozone and UV inactivation
procedures on Streptomycetes. Values obtained are listed on table 19. In general it could
be stated that 0.7 log removal of Streptomycetes was obtained after 30 minutes of UV
light exposure. Streptomycetes were exposed to 10.27 µW/cm2 For Ozone, 0.9 log
removal of Streptomycetes was obtained after 15 minutes. This procedure started with an
initial concentration of 1.85 mg/l of Ozone, which was measured indirectly, by measuring
UV absorbency. Finally for monochloramine, 1 log removal was obtained after 20
minutes of exposure to the chemical.
PIPE – LOOP EXPERIMENTS
The pipe –loop experiments were divided into four different assays. The first two set
were conducted without chlorine addition into the pipe – loop system. The last two sets
were carried out with chlorine been pumped into the pipe - loop set up. Streptomyces
griseous subsps. griseous ATCC ® 3343 were seeded into the system before every assay.
Water samples were taken and analyzed for Actinomycetes before spiking streptomyces
into the system. Figures 16 and 17 show the results obtained after the first two runs. It
can be seen, that the first assay showed no Streptomycetes present within the system
before they were spiked into the pipe-loop. Nevertheless. In contrast, figure 17 shows
that samples taken on the first day of the second run Streptomycetes appeared on the
61system in the order of almost 1500 colony forming units per plate. This is logical,
because the system also contained previously seeded Streptomycetes from the previous
experiment. During these first two assays, the average free chlorine available within the
system was 0.02 mg/L. As it was stated before, this pipe-loop system run with water
treated by the City of Tempe.
After the first run, the maximum number of colonies observed on the a single Petri dish
was above 1150 CFU. This value was reached after day 5. For the second run, the peak
was reached on day 3, en the value attained was above 2000 CFU. After the peak was
reached , the number of colony forming units observed in both cases diminished steadily.
The maximum geosmin concentration reached was 6.63 ng/L, while the maximum MIB
concentration was 125.23 ng/L, both obtained at day third.
After obtaining the first results from the pipe-loop assays, chlorine was added to the
system. Chlorine consumption rates were obtained for the third and fourth assays. This
rate was used to determine the pumping rate required to inject chlorine into the system.
This information is very important since it help maintain a constant chlorine level within
the pipe loop. Both total and free chlorine were measure at each sampling port. Combined
chloramines formation within the system was determine. by subtracting free chlorine
values, from total chlorine values. These results were then plotted against the number of
Streptomycetes counts at each of the port, and the results are shown on figures 20 and 21.
These two figures have very similar characteristics. On both of them could be observed a
62steady decay of chloramines along with a decay in CFU. Nevertheless, in figure 21 it
could be seen that the number on CFU is higher in the fourth run than in the third one.
Figures 20 and 21 present chloramines formation and it can be seen that the highest
concentration is reached a 120 minutes after chlorine was added to the system. In both
cases a sharp bacterial decay is observed during the first 120 minutes. After this time,
counts were rather constant during the last 180 minutes of the experiment. A final
observation is that for higher chloramines residual within the system, higher bacterial
decay is achieved.
63CONCLUSIONS
INTRODUCTION
This final chapter lists the conclusions reached after the termination of this thesis.
ACTINOMYCETES, STREPTOMYCETES AND THE MEDIA
The Streptomyces griseous subsps. griseous ATCC ® 3343 used for this project generated
a very strong odor. As successive platings were done the foulness of the smell
diminished.
The type of media used affected physical properties such as shape, size and odor
production. Starch-Casein should be use only to detect the presence of Actinomycetes,
since it does not provide the conditions that are necessary to maintain natural
Actinomycetes properties.
Stressful conditions, such as the one experienced within the pipe – loop affected
Streptomycetes’ shape , size and odor production. Consecutive plating and the age of the
culture also affect the same properties.
INACTIVATION EXPERIMENTS
Various levels of inactivation were achieved with the different disinfectants and
inactivation procedures used throughout this project.
64Streptomyces spiked onto previously autoclaved PBS diminished an average of 43% of
its original concentration after 5 days without any other type of inactivation.
