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UNIVERSllY OFHAWAU UBR1\RY

DIVERSITY AND TRANSPORT OF BACTERIA AND VIRUSES IN SOILS

FOLLOWING IRRIGATION WITH RECYCLED WATER

A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THEUNIVERSITY OF HAWAI'I IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF

MASTER OF SCIENCE

IN

CIVIL ENGINEERING

DECEMBER 2003

ByTieshi Huang

Thesis Committee:

Roger Babcock, ChairpersonChittaranjan Ray

Albert Kim

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my professors and friends who have

helped me during my study at the University of Hawaii at Manoa. My advisor Dr. Roger

Babcock has given me the detailed instructions and suggestions regarding my thesis

research. Dr. Chittaranjan Ray and Dr. Albert Kim provided very helpful suggestions for

me. I would like to thank Ms. Bunnie Yoneyama for her help in laboratory. The help

from my laboratory mates Sumon Kanpirom is also appreciated.

I would like to give special thanks to my wife Jing Meng and my son Jianxin Huang.

Their love and support are always with me throughout my studies.

iii

ABSTRACT

Field and laboratory analyses were conducted to study bacteria and virus transport and

diversity in surface soils following irrigation with recycled water. Field test plots were

irrigated with R-l (oxidized, filtered, disinfected) water and tap water, and pan lysimeters

were used to collect leachate water samples. R-l, R-2 (oxidized, disinfected), R-3

(oxidized only) and tap water were applied to soil columns. Fecal coliform and coliphage

were only found in the leachate from the R-3 soil column. Polymerase chain reaction

denaturing gradient gel electrophoresis analysis (PCR-DGGE) of 16S rRNA genes was

used to analyze the bacterial population in leachate and soil samples from different

depths. The R-l field test plot had a more diversified bacterial community than a tap

water control plot. Surface soils from field test plots had more bacteria species than that

of bottom soils. Different DGGE banding profiles were found at different depths in R-l,

R-2 and R-3 effluent soil columns. UV dosage did have some effect on soil microbial

diversity.

iv

TABLE OF CONTENTS

ACKNOWLEDGEMENTS .iii

ABSTRACT .iv

TABLE OF CONTENTS v

LIST OF TABLES vii

LIST OF FIGURES viii

LIST OF PHOTOS .ix

CHAPTER 1. INTRODlJCTION 1

1.1 General 1

1.2 Introduction to Denaturing Gradient Gel Electrophoresis (DGGE) .4

1.3 Objectives 5

CHAPTER 2. MATERIALS AND METHODS 7

2.1 Field Study 7

2.2 Laboratory Study 10

2.3 Soil. 10

2.4 Water Samples 11

2.5 Grass Samples 12

2.6 DNA Extraction and PCR Amplification 12

2.7 DGGE Analysis 13

CHAPTER 3. RESlJLTS AND DISCClJSION 15

3.1 Field Study 15

3.1.1 Lysimeter Water Sample Analysis 15

v

3.1.2 DGGE Analysis 17

3.2 Soil Column Test. 19

3.2.1 R-3 Water Column Leaching Experiment.. 19

3.2.2 Tap Water, R-1 Water and R-2 Water Column Leaching Experiment.. 21

3.2.3 DGGE Analysis 23

CHAPTER 4. CONCLUSIONS 27

APPENDIX A. Physical Conditions of Column Leaching Experiment 29

APPENDIX B. Genomic DNA Isolation protocol.. 31

APPENDIX C. DGGE Reagent 32

APPENDIX D. DGGE Operation .35

REFERENCES 39

vi

LIST OF TABLES

Table Page

Table 2.1: Irrigation Rate of Field Test Plots 7

Table 3.1: Average Water Quality Data of R1 Water and Lysimeter Samples 15

Table 3.2: Fecal Coliform, Total Coliform, and Coliphage Concentrations in

Laboratory Soil Column Applied Waters 22

Table A.1: Physical Conditions of Tap Water Column Leaching Experiment. 29

Table A.2: Physical Conditions of R-3 Column Leaching Experiment. .29

Table A.3: Physical Conditions of R-2 Column Leaching Experiment 29

Table A.4: Physical Conditions of R-1-70 Column Leaching Experiment .30

Table A.5: Physical Conditions of R-1-140 Column Leaching Experiment 30

Table A.6: Physical Conditions of R-1-400 Column Leaching Experiment. 30

Table C.1: 40% Acrylamide/Bis (37.5:1) 32

Table C.2: 50x TAB Buffer. 32

Table C.3: Percentage Acrylamidelbis needed for a Particular Size Range 32

Table C.4: 0% Denaturing Solution 33

Table C.5: 100% Denaturing Solution 33

Table C.6: Formamide & Urea Amounts for Denaturing Solutions Less Than 100% 33

Table C.7: 10% Ammonium Persulfate 34

Table C.8: DCode Dye Solution 34

Table C.9: 2x Gel Loading Dye 34

Table C.1O: Ix TAB Running Buffer 34

vii

LIST OF FIGURES

Figure Page

Figure 3.1: Electrophoresis of PCR Products '" 18

Figure 3.2: DGGE Analysis ofPCR Amplified 16S rDNA Fragments Obtained

from Field Test Plot Samples 19

Figure 3.3: Fecal Coliform and Coliphage in the R-3 Water Laboratory Column

Leachate 21

Figure 3.4: Total Coliform in the Leachate from the Tap Water, R-I-70, R-I-140

and R-2 Soil Columns 22


from R-l, R-2 and R-3 Laboratory Soil Column Samples 24


from Rl Soil Column Samples 25

viii

LIST OF PHOTOS

Figure Page

Photo 2.1: SBWWTP Field Test Plot 8

Photo 2.2: Pan Lysimeter 8

Photo 2.3: Sand Filtration 9

Photo 2.4: UV Disinfection System 9

Photo 2.5: Laboratory Soil Column 11

Photo 2.6: Bead Beater and Microcentrifuge 12

Photo 2.7: DCode System for DGGE Analysis, Horizontal Electrophoresis System,

and PCR Instrument 14

Photo 2.8: Gel Documentation System 14

ix

CHAPTER 1. INTRODUCTION

1.1 General

Water is becoming a limited resource in highly populated metropolitan areas. In such

areas, irrigation with recycled water is increasingly attractive as shortages and/or costs of

fresh water increase. Recycled water is primarily used for irrigation of golf courses,

parks and recreation areas. In Hawaii, nearly all of the drinking water supply is obtained

from unconfined groundwater aquifers under each island. On Oahu, with a population of

approximately one million, all of the drinking water supply is obtained from

groundwater. Approximately 100 million gallons per day is withdrawn from the Pearl

