effect of heavy metals

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The Effect of Heavy Metals on BacteriaBy

Elena Vratonjic


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Columbus State UniversityThe College of science

The Graduate Program In Environmental Science

Effect of Heavy Metals on Bacteria

A Thesis inEnvironmental Science

By

Elena Vratonjic

Submitted in Partial Fulfillmentof the requirementsfor the Degree ofMaster of Science

December 2003

@2003 by Elena Vratonjic


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11

I have submitted this thesis in partial fulfillment of the requirements for thedegree of Master of Science

Date Elena Vratonjic

We approve the thesis of Elena Vratonjic as presented here.

IDate Davis K. John, AssociateProfessor of Biology, ThesisAdvisor

hi W J 03 c ^iA :Date Charles A. Lovelette,

Associate Professor ofChemistry

Date Thomas B. Hanley, Professorof Geology


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IllABSTRACT

Heavy metals have become devastating environmental contaminants due totheir widespread use in the manufacture of electronics, plastics, batteries anddyes. The discharge of heavy metals into the environment has caused concernabout their effects on the ecosystem. Many metals are essential for growth insmall amounts. However, at higher concentrations heavy metals are toxicbecause they bind to organic compounds. This accounts for their effects onsome important parts of the cell structure, like their ability to denatureprotein molecules. The initial objective of this study was to find out the growthrate of E.coli and M.roseus in the presence of 1mM and 10 uM concentrations ofcadmium and lead. E.coli and M.roseus were used due to their ability to growin a controlled environment and ease of usage. The growth rates of thebacteria and viable counts were obtained via light scattering. Bacteria weregrown on rich media (LB broth) as well as mineral M9 media supplemented withand without casamino acids and growth rates were compared. Anotherobjective in this study was to identify genes in E. coli mutants that were moreresistant or sensitive to cadmium. E.coli was mutagenized with a transposon toidentify mutants with altered sensitivities to Cd. The growth rates of themutants and wild type were obtained with and without Cd. Results suggestthat Cd +2 , at 10^iM and 1mM concentrations as well as Pb+2 at 1mMconcentration, had a significant effect on E.coli and M.roseus in all mediatypes. Insignificant effects were found in M.roseus and E.coli growth rateswhen exposed to lO^iM Pb. The responses of bacteria to cadmium salts were100 times more toxic in comparison to lead salts, suggesting slightly differentbiological effect of these heavy metals. The isolation of mutants resistant tospecific toxic agents such as Tc and Rif antibiotics was not a very useful geneticapproach in this study. It was unfortunate that it cannot be proven that realmutants were created. Instead, the donor was already cadmium sensitive, andthe Rif donor appeared to be even more sensitive to cadmium and thereforebehaved like a mutant. UV-light exposure, % survival, growth rates, andappearance of plasmids confirmed exactly the same behavior of Rif resistantdonor and suspected mutant. Therefore it seemed like our donor strain wasalready a mutant that has very high cadmium sensitivity. A differentapproach was taken In order to identify E. coli genes involved with cadmiumresistance, by simply increasing concentrations of Cd +2 . E.coli strain was foundthat is resistant to 4.5 mM Cd+2 .


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VRESULTS 23E. coli and M. roseus growth rates with glucose and lactose at 231x 10 3 M cadmium and leadM. roseus growth rates in different media at 1x 10 3 M and 241x 10' 5 M cadmium and leadE. coli growth rates in different media at 1x 10 3 M and 241x 10 5 M cadmium and leadMutagenesis 25

DISCUSSION AND CONCLUSION 99

REFERENCES 106APPENDIX A - E. coli doubling times 109APPENDIX B - M. roseus doubling times 199APPENDIX C - Suspected mutant, donor and recipient doubling times 278