Values obtained for k using Hom’s Model vary greatly depending on the water’s pH and
of the disinfectant used.
Coefficients K m n
Chlorine pH 5 0.0102 0.5826 5.947 Chlorine pH 7 0.0589 0.7820 2.022 Chlorine pH 9 0.05526 0.7968 2.118
Monochloramine 0.01947 1.1382 2.4576 Ozone 0.00585 1.0084 6.2715
Ultra-Violet inactivation
0.1305 [1/minutes]
Similarities were observed from the graphs obtained for Streptomycetes decay for the
experiments set at pH7 and pH 9. A shoulder could be appreciated during the first five to
ten minutes of disinfection, then a sharp decay for the next 20 to 30 minutes and finally a
tailing off.
All three graphs obtained for chlorinated water set at pH 5, pH 7 and pH 9 start tailing
starts after 30 minutes of inactivation. The showed first a very fast decay between 0 and
30 minutes, then a slower rate between 30 and 60 minutes of contact time between the
Streptomycetes and the disinfectant.
In general it could be stated that 0.7 log removal of Streptomycetes was obtained after 30
minutes of UV light exposure. Streptomycetes were exposed to 10.27 µW/cm2.
650.9 log removal of Streptomycetes was obtained after 15 minutes using ozone. This
procedure started with an initial concentration of 1.85 mg/l of Ozone, which was
measured indirectly, by measuring UV absorbency.
1 log removal was obtained after 20 minutes of exposure to monochloramine.
PIPE LOOP ASSAYS
After the first streptomyces were added into the pipe – loop system an average of 1156
colony forming units (CFU) were counted on a single Petri dish. This was the maximum
number of CFU, and it was reached after 5 days.
The maximum geosmin concentration reached was 6.63 ng/L, while the maximum MIB
concentration was 125.23 ng/L, both obtained at day three.
After the last two chlorine assays perfomed on the pipe loop set, bacteria, different from
the seeded Streptomycetes appeared on the Petri dishes. Probably these bacteria were
present on the PVC pipe – loop and detached from the pipe walls after chlorine was
added to the system.
Since the media used to plate the samples obtain from the pipe loop was Starch – casein,
which is selective for Actinomycetes, Bacteria that appear on pictures C and D could be
Actinomycetes, but from different subspecies than the ones seed during this project.
66It was not confirmed that the reddish bacteria present on the after chlorination plates
was in fact Nocardia, because that goes beyond the scoop of the present project.
Nevertheless, Nocardia was listed as one of the bacteria present within the pipe – loop
system during a previous research (Ghatpande, 2002).
RECOMMENDATIONS FOR FUTURE RESEARCH
Streptomycetes and Actinomycetes in general are not considered as harmful bacteria.
Nevertheless, the fact that they generate metabolites such as MIB and Geosmin is a fact
that interests water utilities. For this reason and even though several studies correlate
Actinomycetes, their concentration and the amount of Geosmin and MIB produced, pilot
studies have not been done so far. This type of studies could help to determine what
minimum concentration of Actinomycetes could generate enough metabolites to affect
water’s organoleptic properties.
Actinomycetes have been found within several distribution systems, but no real
correlation between available organic carbon, nitrogen presence and Actinomycetes have
been presented. The knowledge of these interactions might help in preventing
actinomycetes attachment to water distribution system pipe walls.
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74
APPENDIX A
STARCH - CASEIN MEDIA
75
STARCH -CASEIN MEDIA
Make the following stock solutions:
Cyclohexamide-add 10 mg of cyclohexamide to 100 ml of reagent water. After
cyclohexamide has dissolved, filter sterilize. Store 2-8°C.
Ferrous Sulfate-add 1 gm Ferrous Sulfate to 100 ml of reagent water. Store 2-8°C.
Magnesium Sulfate•7H2O-add 5 gm to 100 ml of reagent water. Store at room temp.