Harbor Aquifer, much of which is overlain with prime agricultural land. Oahu also

currently has 35 golf courses in operation. Population expansion has placed pressure on

water supplies such that there is a push (by the State Department of Health, DOH) to

implement large scale recycling of treated wastewater for agricultural and golf course

irrigation on Oahu. The DOH recognizes three types of recycled water; R-3 is

undisinfected secondary effluent, R-2 is disinfected secondary effluent with fecal

coliform < 23 CFU/lOO ml, and R-l is filtered and disinfected secondary effluent with

fecal coliform < 1 CFU/lOO mI.

Application of wastewater to soil could result in the contamination of groundwater

and soil with pathogens. Pathogen exposure is one of the most important concerns

related to the risk of recycled water irrigation (Quanrud et aI., 2003). Pathogens in

secondary effluent include the environmentally resistant oocysts of Crytosporidium

parvum, cysts of Giardia lamblia, and enteric pathogenic bacteria and viruses. Fecal

1

coliform, total coliform, Escherichia coli and enterococci are some commonly used

indicators to detect human pollution (Desmarais et aI., 2002). Coliphage virus is a

commonly occurring virus in wastewater, and is not commonly found as background in

the environment. In general, fecal coliform bacteria and coliphage virus are the most

representative indicators for pathogen presence, and simple standardized methods exist

for their enumeration. However, an absolute relationship between the enumeration of

fecal coliform and other pathogens does not exist.

Soil is an effective filter for pathogen removal. Pathogen removal in soil is mostly

dependent upon the degree of adsorption. Up to 95% of pathogens concentrate near the

soil surface, while most of the remainder concentrates in subsurface soils (Pettygrove and

Asano 1985). There are many field and laboratory studies investigating the mobility and

removal of bacteria and virus in soils, ground waters and soil columns. The studies

indicate that significant reduction in numbers of bacteria and viruses occurs during the

passage of wastewater through the soil and that removal is controlled by several factors.

The factors that influence virus persistence in the subsurface soil are temperature, virus

type, soil type, infiltration rate and microbial activity (Yates et aI., 1985; Nasser and

Oman 1999; Schijven and Hassanizadeh 2000; Nasser et aI., 2002; Quanrud et aI., 2003).

Among these factors, virus type is the most important factor in virus adsorption to soil

(Nasser et aI., 2002). Besides the physical factors, the effect of soil microbial activity on

virus survival is also important. Clay minerals in soil may protect viruses from biological

biodegradation (Lopez-Torres et al., 1987). Antiviral activity in soil attributed to aerobic

microorganisms could result in three fold increases in virus inactivation (Hurst 1988).

Furthermore, it has been demonstrated that bacteria could utilize viruses as growth

2

substrates (Lipson and Stotzky 1985). The factors that influence bacteria removal

include soil type (Yamaura 1989), infiltration rate, and the addition of flocculent and

adsorbing materials (Entry et aI., 2003; Parkpian et al., 2002). Polyacrylamide (PAM) is

an effective flocculent, which could reduce bacteria transport in percolating water. It is

reported that with proper application PAM + A!z(S04)3 and PAM + CaO can reduce total

coliform bacteria and fecal coliform bacteria by up to 50% (Entry et aI., 2003).

Estimates of microbial diversity within an environment include culture-dependent and

culture-independent methods. Culture -dependent methods are tedious and time

consuming due to the need for selective media for all species of interest. It is difficult to

detect more than about 1% of the bacteria species by any conventional cultivation

techniques (Ward et aI., 1990). Recent advances in molecular fingerprinting techniques

have overcome these difficulties. Analysis of total community DNA extracted from an

environment using culture-independent methods permits the detection of uncultured

organisms. Denaturing gradient gel electrophoresis (DGGE) separation of 16S rRNA

gene amplification products is one of the powerful tools to characterize the microbial

community and monitor the dominant population. To date, the16S rRNA genes have

been widely used for microbial diversity analysis of natural environments since the

nucleotide sequences of 16S rRNA genes are very conservative and change much more

slowly than whole genome sequences. The eight variable regions of the16S rRNA gene

are commonly used for microbial diversity analysis at the species scale. DGGE has been

largely used for the description of several environments at the molecular level (Muyzer et

aI., 1993; Juck et aI., 2000; Lapara et aI., 2000). Using DGGE, PCR products of the same

length but with different sequences can be separated. The total number of DGGE bands

3

provides an estimate of the microbial diversity within a given environment (Muyzer et

al.,1993).

The Schofield Army Barracks Wastewater Treatment Plant (SBWWTP) provides

secondary treatment using the conventional activated sludge process and chlorine

disinfection with NaGCl. At present, the treated effluent from SBWWTP is discharged

to an irrigation ditch operated by the Dole Foods Company (OFC). The effluent is mixed

with water drawn from the Wahiawa Reservoir and used for irrigation of diversified

agricultural crops such as coffee, papaya, corn, and macadamia nuts. Prior to this, the

water was used for about 70 years to irrigate sugar cane on the same lands.

1.2 Introduction to Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis (DGGE) is an electrophoretic method to

identify single base changes in a segment of DNA. In a denaturing gradient acrylamide

gel, double-stranded DNA is subjected to an increasing denaturant environment and will

melt in discrete segments called "melting domains". The melting temperature (TnJ of

these domains is sequence-specific. When the Tm of the lowest melting domain is

reached, the DNA reduces its mobility in a polyacrylamide gel. Since the Tm of a

particular melting domain is sequence-specific, the presence of mutation will alter the

melting profile of that DNA when compared to wild-type. DNA containing mutations

will encounter mobility shifts at different positions in the gel than the wild-type. If the

fragment completely denatures, then migration again becomes a function of size.

