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