To make Starch Casein Agar add the following to 1 liter of reagent water:
Soluble Starch 10.0g
Casein 0.3 g
Potassium nitrate, KNO3 2.0 g
Sodium Chloride, NaCl 2.0 g
Dipotassium hydrogen phosphate, K2HPO4 2.0 g
Calcium Carbonate, CaCO3 0.02 g
Magnesium Sulfate stock solution 1.0 ml
Ferrous Sulfate Stock solution 1.0 ml
Agar 15 gm
No pH adjustment is necessary. Heat media until agar goes into solution. Then dispense
250-300 mL into 500 ml Pyrex bottles No. 1395. Cap bottles with autoclavable orange
76cap and autoclave at 121°C for 15 minutes. After sterilizing allow to solidify at room
temperature then store a 2-8°C.
Note: this recipe is based on the one listed on the Standard methods for the examination
of water and wastewater (18th Edition) and it was provided by Terry Kitchen from the
City of Phoenix Laboratory.
77
APPENDIX B
3 X PHOSPHATE BUFFER SALINE
783 x Phosphate Buffer Saline
Materials
NaCl
Na2HPO4.
NaH2PO4 . H2O
Nanopure water
23 ml of 5M NaCl
18 ml of 0.5M NaPO4 Buffer
258.6 nanopure water.
Methods
Prepare 0.5 M of Sodium Phosphate monobasic (NaH2PO4 . H2O) solution using
nanopure water.
Prepare 0.5 M of Sodium Phosphate dibasic (Na2HPO4.) solution using nanopure water.
Stir 0.5M Na2HPO4, then add 0.5M Na2HPO4 and adjust the pH
Transfer 23 ml of 5M NaCl into a bottle, and add 18 ml of 0.5M NaPO4 Buffer. Then add
258.6 nanopure water to the bottle
To prepare 1 x Phosphate Buffer Saline add 600 ml of nanopure water to the previous
solution.
Note: this recipe was provided by Raghunatha Komaragiri, graduate student at Arizona
State University.
79
APPENDIX C
DATA OBTAINED FROM CHLORINE EXPERIMENTS
80Chlorine Experiments
Average, Standard Deviation and Standard Error for experiment B set at pH 5 Time
(minutes) n1 n2 n3 average standard deviation standard error %
0 1.78E+12 1.56E+12 1.90E+12 1.75E+12 1.72E+11 1.40E-01 10% 2 1.92E+11 1.52E+11 1.78E+11 1.74E+11 2.03E+10 1.52E-02 12% 5 3.86E+09 3.96E+09 3.68E+09 3.83E+09 1.42E+08 2.31E-04 4%
10 4.24E+07 4.02E+07 4.90E+07 4.39E+07 4.58E+06 3.61E-06 10% 30 7.60E+06 6.80E+06 6.60E+06 7.00E+06 5.29E+05 4.98E-07 8% 60 4.90E+06 3.92E+06 4.34E+06 4.39E+06 4.92E+05 3.75E-07 11%
Average, Standard Deviation and Standard Error for experiment C set at pH 5 Time
( minutes) n1 n2 n3 average standard deviation standard error %
0 1.92E+12 1.74E+12 1.44E+12 1.70E+12 2.42E+11 2.02E-01 14% 5 1.96E+11 1.70E+11 1.58E+11 1.75E+11 1.94E+10 1.86E-02 11%
15 4.70E+08 3.96E+08 4.50E+08 4.39E+08 3.83E+07 4.31E-05 9% 30 3.78E+07 3.56E+07 3.14E+07 3.49E+07 3.25E+06 3.50E-06 9% 60 1.90E+06 2.46E+06 2.22E+06 2.19E+06 2.81E+05 2.47E-07 13%
Average, Standard Deviation and Standard Error for experiment B set at pH 7 Time
( minutes) n1 n2 n3 average standard deviation
standard error %