In DGGE, the denaturing environment is created by a combination of uniform

temperature, typically between 50 and 65°C and a linear denaturant gradient formed with

4

urea and formamide. A solution of 100% chemical denaturant consists of 7 M urea and

40% formamide. The denaturing gradient may be formed perpendicular or parallel to the

direction of electrophoresis. A perpendicular gradient gel, in which the gradient is

perpendicular to the electric field, typically uses a broad denaturing range, such as 0

100% or 20-70%. In parallel DGGE, the denaturing gradient is parallel to the electric

field, and the range of denaturant is narrowed to allow better separation of fragments.

When running a denaturing gradient gel, both the mutant and wild-type DNA

fragments are run on the same gel. This way, mutations are detected by differential

migration of mutant and wild-type DNA. The mutant and wild-type fragments are

typically amplified by the polymerase chain reaction (PCR) to make enough DNA to load

on the gel. Optimal resolution is attained when the molecules do not completely denature

and region screened is in the lowest melting domain. The addition of a 30-40 base pair

GC clamp to one of the PCR primers insures that the region screened is in the lower

melting domain and that the DNA will remain partially double-stranded. The size of the

DNA fragments run on a denaturing gel can be as large as 1 kb in length, but only the

lower melting domains will be available for mutation analysis. For complete analysis of

fragments over 1 kb in length, more than one PCR reaction should be performed.

1.3 Objectives

The objective of this study was to evaluate the potential impacts on the underlying

groundwater of using treated effluent from the SBWWTP for future golf course

irrigation, with an emphasis on evaluating the movement of fecal coliform bacteria and

coliphage virus through the shallow soil layer and deep underlying unsaturated saprolitic

5

layers, and estimating bacterial diversity in surface soils following irrigation with

recycled water. In this study, both a field test and laboratory soil column tests were

operated to evaluate the potential impact of irrigating with SBWWTP effluent. Using

both the traditional plate counting method and DGGE separation of 16S rRNA gene

amplification products, the study emphasized estimating bacterial diversity in surface

soils following irrigation, evaluation of the movement of fecal coliform bacteria and

coliphage virus through the shallow soils, and estimating the ultraviolet radiation (UV)

dosages necessary to reduce the fecal coliform bacteria and coliphage virus counts in the

shallow soils to be irrigated with SBWWTP effluent.

6

CHAPTER 2. MATERIALS AND METHODS

2.1 Field Study

Two field test plots were installed at SBWWTP and irrigated with either R-l water or

tap water (control). R-l water was produced using R-3 effluent from SBWWTP followed

by bunch-scale sand filtration and UV disinfection. The UV disinfection dosage was 140

mW-s/cm2 with 3 UV units in series. Each field test plot was 5x5 feet, and was planted

with turf grass. The field test plots were outfitted with a drip irrigation system. Irrigation

rates are shown in Table 2.1. The irrigation rate was gradually increased in an attempt to

obtain more frequent and larger-volume samples. Pan lysimeters were used to allow

collection of coliform bacteria. Three pan lysimeters were installed in the R-l plot at

depths of 12 inches, 18 inches, and 24 inches. Two pan lysimeters were installed in the

tap water plot at depths of 12 inches and 18 inches. The field plots were operated for 9

months (April 2002-Jan. 2002), and lysimeter samples were collected approximately

twice monthly. Samples were analyzed for fecal coliform, coliphage, chemical oxygen

demand (COD), nitrate, phosphate, and total dissolved solids (IDS).

Table 2.1. Irrigation Rate of Field Test Plots

Dates Irrigation Rate Percent of Agronomic(in/yr) Rate (%)

April 23, 2002 - June 12,2002 72 150

June 13, 2002 - July 8, 2002 108 225

July 9,2002 - Aug. 20, 2002 144 300

Aug. 21, 2002 - Oct. 15,2002 180 375

Oct. 16,2002- Jan. 31,2003 216 450

7

Photo 2.1. SBWWTP Field Test Plot

Photo 2.2. Pan Lysimeter

8

Photo 2.3. Sand Filtration

Photo 2.4. UV Disinfection System

9

2.2 Laboratory Study

Soil column tests were conducted to more accurately evaluate the UV and chlorine

dosages necessary to impact the fecal coliform bacteria and coliphage virus counts in

Oahu soils.

Stainless steel columns, 6 inches in length with an inner diameter of 1.8 inches, were

packed with Wahiawa soil collected from a DFC pineapple field. Each soil column was

packed to a bulk density between 1.01 and 1.03 g1cm3• The porosity of soil columns

were 0.67-0.68. A nylon fabric membrane (Soil Measurement Systems) was placed at the

bottom of the column whereas a #1 filter paper (Whatman) was placed on the top end of

the column. The nylon fabric membrane has no resistance to the flow. It was used to

prevent the soil particles from being washed out of the column cell. The #1 filter paper

was used to distribute the water uniformly over the surface of soil columns. One hundred

pore volumes of tap water, R-l water (with UV doses of either 70, 140 or 400 mW

s/cm2), R-2 water, and R-3 water were applied to the top of soil columns at a rate of 1

mlImin (approximately 40 in/d). Applied water flowed down through the soil columns by

gravity under unsaturated conditions. Leachate samples were collected and analyzed for

fecal coliform, total coliform, coliphage, and PCR-DGGE. Soil samples at depths of 0

inch (top), 3 inches (middle), and 6 inches (bottom) were used for PCR-DGGE.

2.3 Soil

The soil selected for this experiment belongs to the Wahiawa series. It was collected

between depths of 20 to 40 cm from DFC pineapple fields. The Wahiawa soil contained

83% clay, 12% silt, 4.2% sand and 0.8% organic carbon. Based on the results obtained

10

by Miller (Miller 1987), the particle densities of Wahiawa soil samples ranged from 2.78

to 3.23 g/cm3• An average particle density of 3 g/cm3 was used throughout the

experiment to determine the porosity of soil columns (0.67-0.68).