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LIST OF FIGURES VI

Figure Page1 -1 Average doubling times of E. coli vs. Cd and Pb from 33

absorbance vs. time1-2 Average doubling times of E. coli vs. glucose or lactose 34

from absorbance vs. time1-3 Summary of average doubling times of E. coli from 35

absorbance vs. time1-4 Average doubling times of M. roseus vs. Cd and Pb from 41

absorbance vs. time1 -5 Average doubling times of M. roseus vs. glucose or lactose 42

from absorbance vs. time1 -6 Summary of average doubling Times of M. roseus from 43

absorbance vs. time1-7 Average doubling times of E. coli vs. Cd and Pb from 49

cell count vs. time1-8 Average doubling times of E. coli vs. glucose or lactose 50

from cell count vs. time1 -9 Summary of average doubling times of E. coli from 51

cell count vs. time2-1 Average absorbance of E. coli vs. Cd and Pb from 57

cell count vs. absorbance2-2 Average absorbance of E. coli vs. glucose or lactose 58

from cell count vs. absorbance

2-3 Summary of average absorbance's of E. coli from 59cell count vs. absorbance2-4 Average doubling times of M. roseus vs. Cd and Pb from 65

cell count vs. time2-5 Average doubling times of M. roseus vs. glucose or lactose 66

from cell count vs. time


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Vll

Figure Page2-6 Summary of average doubling times of M. roseus from 67

cell count vs. time2-7 Average absorbance of M. roseus vs. Cd and Pb from 73cell count vs. absorbance2-8 Average absorbance of M. roseus vs. glucose or lactose 74

from cell count vs. absorbance2-9 Summary of average absorbance's of M. roseus from 75

cell count vs. absorbance3-1 Average doubling times of M. roseus vs. Cd and Pb from 81

absorbance vs. time3-2 Average doubling times of M. roseus vs. different media 82

from absorbance vs. time3-3 Summary of average doubling times of M. roseus from 83

absorbance vs. time3-4 Average doubling times of E. coli vs. Cd and Pb from 89

absorbance vs. time3-5 Average Doubling Times of E. coli vs. different media 90

from absorbance vs. time3-6 Summary of average doubling times of E. coli from 91

absorbance vs. time3-7 Average % survival for wild type, Tc r donor, Rf r donor 96

and mutant


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VlllLIST OF TABLES

Table Page1-1 Doubling times for E. coli absorbance vs. time in M9 28

media with glucose or lactose exposed to 1x10 3 MCd and Pb

1 -2 ANOVA: Two-Factor With Replication for E. coli 29absorbance vs. time grown in M9 media with glucoseor lactose exposed to 1x10' 3 M Cd and Pb

1-3 Duncan's Multiple Range Test for E. coli absorbance 30vs. time grown in M9 media with glucose or lactoseexposed to 1x10 3 M Cd and Pb

1-4 Mean Comparisons and Rp Comparisons for E. coli 31absorbance vs. time grown in M9 media with glucoseor lactose exposed to 1x10 3 M Cd and Pb

1 -5 Summary of doubling times for E. coli absorbance vs. 32time grown in M9 media with glucose or lactoseexposed to 1x1 0~ 3 M Cd and Pb

1-6 Doubling times for M. roseus absorbance vs. time 36in M9 media with glucose or lactose exposed to1x10 3 MCdandPb

1-7 ANOVA: Two-Factor With Replication for M. roseus 37absorbance vs. time grown in M9 media with glucoseor lactose exposed to 1x10 3 M Cd and Pb

1 -8 Duncan's Multiple Range Test for M. roseus 38absorbance vs. time grown in M9 media with glucoseor lactose exposed to 1x10' 3 M Cd and Pb

1-9 Mean Comparisons and Rp Comparisons for M. roseus 39absorbance vs. time grown in M9 media with glucoseor lactose exposed to 1x10 3 M Cd and Pb

2-1 Summary of doubling times for M. roseus absorbance 40vs. time in M9 media with glucose or lactoseexposed to 1x10' 3 M Cd and Pb


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ixTable Page2-2 Doubling times for E. coli cell count vs. time in M9 44

media with glucose or lactose exposed to 1x10 3 MCd and Pb

2-3 ANOVA: Two-Factor With Replication for E. coli 45cell count vs. time grown in M9 media with glucoseor lactose exposed to 1x10 3 M Cd and Pb

2-4 Duncan's Multiple Range Test for E. coli cell count 46vs. time grown in M9 media with glucose or lactoseexposed to 1x1 0~ 3 M Cd and Pb

2-5 Mean Comparisons and Rp Comparisons for E. coli 47cell count vs. time grown in M9 media with glucoseor lactose exposed to 1x10'

3

M Cd and Pb2-6 Summary of doubling times for E. coli cell count vs. 48

time grown in M9 media with glucose or lactoseexposed to 1x10' 3 M Cd and Pb

2-7 Doubling times for E. coli cell count vs. absorbance 52in M9 media with glucose or lactose exposed to1x10 3 MCdandPb