0 1.34E+11 1.42E+11 1.52E+11 1.43E+11 9.02E+09 8.94E-02 6% 5 1.64E+11 1.44E+11 8.60E+10 1.31E+11 4.05E+10 2.90E-01 31%
10 1.60E+10 8.60E+09 7.80E+09 1.08E+10 4.52E+09 3.20E-02 42% 15 1.78E+09 1.52E+09 1.10E+09 1.47E+09 3.43E+08 2.49E-03 23% 30 1.12E+08 1.52E+08 1.34E+08 1.33E+08 2.00E+07 1.52E-04 15% 60 7.20E+06 8.20E+06 6.20E+06 7.20E+06 1.00E+06 7.70E-06 14%
81Average, Standard Deviation and Standard Error for experiment C set at pH 7
Time ( minutes) N1 n2 n3 average standard
deviation standard
error %
0 1.48E+12 1.74E+12 1.86E+12 1.69E+12 1.94E+11 1.62E-01 11% 5 1.92E+11 1.44E+11 8.60E+10 1.41E+11 5.31E+10 3.28E-02 38%
10 1.34E+10 1.06E+10 1.50E+10 1.30E+10 2.23E+09 1.58E-03 17% 15 1.56E+09 1.70E+09 1.38E+09 1.55E+09 1.60E+08 1.41E-04 10% 30 1.78E+08 1.66E+08 1.26E+08 1.57E+08 2.72E+07 1.93E-05 17% 60 1.56E+07 1.62E+07 1.38E+07 1.52E+07 1.25E+06 1.27E-06 8%
Average, Standard Deviation and Standard Error for experiment B set at pH 9 Time
(minutes) n1 n2 n3 average standard deviation
Standard error %
0 1.34E+11 1.58E+11 1.44E+11 1.45E+11 1.21E+10 1.17E-01 8% 5 1.16E+11 1.26E+11 1.58E+11 1.33E+11 2.19E+10 1.69E-01 16%
10 1.24E+09 1.48E+09 1.30E+09 1.34E+09 1.25E+08 1.15E-03 9% 15 1.58E+08 1.70E+08 1.78E+08 1.69E+08 1.01E+07 1.19E-04 6% 30 1.62E+07 1.44E+07 1.34E+07 1.47E+07 1.42E+06 1.29E-05 10% 60 1.18E+07 1.36E+07 1.52E+07 1.35E+07 1.70E+06 1.40E-05 13%
Average, Standard Deviation and Standard Error for experiment C set at pH 9 Time
(minutes) n1 n2 n3 average standard deviation
Standard error %
0 1.02E+11 1.20E+11 1.08E+11 1.10E+11 9.17E+09 1.41E-01 8% 5 8.60E+10 9.80E+10 8.40E+10 8.93E+10 7.57E+09 1.16E-01 8%
10 1.78E+10 1.90E+10 2.20E+10 1.96E+10 2.16E+09 3.06E-02 11% 15 1.16E+09 1.14E+09 8.60E+08 1.05E+09 1.68E+08 2.21E-03 16% 30 9.40E+07 7.00E+07 8.00E+07 8.13E+07 1.21E+07 1.60E-04 15% 60 1.34E+07 1.14E+07 8.60E+06 1.11E+07 2.41E+06 3.07E-05 22%
82
APPENDIX D
DATA OBTAINED FROM MONOCHLORAMINE, OZONE AND UV
EXPERIMENTS
83Average, Standard Deviation and Standard Error for assay using monochloramine.
Time (minute)
Concentration (mg/L) n1 n2 n3 average standard
deviation 1/dilution N
0 2.04 176 80 109 121.7 49.2 1.00E+06 2.43E+08 5 1.95 244 340 292 292.0 48.0 1.00E+05 5.84E+07
15 1.39 73 40 34 49.0 21.0 1.00E+05 9.80E+06 30 1.15 107 116 45 89.3 38.7 1.00E+02 1.79E+04 60 0.87 260 232 224 238.7 18.9 1.00E+01 4.77E+03
Average, Standard Deviation and Standard Error for assay using ozone.
Time (minute)
Concentration (mg/L) n1 n2 n3 average standard
deviation 1/dilution N
0 1.86 67 70 73 70.0 3.0 1.00E+07 1.40E+09 5 1.42 53 47 44 48.0 4.6 1.00E+06 9.60E+07
15 1.1 60 56 64 60.0 4.0 1.00E+05 1.20E+07 30 0.85 42 52 51 48.3 5.5 1.00E+05 9.67E+06
Average, Standard Deviation and Standard Error for experiment using ultraviolet radiation.