Photo 2.5. Laboratory Soil Column

2.4 Water Samples

Fecal coliform and total coliform were enumerated by the membrane filter technique,

and coliphage was enumerated by the double-layer technique (American Public Health

Association 1998). COD, nitrate, phosphate, and TDS were measured by standard

methods (American Public Health Association 1998).

11

2.5 Grass Samples

10 grams of grass samples from the R-1 and tap water plot were suspended in 100 ml

sterilized ddHzO. After shaking the mixture of grass and sterilized ddHzO for 5 min,

analyze fecal coliform and coliphage concentrations.

2.6 DNA Extraction and peR Amplification

Water samples (1-2 L) were applied to 0.45 Jlm pore-size Nitrocellulose filters

(Fisher Scientific, Pittsburgh, PA). Total genomic DNA was purified directly from the

filters using FastDNA Spin Kit (BIO 101; Vista, CA). Water samples and soil samples

were incubated on heterotrophic media plate for 5 days at 20DC. Colonies, which were

washed off with sterile water, were used to isolate genomic DNA using FastDNA Kit

(BIO 101; Vista, CA).

-.,.

~ - -. ~ . "

'i--~!,:1 r,F~!l. ~ ':: ~ ~ ,

" .- ...............- ~--

Photo 2.6. Bead Beater and Microcentrifuge

12

The V3 region of 165 rRNA genes was amplified by PCR using the forward primer

P338f (5'-ACT CCT ACG GGA GGC AGC AG-3') (Lane 1991) and the reverse primer

P518r (5' -ATT ACC GCG GCT GCT 00-3') (Muyzer et aI., 1993; 0vreas et aI., 1997).

A GC clamp of 40 bp was added to the forward primer (Muyzer et al., 1993; 0vreas et

aI., 1997). The length of the expected amplified fragment with the GC clamp was 236

bp.

The 50 ~l PCR mixtures contained: 0.5 ~M each primer, 200 ~M each

deoxynucleoside triphosphate, 5 ~l of thermophilic DNA polymerase, lOx reaction buffer

(w/15 mM MgC!z), 1.25 U of Taq DNA polymerase (Promega; Madison, WI), 400 ng of

bovine serum albumin (promega; Madison, WI) per ~l, and 0.75 ~l of extracted DNA.

PCR amplifications were performed in iCycler (Bio-Rad Laboratories, Hercules, Calif)

with an initial denaturation at 94°C for 5 min; 35 cycles of denaturation (45 sec at 94°C),

annealing (45 sec at 55°C), and extension (45 sec at 72°C); and a final extension at 72°C

for 10 min. Amplified DNA was examined by horizontal electrophoresis in 1.5% agarose

with 5 ~l aliquots of PCR products.

2.7 DGGE Analysis

DGGE was performed as described by Muyzer et aI., 1993. PCR products were

loaded onto 8% (wtivol) polyacrylamide gels in Ix TAB (20 mM Tris, 10 mM acetate,

0.5 mM EDTA, pH 7.4). The denaturant gradient of urea and formamide was 40-60%

(where 100% denaturant contains 7 M urea and 40% formamide). The gels were run by

DCode system (Bio-Rad Laboratories, Hercules, Calif) for 210 min at 60°C and 130V.

After electrophoresis, the gels were incubated for 10 min in ethidiun bromide (1.0 mgll),

13

rinsed for 10 min in lxTAE buffer, and then documented using Gel Doc 2000 (Bio-Rad

Laboratories, Hercules, Calif). DGGE analyses were used to examine bacterial diversity

in this study, and no attempt was made to identify individual bacteria species.

Photo 2.7. DCode System for DGGE Analysis, Horizontal Electrophoresis System, andPCR Instrument

Photo 2.8. Gel Documentation System

14

CHAPTER 3. RESULTS AND DISCUSSION

3.1 Field Study

3.1.1 Lysimeter Water Sample Analysis

The DOH regulations for R-l water with ultraviolet (UV) disinfection require that the

influent turbidity be less than 2 NTU to maintain disinfection performance. In this study,

the turbidity of R-3 effluent was reduced from 2.3 to 1.6 using sand filtration. Fecal

coliform and coliphage concentrations after sand filtration were approximately 25,000

CFU/lOOmI and 3,000 PFU/lOOml, respectively. However, after UV disinfection no

fecal coliform or colophage were ever detected in the R-l water.

All five pan lysimeters performed well for the duration of experimentation.

Lysimeter sample volume ranged from 40-200 ml. Due to the limited amount of some

lysimeter samples, COD was selected for analysis rather than biochemical oxygen

demand (BOD). Water quality data for applied waters and lysimeter samples, averaged

over the period of experimentation are shown in Table 3.1.

Table 3.1. Average Water Quality Data of Rl Water and Lysimeter Samples

Parameter Rl Tap Rl,24 Rl,18 Rl,12 Tap, 18 Tap, 12water water inches inches inches inches inches

Fecal <1 0 <1 <1 <1 <1 <1coliform(CFU/lOO ml)Coliphage <1 0 <1 <1 <1 <1 <1(PFU/IOO mI)COD (mg/l) 21 0 9 10 15 20 18Nitrate (mg/l) 23 0 2 10 6 6 3Phosphate 9.5 0 0.13 0.06 0.07 0.59 0.82(mg/l)TDS (mg/l) 357 164 710 440 360 309

15

The data in Table 3.1 show that no fecal coliform or coliphage were found in any

lysimeter samples during 9 months of sampling while applying water in excess of

agronomic needs. This seems to indicate little potential exists for transport of fecal

indicators through the deep Vadose Zone (400-600 ft) and into underlying drinking water

on Oahu. However, fecal coliform were found in grass samples from both the R-l and

tap water plots. Coliphage was also found in a grass sample from the R-l plot. The fecal

coliform values for the R-l and tap water plot grass samples were 20 and 6 CPU/IOO ml,

respectively. The coliphage value for the Rl plot grass sample was 200 PPU/IOO mI.