2-8 ANOVA: Two-Factor With Replication for E. coli 53cell count vs. absorbance grown in M9 media withglucose or lactose exposed to 1x10 3 M Cd and Pb

2-9 Duncan's Multiple Range Test for E. coli cell count 54vs. absorbance grown in M9 media with glucose orlactose exposed to 1x10 3 M Cd and Pb

3-1 Mean Comparisons and Rp Comparisons for E. coli 55cell count vs. absorbance grown in M9 media withglucose or lactose exposed to 1x10 3 M Cd and Pb

3-2 Summary of doubling times for E. coli cell count vs. 56absorbance grown in M9 media with glucose orlactose exposed to 1x1 0~ 3 M Cd and Pb

3-3 Doubling times for M. roseus cell count vs. time in 60M9 media with glucose or lactose exposed to1x10 3 MCdandPb


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Table Page3-4 ANOVA: Two-Factor With Replication for M. roseus 61

cell count vs. time grown in M9 media with glucoseor lactose exposed to 1x10 3 M Cd and Pb

3-5 Duncan's Multiple Range Test for M. roseus cell 62count vs. time grown in M9 media with glucose orlactose exposed to 1x10 3 M Cd and Pb

3-6 Mean Comparisons and Rp Comparisons for M. roseus 63cell count vs. time grown in M9 media with glucoseor lactose exposed to 1x10 3 M Cd and Pb

3-7 Summary of doubling times for M. roseus cell count 64vs. time grown in M9 media with glucose or lactoseexposed to 1x10 3 M Cd and Pb

3-8 Doubling times for M. roseus cell count vs. 68absorbance in M9 media with glucose orlactose exposed to 1x10 3 M Cd and Pb

3-9 ANOVA: Two-Factor With Replication for 69M. roseus cell count vs. absorbance grownin M9 media with glucose or lactose exposedto1x10 3 MCdandPb

4-1 Duncan's Multiple Range Test for M. roseus 70cell count vs. absorbance grown in M9 media withglucose or lactose exposed to 1x10 3 M Cd and Pb

4-2 Mean Comparisons and Rp Comparisons for E. coli 71cell count vs. absorbance grown in M9 media withglucose or lactose exposed to 1x10 3 M Cd and Pb

4-3 Summary of doubling times for M. roseus 72cell count vs. absorbance grown in M9 mediawith glucose or lactose exposed to 1x10' 3 MCd and Pb

4-4 Doubling times for M. roseus absorbance vs. time 76in M9 and LB media exposed to 1x10 3 M and 1x10' 5 MCd and Pb


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XITable Page4-5 ANOVA: Two-Factor With Replication for 77

M. roseus absorbance vs. time grown in M9 and LBmedia exposed to 1x10 3 M and 1x1 0~ 5 M Cd and Pb

4-6 Duncan's Multiple Range Test for M. roseus 78absorbance vs. time grown in M9 and LB mediaexposed to 1x10' 3 M 1x10' 5 M Cd and Pb

4-7 Mean Comparisons and Rp Comparisons for M. roseus 79absorbance vs. time grown in M9 and LB media withglucose or lactose exposed to 1x10 3 M Cd and Pb

4-8 Summary of doubling times for M. roseus 80absorbance vs. time grown in M9 and LB mediaexposed to 1x10 3 M 1x10 5 M Cd and Pb

4-9 Doubling times for E. coli absorbance vs. time 84in M9 with and without casamino acids and LB mediaexposed to 1x10 3 M and 1x10 5 M Cd and Pb

5-1 ANOVA: Two-Factor With Replication for 85E. coli absorbance vs. time grown in M9 with andwithout casamino acids and LB media exposed to1x10 3 M and 1x10 5 M Cd and Pb

5-2 Duncan's Multiple Range Test for E. coli absorbance 86vs. time grown in M9 with and without casaminoacids and LB media at 1x10 3 M and 1x10 5 M Cd andPb

5-3 Mean Comparisons and Rp Comparisons for E.coli 87absorbance vs. time grown in M9 with and withoutcasamino acids and LB media at 1x10 3 M and 1x10 5M Cd and Pb