Time (minutes)
Dose (uW/cm2) 1 2 3 average standard
deviation 1/dilution N
0 10.27 456 400 428.0 39.6 1.00E+05 8.56E+07 5 10.27 328 344 400 357.3 37.8 1.00E+05 7.15E+07
15 10.27 192 160 208 186.7 24.4 1.00E+04 3.73E+06 30 10.27 296 352 384 344.0 44.5 1.00E+03 6.88E+05 60 10.27 360 384 296 346.7 45.5 1.00E+02 6.93E+04
84APPENDIX E
STREPTOMYCETES DECAY ON PBS
85Streptomycetes decay on PBS
Time ( days) N1 average 1/dilution N Log (N/No)
pH 7 Set A
1 17 17.0 1.00E+01 340.00 0.00 100.00 2 16 16.0 1.00E+01 320.00 -0.03 94.12 3 14 14.0 1.00E+01 280.00 -0.08 82.35 4 12 12.0 1.00E+01 240.00 -0.15 70.59 5 9 9.0 1.00E+01 180.00 -0.28 52.94
Time ( days) N1 average 1/dilution N Log (N/No)
pH 7 Set B
1 23 23.0 1.00E+01 460.00 0.00 100.00 2 16 16.0 1.00E+01 320.00 -0.16 69.57 3 13 13.0 1.00E+01 260.00 -0.25 56.52 4 10 10.0 1.00E+01 200.00 -0.36 43.48 5 8 8.0 1.00E+01 160.00 -0.33 34.78
Time ( days) N1 average 1/dilution N Log (N/No)
pH 7 Set C
1 25 25.0 1.00E+01 500.00 0.17 100.00 2 21 21.0 1.00E+01 420.00 0.09 84.00 3 17 17.0 1.00E+01 340.00 0.00 68.00 4 13 13.0 1.00E+01 260.00 -0.12 52.00 5 10 10.0 1.00E+01 200.00 -0.23 40.00
APPENDIX F
PVC PIPE-LOOP: COLONY COUNTS, MIB AND GEOSMIN
87
Colony counts, MIB and Geosmin values for the 1st Pipe-Loop Run
Days Colony Counts
Colony Counts
Colony Count
Average
MIB Sample
A
MIB Sample
B
MIB Average
Geosmin Sample
A
Geosmin Sample
B
Geosmin Average
1 0 0 0 23.23 22.40 22.82 4.10 3.10 3.60 2 144 113 128.5 23.92 22.32 23.12 4.55 3.56 4.06 3 86 98 92 81.75 168.70 125.23 5.87 7.39 6.63 4 664 1128 896 24.00 24.20 24.10 4.16 3.73 3.95 5 1248 1064 1156 21.00 24.09 22.55 3.54 4.46 4.00 6 1216 1016 1116 15.41 15.98 15.70 3.36 3.13 3.25 7 920 944 932 11.39 12.87 12.13 2.96 3.25 3.11 8 912 904 908 7.97 8.84 8.41 2.22 2.17 2.20
10 888 896 892 7.87 7.82 7.85 2.02 2.15 2.09 15 840 720 780 7.32 6.63 6.98 1.58 1.20 1.39 20 680 632 656 6.76 5.44 6.10 1.13 0.24 0.69
Colony counts, MIB and Geosmin values for the 2nd Pipe-Loop Run
Days Free Chlorine (Average) Total Chlorine Colony Counts Colony Count (Average) 0 0.020 0.02 1496 1496.00 1 0.025 0.04 1600 1680 1640.00 2 0.030 0.04 2000 2160 2080.00 3 0.025 0.04 2048 2104 2076.00 4 0.020 0.04 2032 1888 1960.00 5 0.015 0.05 1600 1680 1640.00 6 0.015 0.04 1520 1680 1600.00 7 0.015 0.04 1200 1312 1256.00
88
Colony counts and chlorine values for the3rd Pipe-Loop Run
Free Chlorine Sample Ports