This is consistent with the study that fecal indicator bacteria, which include fecal

coliform, Escherichia coli, and enterococci, are present in natural environments in the

tropics, such as grass, soil, and freshwater streams (Byappanahalli and Fujioka 1998;

Murakami and Ray 2000; Fujioka and Hardina 1995; Lopez-Torres et aI., 1987;

Desmarais et aI., 2002). The tropical soil and grass environment is suitable for the

growth of fecal indicator bacteria. Birds and other animal feces are assumed to be the

sources of these fecal indicator bacteria in the soil and grass samples collected herein.

All the COD values for lysimeter samples were lower than that of the R-l water. The

deeper the field test plot lysimeter depth, the lower the COD values. Most of the organic

matter in the Rl water is expected to be consumed by aerobic bacteria near the soil

surface, thus causing a decrease in COD values. The COD values for the tap water plot

were slightly higher than those for the R-l plot possibly due to leaching of surface soil

COD.

Data obtained for nitrate and phosphate indicate that nitrate in the Rl water was

removed by 50 to 90%, and that about 99% of the phosphate in R-1 effluent was removed

16

in the field site turf grass-soil system. Most of the phosphate is assumed to be removed

by the grass, which was supported by visual observation of better grass growth and

greener hue in the R-l plot. The nitrate values from the tap water plot were about the

same as those of the R-l plot. However, the phosphate values from the Rl plot were only

about one tenth that of the tap water plot. This was possible due to leaching of grass root

of tap water plot.

The TDS values of lysimeter water samples were higher than those of R-l and tap

water. The TDS values increased with increasing lysimeter depth. This was due to the

phenomenon of evapotranspiration.

3.1.2 DGGE Analysis

Samples of soil were obtained from each field test plot. A few grams of soil were

collected at each pan lysimeter location (bottom soil) as well as at the root zone of the

grass (surface soil). Figure 3.1 shows the electrophoresis ofPCR products. All the PCR

products are 236 bp. Figure 3.2 shows the result of the PCR-DGGE analysis of field test

plot lysimeter and soil samples. At least 13 and 12 detectable DGGE bands were

observed for the R-l plot and tap water plot lysimeter samples, respectively, using the

direct DNA isolation method. There are 10 and 6 detectable DGGE bands for R-l and

tap water plot lysimeter samples, respectively, using the culturing DNA isolation method.

There are 6, 13, 10, and 12 detectable DGGE bands for the R-l plot bottom soil, R-l plot

surface soil, tap water plot bottom soil, and tap water plot surface soil, respectively.

These results mostly indicate that the R-l plot has a more diversified microbial

community than that of the tap water plot. This potentially indicates that the organic

17

matter and nutrients in the R-l water has the potential to enhance the growth of more soil

bacteria. Results also show that surface soils have more and different bacteria species

than that of the bottom soils. DGGE banding profiles of lysimeter samples are different

using culturing and direct DNA isolation methods, which indicate that dominant viable

species in water samples are not necessarily the dominant species of the whole microbial

population.

Figure 3.1. Electrophoresis of PCR Products. Lane 1, DNA marker(1,000, 750, 500, 300,150, 50 bp); lane 2-8, PCR products.

So far, no research studied the microbial diversity of soil exposed to recycled water using

DGGE method. Some researches using DGGE method have studied the microbial

diversity of either different type of soil, or soil exposed to some contaminants. Organic

soil exhibited a higher microbial diversity than the sandy soil (0vreas and Torsvik 1998).

18

It is reported that the microbial diversity of petroleum hydrocarbon-contaminated soils

maintained or increased as compared to uncontaminated controls (Juck et al., 2000).

1 2 3 4 5 6 7 8

Figure 3.2. DGGE Analysis of peR Amplified I6S rDNA Fragments Obtained fromField Test Plot Samples. Lane 1, R-I plot lysimeter (0.45 urn membrane);lane 2, tap water plot lysimeter (0.45 urn membrane); lane 3, R-l plotlysimeter(culture); lane 4, tap water plot lysimeter (culture); lane 5, R-Iplot (bottom soil); lane 6, R-l plot (surface soil); lane 7, tap water plot(bottom soil); lane 8, tap water plot (surface soil).

3.2 Soil Column Test

3.2.1 R-3 Water Column Leaching Experiment

19

Figure 3.3 shows fecal colifonn and coliphage breakthrough at approximately 5 pore

volumes and the breakthrough curve reached a fonn of equilibrium after about 30 pore

volumes. The fecal colifonn breakthrough curve and the coliphage breakthrough curve

look quite similar. The fecal colifonn and coliphage concentrations in R-3 water were 1.8

x 105 and 2.0 x 103, respectively. These results indicate that there is much lower removal

efficiency for coliphage than for fecal colifonn. A previous study in the laboratory on

virus transport through sterile Oahu soils showed that a pure culture of MS-2 virus could

not leach all the way through soil columns of the same size containing the same soil

(Wong 2001). However, in the present study coliphage was found in the leachate from

the R-3 soil column. Since the soils in the present study were not sterilized, the soil

environment may have been more conducive to coliphage survival and less conducive to

adsorption. One possible explanation for this difference is that the coliphage viruses can

be transported together with bacteria since viruses have to infect host bacteria to

reproduce. Moreover, the high concentration of clays (83%) in the soil samples may

protect coliphage from biological biodegradation (Gerba 1987), which enhances

coliphage transport.

The transport of fecal colifonn and coliphage through laboratory soil column seems

to indicate the potential contamination of groundwater with pathogens. However, due to

the high porosity (0.67-0.68) of laboratory soil column, the removal efficiency of fecal

colifonn and coliphage in laboratory soil column should be much greater than in field.

The modeling part of this study (data not shown) suggests that the adsorption of fecal

colifonn and coliphage are very high in shallow soils of central Oahu, and that the

20

movement of fecal coliform and coliphage are limited to six feet below soil surface after

83 years of percolation.

250 .,-----------------------,oa_E~ 200 ;------/---+---.,-------------100o~

§ ~ 150 +----------,I/-----'l.0""r.

L&.c.0-CI)E en 100 ;----.Ak-----I-I--IL...... cuoJ::

:!:: a.8 '0 50 -1---1.0

L&.