5-4 Summary of doubling times for E. coli absorbance 88vs. time grown in M9 with and without casaminoacids and LB media exposed to 1x10 3 M and 1x1 0~ 5 MCd and Pb

5-5 Exposure of Rf r wild type, Tc r , Rf r donor, 92and mutant strain to UV-light


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XllTable Page5-6 % Survival of wild type, Tc r donor, Rf r donor, 93

and mutant strain5-7 Single Factor ANOVA for % survival of wild type, 94

Tc r donor, Rf r donor and mutant strain5-8 Analysis of Variance for % survival of wild type, 95

Tc r donor, Rf r donor and mutant strain5-9 Duncan's Multiple Range Test for % survival of wild 97

type, Tc r donor, Rf r donor and mutant strain6-1 Mean Comparisons and Rp Comparisons for % survival 98

of wild type, Tc r donor, Rf r donor and mutant strain


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INTRODUCTION

The effect of heavy metals on bacteria

Heavy metals have become significant environmental contaminants due totheir widespread use in the manufacture of electronics, plastics, batteries anddyes. The discharge of heavy metals into the environment has caused concernabout their effects on the ecosystem (Shapiro and Keasling, 1996). Thoughmany metals are essential for growth in small amounts, at higherconcentrations heavy metals are toxic because they bind to organiccompounds. This accounts for their effects on some important parts of the cellstructure, like their ability to denature protein molecules (Shapiro andKeasling, 1996, Morozzi et al., 1982). Because of their intrinsically persistentnature and toxicity of the metals to living systems, the mechanisms ofresistance and repair of damage caused by heavy metals is an important andunresolved problem (Pazirandeh, 1996). Some of the earliest attempts tocontrol microorganisms by chemical means were based on the use of heavymetals. For example, copper sulphate was used as a plant fungicide, andmercury salts were used in the treatment of certain infectious diseases (Sadlerand Trudinger, 1967).

Data from numerous studies indicates that the presence of highconcentrations of metals provides a selective medium favoring metal resistantbacteria (Mietz and Sjogren, 1982). Some bacteria are resistant toconcentrations of heavy metals that are toxic to higher organisms. These


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2bacteria may concentrate or change the valence of heavy metals and removehem from the environment. Each species may be affected in different ways, sothat a particular concentration of a metal may exert no, little, or a dramaticeffect, depending on the microorganism's characteristics (Gadd, 1990).

Most bacteria exhibit a biphasic response to a number of heavy metals. Atlow concentrations of the metal there is a stimulation of growth, but as themetal concentration is increased growth becomes progressively inhibited andfinally ceases. The cross-over zone between stimulation and inhibition bymetals is generally over a very narrow range. The actual concentrations atwhich the responses occur depend upon the organism, the form of the metal,and the chemical and physical composition of the medium in which theresponses are determined (Gadd, 1990). An understanding of the mechanisms

by which microbes survive heavy metal exposure is critical for understandingheavy metal toxicity in all organisms and for the use of such organisms for theremoval of metals from the environment (Shapiro and Keasling, 1996).Furthermore, the biological effects of such pollutants on microorganisms andthe frequency of appearance of heavy metal resistant bacteria can be useful asan indicator in bioassay systems (Gelmi et al., 1994).

An increasing frequency of appearance of bacteria resistant to toxicmetals such as cadmium seems to be correlated with increasing loads of metalsin the environment (Gelmi et al., 1994). However, the interaction of inorganiclead (Pb) and cadmium (Cd) with well-studied bacteria such as Escherichia colihas received little attention. One reason for this may be because E. coli is not


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a normal member of soil flora, so it is usually not isolated from metalcontaminated sites. Another reason for the lack of study of effects of heavymetals on E. coli may be that phosphate found in most standard media combinewith many metal ions to form an insoluble precipitate. Metal solubility iscritical for uptake into the bacterial cell. Avoidance of precipitation by thephosphate found in most standard media is necessary prior to analysis ofgrowth inhibition. Metals reduce growth if a modified medium containingglycerol-2-phosphate is used to lower the concentration of precipitate (LaRossaet al., 1994). Chelating compounds, such as citrate and EDTA have been shownto slightly increase the toxicity of heavy metals (Gadd and Griffiths, 1978).