minutes a b C d e 30 2.14 1.88 0.22 0.74 1.96 60 1.87 1.80 1.08 1.41 1.62 90 1.45 1.56 0.77 1.25 1.36
120 1.33 1.06 1.22 0.64 1.05 150 1.11 1.09 0.77 1.22 0.78 180 0.90 0.89 0.56 0.94 0.87 200 0.83 0.76 0.49 0.73 0.67 240 0.53 0.45 0.37 0.59 0.38 300 0.08 0.15 0.03 0.05 0.08
Total Chlorine Sample Ports
minutes a b c d e 30 2.42 2.15 0.4 0.94 2.46 60 2.07 1.93 1.33 1.58 1.96 90 1.6 1.66 1.25 1.45 1.74
120 1.34 1.41 1.32 0.79 1.44 150 1.17 1.15 0.82 1.29 1.09 180 1.06 1.09 0.72 1.11 1.04 200 0.87 0.92 0.58 0.83 0.93 240 0.62 0.56 0.46 0.61 0.63 300 0.12 0.19 0.13 0.24 0.17
Combine Chloramines Concentration (mg/L) Sample Ports
colony counts
minutes a b c d e 1 2 3 average standard deviation
30 0.28 0.27 0.18 0.20 0.50 2000 1880 1960 1946.7 61.1 60 0.20 0.13 0.25 0.17 0.34 1360 1480 1408 1416.0 60.4 90 0.15 0.10 0.48 0.20 0.38 1280 1496 1408 1394.7 108.6
120 0.01 0.35 0.10 0.15 0.39 960 1080 920 986.7 83.3 150 0.06 0.06 0.05 0.07 0.31 904 984 936 941.3 40.3 180 0.16 0.20 0.16 0.17 0.17 824 880 920 874.7 48.2 210 0.04 0.16 0.09 0.10 0.26 808 816 800 808.0 8.0 240 0.09 0.11 0.09 0.02 0.25 784 760 744 762.7 20.1 300 0.04 0.04 0.10 0.19 0.09 744 728 712 728.0 16.0
89
Colony counts and chlorine values for the4th Pipe-Loop Run
Free Chlorine Sample Ports
Time (minutes) a b C D E
30 1.14 1.25 0 0.61 1.04 60 0.94 0.93 0 0.69 0.72 90 0.79 0.73 0.44 0.68 0.49
120 0.46 0.41 0.31 0.6 0.4 150 0.35 0.36 0.21 0.35 0.33 180 0.24 0.25 0.13 0.26 0.19 210 0.20 0.15 0.1 0.22 0.17 240 0.08 0.13 0.09 0.13 0.11 300 0.07 0.11 0.05 0.08 0.08
Total Chlorine Sample Ports
Time (minutes) a b C d E
30 1.37 1.33 0 0.86 1.08 60 1.15 1.01 0.05 0.77 0.87 90 0.86 0.77 0.63 0.71 0.76
120 0.65 0.66 0.51 0.66 0.58 150 0.54 0.52 0.42 0.59 0.52 180 0.45 0.46 0.34 0.41 0.4 210 0.39 0.35 0.2 0.38 0.31 240 0.32 0.32 0.26 0.36 0.29 300 0.27 0.23 0.17 0.25 0.21
Combine Chloramines Concentration (mg/L) Sample Ports
colony counts
Minutes a b c d E 1 2 3 average standard deviation
30 0.23 0.08 0.00 0.25 0.04 1600 1576 1696 1624.0 63.5 60 0.21 0.08 0.05 0.08 0.15 1464 1416 1496 1458.7 40.3 90 0.07 0.04 0.19 0.03 0.27 824 872 768 821.3 52.1 120 0.19 0.25 0.20 0.06 0.18 712 648 680 680.0 32.0 150 0.19 0.16 0.21 0.24 0.19 696 704 720 706.7 12.2 180 0.21 0.21 0.21 0.15 0.21 640 608 664 637.3 28.1 200 0.19 0.20 0.10 0.16 0.14 616 576 584 592.0 21.2 240 0.24 0.19 0.17 0.23 0.18 584 560 568 570.7 12.2 300 0.20 0.12 0.12 0.17 0.13 552 544 560 552.0 8.0