O+-......-,-----,.--r----.---,-----,.--.,.---r-----.----i

o 10 20 30 40 50 60 70 80 90 100

Time (Pore volume)

1-.-F.coliform~Coliphage IFigure 3.3. Fecal Coliform and Coliphage in the R-3 Water Laboratory Column Leachate

3.2.2 Tap Water, R-l Water and R-2 Water Column Leaching Experiment

Table 3.2 shows the fecal coliform, total coliform, and coliphage concentrations in

waters applied to the soil columns. Only total coliform in leachate was plotted in Figure

3.4 since no fecal coliform and coliphage were found in the leachate from these soil

columns. The results indicate that total coliform is indigenous in the soil samples, and can

be washed out by the applied tap water. The total coliform number in the R-I-140 soil

column leachate is only slightly higher than that of tap water leachate. However, the total

coliform numbers in the R-I-70 and R-2 soil column leachates are much higher than that

of the R-I-140 and tap water soil column leachates. This is presumably due to the higher

21

total coliform number in the R-1-70 and R2 applied waters. The R-1-140 and tap water

column data may represent washout of total coliform from the soil, whereas the R-1-70

and R-2 column data may represent this same washout enhanced by some transport of

applied coliform. Compared to UV-70 and chlorine, UV-140 seems to provide more

protection from coliform leaching since higher UV dosage can inactivate more bacteria

than lower UV dosage.

Table 3.2. Fecal Coliform, Total Coliform, and Coliphage Concentrations in LaboratorySoil Column Applied Waters

Sample Fecal coliform Total coliform Coliphage(CFU/100 mil (CFU/100 ml) (PFU/100 ml)

Tap water 0 0 0

R-1-70 20 300 0

R-1-140 0 100 0

R-2 effluent 0 172 0

_ 120

E8 100'I"'":s 80u.0-E 60....g

40(5u

S 200... 0

0 20 40 60 80 100 120

Time (Pore volume)

I~Tap water -+- R1-70 --- R1-140 .......- R21Figure 3.4. Total Coliform in the Leachate from the Tap Water, R-1-70, R-I-140

and R-2 Soil Columns

22

3.2.3 DGGE Analysis

Figure 3.5 shows the DGGE banding profiles for R-l, R-2, and R-3 soil columns.

The results show that the DGGE banding profiles of the soil columns which received

wastewater are different from that of the control soil, which means the dominant viable

bacteria species of the control soil changed after the application R-l, R-2 and R-3 waters.

R-l soil column had a more diversified microbial population than the R-2 and R-3 soil

columns based on the DGGE banding profiles.

One DGGE band was found in the control soil and the top layer of all soil columns,

which indicates that the microorganism represented by this DGGE band is likely an

aerobic microorganism. Another DGGE band was found in the control soil, the R-l

column and the R-3 column. However, this DGGE band was only found in the bottom

layer of the R-2 soil column. This may be due to presence of chlorine residual in the top

layer of the R-2 column.

Compared to the control soil, the populations change due to recycled water

application. The changes in bacterial populations are dependent upon the type of

treatment performed on the recycled water. The use of chlorine as a disinfectant seems to

reduce bacterial diversity and the use of a high dose of UV disinfectant seems to allow an

increase in bacterial diversity (R-I-140 and R-2 lanes) compared to undisinfected (R-3

lanes). Comparison of lanes 11 and 12 seems to indicate that bacterial

diversity/speciation does not change as a result of water passage through the soil column.

Comparison of lanes 11 and 12 with the other lanes seems to show that the

23

microorganisms that flourish due to wastewater percolation do not come from the applied

water; instead they were already present in the soil perhaps in a dormant state.

1 2 3 4 5 6 7 8 9 10 11 12 13

Figure 3.5. DGGE Analysis of PCR Amplified 16S rDNA Fragments Obtained fromR-l, R-2 and R-3 Laboratory Soil Column Samples. Lane 1, control soil;lane 2, R-I-140 column (top); lane 3, R-I-140 column (middle); lane 4, R1-140 column (bottom); lane 5, R-2 column (top); lane 6, R-2 column(middle); lane 7, R-2 column (bottom); lane 8, R-3 column (top); lane 9,R-3 column (middle); lane 10, R-3 column (bottom); lane 11, R-3 waterapplied; lane 12, R-3 soil column percolate water; lane 13, Control soil

Figure 3.6 shows the DOGE banding profiles of R-I-70, R-I-140, and R-I-400

soil columns. The banding profiles look similar but do have some differences. The

results show that the top layers of each column have more bacteria species, and that the

banding profiles for the R-I-140 and R-I-400 columns are more diverse than the R-I-70

24

column. The results seem to indicate the UV dosage does have some impacts on bacterial

diversity.

1 2 3 4 5 6 7 8 9 10 11

Figure 3.6. DGGE Analysis ofPCR Amplified 16S rDNA Fragments Obtained fromRI Soil Column Samples. Lane I, control soil; lane 2, R-I-70 column(top); lane 3, R-I-70 column (middle); lane 4, R-I-70 column (bottom);lane 5, R-I-140 column (top); lane 6, R-I-140 column (middle); lane 7, R1-140 column (bottom); lane 8, R-I-400 column (top); lane 9, R-I-400column (middle); lane 10, R-I-400 column (bottom); lane 11, Control soil.

PCR-DOGE analysis of PCR products allowed a rapid assessment of bacterial

community change at the species level. By using this approach, we were able to study

the distribution of the microbial community along a depth profile of a field test plot and

25

laboratory soil columns. Although the DGGE band profile represents the complexity of

the microbial community, it does not provide quantitative information about the

individual species in the whole community, and it does not represent the exact total

number of species in the sample. Bands at identical positions in the DGGE profile are

not necessary derived from the same species (Muyzer et aI., 1993; Kurisu et aI., 2002).

To solve this problem, a gel with a narrower gradient can be used to get a high-resolution

DGGE profile of particular sections of the original profile. It is also of note that as more

than one copy of 16S rRNA genes exists in most of the bacteria, a single organism can

produce multiple DGGE bands (Ntibel et aI., 1996; Fogel et al., 1999). However, in

microbial diversity analysis one DGGE band is generally considered as representing one

bacterium species, and in this study, we assume that one DGGE band represents one

microorganism.