The extent to which a metallic cation interacts in soil environments withorganic and inorganic surfaces determines the concentration of metal in

solution. Bacteria have a high surface area to volume ratio and a high capacityfor sorbing metals from solution. Many studies have shown that largequantities of metallic cations are complexed by bacteria (Mullen et al., 1989).Previous studies concluded that cell walls of gram-positive bacteria such as B.subtilis bind larger quantities of several metals than cell envelopes of the E.

coli (gram-negative bacterium) (Beverage and Fyfe, 1985).The objective of a microbe study by Mullen et al. (1989) was to determine

the metal binding capacities of whole cells of two-gram positive bacteria andtwo-gram negative bacteria. Concentrations of 1uM - 1mM Ag + , Cd 2+ , Cu 2+ ,and La 3+ were used as metal salt solutions. Examinations of the values of Cdsorption showed gram-negative E. coli and P. aeruginosa were more efficient at


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4Cd sorption than gram-positive bacteria. On the other hand, B. subtilisremoved the most Cu 2+ at an equilibrium concentration of 1uM. Highequilibrium concentrations affected gram-negative bacteria the most andgram-positive bacteria the least. This research evaluated the sorptioncapabilities of selected bacteria (Mullen et al., 1989).

Traditionally, adsorption, chemisorption, and ion exchange have been usedto describe the removal of various cations. When using intact bacteria cells weshould also consider other processes like active uptake of the metal into thecytoplasm through non-specific cation transport systems. Evidence suggeststhat the role of bacteria is not limited to short term metal immobilization andthat bacteria cells are capable of binding large quantities of metal cations(Mullen et al., 1989). There is evidence suggesting that microbial biomasses

have high heavy metal uptake capacities. One possible method ofconcentration may be due to the production of certain metal-binding proteinssuch as metallothioneins. These proteins are cysteine rich and bind heavymetals, such as Cd, with very high affinity (Pazirandeh, 1996). Pazirandeh

(1996) suggests further that E. coli does not have any gene encodingmetallothionein-like proteins. Even without a metallothionein gene, E.colidemonstrates considerable heavy metal resistance (Gelmi et al., 1994). Thestudy of this resistance could identify the genes that will give E. coli improvedability to sequester heavy metals (Pazirandeh, 1996).

Cenci et al. (1985) used total dehydrogenese activity (TDH) as a means ofmeasuring metal toxicity in E. coli. TDH was used since it is a measure of


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5bacterial activity closely related to energetic metabolic systems and beta-galactosidase as a typical inducible enzyme in E. coli. From the results it waspossible to define the order of metal toxicity. The 50 percent inhibition ofTDH-activity was: 7.4, 10, 30, and 50 uM, respectively for Hg, Zn, Cd, and Cu.In the presence of the metals incubation time of over 60 minutes consistentlylead to the inhibition of TDH activity. Experiments were carried out to definesome characteristics of the biological damage to the total cell population. Hg

and Cu markedly inhibited growth in Trypticase soy agar (TSYA) medium, whilethe viability of cells treated with Cd and Zn remained constant in the samemedium. This indicated that treatment with Cd and Zn, unlike that observedfor Hg and Cu, did not cause metabolic injury to E. coli (Cenci et al., 1985).

Continued pollution of the biosphere with toxic heavy metals makes itclear that microbe-based technologies will have an important role inenvironmental remediation (Gadd, 1990). Technological applications ofmicrobial metal concentration may depend on the ease of metal recoveryeither by biomass regeneration or by reclamation. If inexpensive biomass isused to reclaim valuable metals, then destructive recovery (incineration or

dissolution in acids or alkalis) may be economically feasible. Microorganismswith high capacity for metal uptake removal such as Staphylococcus aureus,Bacillus sphaericus, Bacillus licheniformis and Arthrobacter species can beused as both biological monitors of environmental contamination and forremediation (El-Bestawy et al., 1998). These species were isolated from waterheavily polluted with heavy metals. Mutagenesis, both physical and chemical,


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6and gradual increase in concentration of heavy metals significantly increasedbacterial resistance against a wide range of metals and led to high efficienciesfor their removal from soil. Physical mutation was induced by u.v light (254nm) at 30 cm height, and at the dose that resulted in 90% mortality. Agar welldiffusion technique was used for chemical mutation that was induced by 1%ethidium bromide. In this study, the highest removal efficiency achieved bythe different selected mutants was Cd (89.9-100%), Cr (87.3-99.7%) and Pb

(40.2-51%) (El-Bestawy et al., 1998).Microbes are generally the first organisms exposed to heavy metals present

in the environment. Although the presence of heavy metals is detrimental tothem, toxic metals select resistant variants that confer the ability to toleratehigher levels of the toxic compounds (Cervantes et al., 1994). Heavy metalresistance has been shown to be plasmid mediated in some cases, and thegenetic determinants responsible for heavy metal resistance often reside onplasmids that also mediate antibiotic resistance (Mietz and Sjogren, 1982).