26

CHAPTER 4. CONCLUSIONS

Both field and laboratory experiments were conducted to study the diversity and

transport of bacteria and viruses through surface soils following irrigation with recycled

water at SBWWTP on the Island of Ohau. The following conclusions can be drawn:

[1] There was no transport of fecal coliform and coliphage through the top soil layer

(12-24 inches) of a field test plot even during excessive over-irrigation with R-I

recycled water, which contains no fecal coliform or coliphage.

[2] Residual COD in R-I effluent was not effectively removed, but more COD was

removed with increasing depth of transport. Nitrate and phosphate removals in the

R-I test plot were 50-90% and 99%, respectively.

[3] Based on PCR-DGGE analysis, the R-I test plot had a more diversified microbial

community than that of the tap water test plot, and surface soils had more bacteria

species than bottom soils (12-24 inches depth).

[4] Both fecal coliform and coliphage in R-3 water could be transported through a 6

inch recompacted soil column in the laboratory, and fecal coliform removal was

higher (approx. 3-log) than coliphage removal (approx. I-log).

27

[5] Total coliform was indigenous in the tropical soil samples, and could be washed off

by the applied tap water. The total coliform number in R-1-70 and R-2 column

leachates was much higher than that of R-1-140 and tap water column leachates.

[6] Based on PCR-DGGE analysis, the dominant viable bacteria species in the control

soil changed after the application of R-l effluent, R-2 effluent, and R-3 effluent.

Different DGGE banding profiles were observed at different depths in lab soil

columns. UV dosage did have some effects on soil microbial diversity. The

dominant viable species in applied waters did not become dominant in the soil

columns. Also, diversity and speciation in applied waters did not change due to

leaching through the soil columns.

28

Appendix A: Physical Conditions of Column Leaching Experiment

Table AI. Physical Conditions of Tap Water Column Leaching Experiment

Bulk density =0.915 g1cm'J flow rate =1.0 ml/min

porosity =0.685 velocity =3.66 cmlhr

pore volume =188.27 cm3

time for 1 PV =188.27 min

weight of dry soil =251.51 g

Table A2. Physical Conditions of R-3 Column Leaching Experiment

Bulk density =0.970 g1cmj

flow rate =1.0 ml/min

porosity = 0.667 velocity = 3.66 cmlhr




Table A3. Physical Conditions of R-2 Column Leaching Experiment

Bulk density =0.948 g1cm'J flow rate =1.0 ml/min

porosity = 0.674 velocity = 3.66 cmlhr




29

Table A4. Physical Conditions ofR-1-70 Column Leaching Experiment

Bulk density =0.935 glcmj


porosity =0.678 velocity =3.66 cm/hr




Table A5. Physical Conditions ofR-1-140 Column Leaching Experiment





time for 1 PV = 186.94 min


Table A6. Physical Conditions of R-1-400 Column Leaching Experiment







30

Appendix B: Genomic DNA Isolation protocol

1. Sample Processing.

a. Prepare appropriate Lysing Matrix for the sample to be processed.

b. Choose appropriate CLS and add to tube with Lysing Matrix. For bacteria cells,

add 1 ml CLS-TC to a tube containing samples and Lysing Matrix.

c. Choose appropriate sample size: Samples consist of 200 III suspension of cells in

water.

2. Homogenize in bead beater. Place tube in bead beater and process for 2 minutes.

Incubate on ice for 10 minutes.

3. Centrifuge to pellet debris. Spin in microcentrifuge for 15 minutes at 14,000 x g to

pellet protein and cell debris. Transfer 600 III of the supernatant to a clean

microcentrifuge tube.

4. Add 600 III of Binding Matrix gently, and incubate for 5 minutes at room temperature.

Spin for 1 minute; discard supernatant. Gently resuspend pellet with 500 III SEWS

M. Spin for 1 minute and discard supernatant. Spin for 10 seconds and remove

residual liquid with a small bore pipet tip.

5. Elute DNA from Binding Matrix by gently resuspending in 100 III DES followed by a

2-3 minutes incubation. Spin for 1 minute at 14,000 x g and transfer supernatant to a

new tube. Be careful to avoid transferring particles of Binding Matrix pellet with your

DNA sample. DNA is now ready for electrophoresis and PCR.

31

Appendix C: DGGE Reagent

Table C.1. 40% AcrylamidelBis (37.5:1)

Reagent Amount

Acrylamide 38.93 g

Bis-acrylamide 1.07 g

dH20 to 100.0 ml

FIlter through a 0.45 I..l fIlter and store at 4 cC.

Table C.2. 50x TAE Buffer

Reagent Amount Final Concentration

Tris base 242.0 g 2M

Acetic acid, glacial 57.1 ml 1M

0.5 M EDTA, pH 8.0 100.0 ml 50mM

dH20 to 1,000.0 ml

Mix. Autoclave for 20-30 minutes. Store at room temperature.

Table C.3. Percentage Acrylamidelbis Needed for a Particular Size Range

Gel Percentage Base Pair Separation

6% 300-1000 bp

8% 200-400 bp

10% 100-300 bp

32

Table C.4. 0% Denaturing Solution

6% Gel 8% Gel 10% Gel

40 Acrylamide/Bis 15 ml 20ml 25ml

50x TAB buffer 2ml 2ml 2ml

dHzO 83 ml 78ml 73ml

Total volume 100ml 100ml 100ml

Degas for 10-15 minutes. Fillter through a 0.45 f.l filter. Store at 4 °C in a brown bottlefor approximately 1 month.

Table C.5. 100% Denaturing Solution

6% Gel 8% Gel 10% Gel

40 Acrylamide/Bis 15 ml 20ml 25ml

50x TAB buffer 2ml 2ml 2ml

Formamide (deionized) 40ml 40ml 40ml

Urea 42g 42 g 42g

dHzO to 100 ml to 100 ml to 100 ml

Degas for 10-15 minutes. Fillter through a 0.45 f.l fIlter. Store at 4 °C in a brown bottlefor approximately 1 month. A 100% denaturant solution requires re-dissolving afterstorage. Place the bottle in a warm bath and stir for faster results.

Table C.6. Formamide and Urea Amounts for Denaturing Solutions Less Than 100%

Denaturing Solution 10% 20% 30% 40% 50% 60% 70% 80% 90%

Formamide (ml) 4 8 12 16 20 24 28 32 36

Urea (g) 4.2 8.4 12.6 16.8 21 25.2 29.4 33.6 37.8

For denaturing solutions less than 100%, use the volumes for acrylamide, TAB and waterdescribed in the 100% Denaturing Solution. Use the amounts indicated above for ureaand formamide.

33

Table C.7. 10% Ammonium Persulfate

Reagent Amount

Ammonium persulfate 0.1 g

dHzO 1.0 ml

Store at -20°C for about a week.

Table C.8. DCode Dye Solution


Bromophenol blue 0.05 g 0.5%

Xylene 0.05 g 0.5%

Ix TAB buffer 10.0 ml Ix

Store at room temperature. ThIS reagent IS suppbed In the DCode electrophoresis reagentkit for DGGE.

Table C.9. 2x Gel Loading Dye


2% Bromophenol blue 0.25 m1 0.05%

2% Xylene cyanol 0.25 ml 0.05%

100% Glycerol 7.0ml 70%

dHzO 2.5 ml

Total volume 10.0 ml

Store at room temperature.

Table C.lO. Ix TAB Running Buffer

Reagent Amount

50x TAB buffer 140ml

dHzO 6,860 ml

Total volume 7,000 m1

34

Appendix D: DGGE Operation

Pre-heating the Running Buffer

1. Fill the electrophoresis tank with 7 L of Ix TAE running buffer.

2. Place the temperature control module on top of the electrophoresis tank. Turn the

power, pump, and heater on.

3. Set the temperature controller to the desired temperature with a temperature ramp rate

of 200 °e.

4. Preheat the buffer to the set temperature.

Assembling the Parallel Gradient Gel Sandwich

1. Lay the large rectangular plate down first, and then place the left and right spacers of

equal thickness along the short edges of the larger rectangular plate.

2. Place the short glass plate on top of the spacers so that it is flush with the bottom edge

of the long plate.

3. Loosen the single screw of each sandwich clamp. Place each clamp by the appropriate

side of the gel sandwich with the arrows facing up and toward the glass plates.

4. Guide the left and right clamps onto the sandwich. Tighten the screws.

5. Place the sandwich assembly in the alignment slot of the casting stand with the short

glass plate forward. Loosen the sandwich clamps and insert an alignment card.

6. Align the plates and spacers. Tighten both clamps just enough to hold the sandwich in

place.

7. Remove the alignment card. Remove the sandwich assembly from the casting stand.

35

Casting Parallel Denaturing Gradient Gels

1. Place the gray sponge onto the front casting slot. Place the sandwich assembly on the

sponge with the shorter plate facing you. Press down on the sandwich and tum the

handles of camshaft down so that the cams lock the sandwich in place.

2. Connect one end of 9 cm Tygon tubing to the Y-fitting and connect a luer coupling to

the other end of 9 cm tubing. Connect luer fittings onto the two long pieces of tubing.

Connect the luer fittings to 30 ml syringes.

3. Label one of the syringes LO and one HI. Attach a plunger cap onto each syringe

plunger head. Position the plunger head in the middle of the plunger cap and tighten

enough to hold the plunger in place. Slide each syringe into a syringe sleeve.

4. Rotate the cam wheel counterclockwise to the start position. Set the desired delivery

volume.

5. From the stocks solutions, pipet out the desired amounts of the high and low density

gel solutions into two disposable test tubes.

6. Add the final concentration of 0.09% (v/v) each of ammonium persulfate and TEMED

solutions. Cap and mix. Withdraw all of the high density solution and low density

solution into the HI and LO syringe, respectively.

7. Push the gel solution to the end of the tubing.

8. Place the LO syringe into the LO side of gradient delivery system syringe holder.

Place the HI syringe into the HI side of gradient delivery system syringe holder.

9. Slide the tubing from the LO and HI syringes over two ends on the Y-fitting.

10. Attach a 19 gauge needle to the coupling. Hold the beveled slide of the needle at the

top-center of the gel sandwich and cast.

36

11. Rotate the cam wheel slowly and steadily to deliver the gel solution.

12. Carefully insert the comb. Let the gel polymerize for about 60 minutes.

13. Rinse the tubing and Y-fitting by placing the tubing and needle into a beaker of water

and reversing the cam.

14. After polymerization, remove the comb by pulling it straight up slowly and gently.

Assembling the Upper Buffer Chamber

1. Lay the inner core flat on a bench.

2. Release the gel sandwich from the casting stand.

3. Position the gel sandwich so that the locating pins on the core are fitted into the

grooves on the outside surface of the sandwich clamps.

4. Gently push the gel sandwich down onto the core with one simple motion.

5. Repeat steps 1-4 to attach the second gel sandwich. If only one gel is to be run,

assemble a set of glass plates without the spacers.

6. Pour 350 ml of running buffer into the upper buffer.

7. When the running buffer has reached the desired temperature, tum the system off.

Place the core and attached gel assemblies into the buffer chamber.

8. Tum on the system. Allow the system to reach the set initial temperature before

loading.

Sample Loading

1. Remove the clear loading lid. Wash the wells with running buffer.

2. Load the samples using a pipetman and sequencing lading tip.

37

3. Place the clear loading lid on top of the temperature control module.

Running the Gel

1. Attach the electrical leads to Bio-Rad's Power Pac 300.

2. Run the gel at 130 volts for 210 min.

Removing the gel

1. After electrophoresis is complete, tum the system off and let the heater to cool for 1

min. Remove the temperature control module

2. Gently remove the assembly.

3. Loosen the single screw of each clamp. Carefully pry off the shorter glass plate.

4. Remove the spacers and cut one comer of the gel to distinguish between gels.

Staining and Photographing the Gel

1. Place the gel into a dish containing 250 ml of Ix running buffer and 25 IJI of 10 mg/ml

ethidium bromide. Stain for 10 min.

2. Using 250 ml of Ix running buffer to destain for 5 min.

3. Place the gel on the UV transilluminator of Gel Doc 2000 and photograph.

38

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