Transposons are useful for making mutations since they can be helpful ingene identification. Transposons are specific DNA segments with the ability tomove as a unit in a more or less random fashion from one genetic point toanother. Complete loss of gene function often results upon insertion of atransposon within a gene. Therefore, transposons can be used as a mutagenicagent, if a replicon that carries the transposon can be efficiently introducedinto the organism to be mutagenized. Selecting for transposon-mediatedantibiotic resistance can easily isolate transposition events. Molecular cloning


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7of prokaryotic genes and manipulation and genetic analysis of bacteria havebeen greatly facilitated by the use of antibiotic-resistance transposons (Simonetal., 1983).

Genetic manipulation of bacteria may allow the development of newstrains with better growth rates and improved resistance to toxic metals.Therefore, manipulation of wild type, mutant, and individual or mixed culturesmay be a useful tool for decontamination of polluted effluents by metal

bioremediation (Brierley et al., 1982).

Bacterial growthThe simplest measure of the effect of heavy metals on bacteria is obtained

by examining their effect on growth rate. Bacterial cultures are exposed todifferent concentrations of various heavy metals and the growth rate isdetermined (Mandelstam and Mc Quillen, 1968).

When bacterial cells are placed in a nutritionally favorable environmentthey will grow and divide. At some point due to exhaustion of nutrients,depletion of oxygen, and/or accumulation of toxic products, the environmentwill become unfavorable for growth. Until this occurs, the bacteria grow in anunhindered manner, and each cell will grow, on the average, at the same rateas its predecessors. This is a geometric doubling called logarithmic growth(Ford and Mitchell, 1992).

Growth parameters most often used to determine growth rate are thosethat can be easily and accurately measured. One parameter easily measured is


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8turbidity of a liquid culture, which depends on the amount of light that isscattered by the organisms and is related to the bacterial mass (Collins andStotzky, 1992). Measuring living or viable cells is also a useful method fordetermining growth rates. The number of viable bacteria is determined bycolony count, which is performed by placing an appropriate dilution of theculture on or in a solid medium where the organisms are known to grow. Sincecolonies arise from single cells, the number of colonies multiplied by thedilution factor equals the number of viable bacteria originally present.Standard curves are used to relate the viable cells to the turbidity (Mandelstamand Mc Quillen, 1968).

E. coli grows well in chemically defined media. Some of the media arevery simple, containing only mineral salts and a single organic component as a

source of carbon and energy. When glucose is the organic component thebacterial mass may double every 45-60 minutes. Growth is slower when othersubstances such as acetate or succinate are used as the carbon energy andsource. The growth rate is increased considerably by the addition of aminoacids, purines and pyrimidines, and vitamins. These are best provided by useof a complex medium containing these substances (Ford and Mitchell, 1992).Since E. coli can take up sugars, proteins and amino acids from the complexmedium and need not synthesize them de novo, growth is much more rapidthan on mineral medium. Many different types of culture media have beenemployed in the study of bacteria, and some common laboratory species willgrow in complex medium. Complex media is generally a solution composed of


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peptones, concentrated meat or yeast extract, and salt. Extracts arecomposed of the water-soluble constituents of meat or yeast, which will diffuseout of the meat or yeast on standing in water, and it supplements the peptoneand helps the growth of bacterial species (Ford and Mitchell, 1992).

The growth rate of bacteria can also be manipulated by altering thetemperature. With E. coli at 37C the growth rate ranges from 18-20 minutesin a complex medium to about 2 hours in media where the sole carbon and

energy source is one of the several sugars which are metabolized inefficiently(Sadler and Trudinger, 1967).

If E. coli is inoculated into an appropriate growth medium in the evening,by the following morning there will be signs of considerable growth. In orderto obtain growing bacteria in log phase, a portion of this overnight culture must

be sub-cultured into fresh medium. Growth will not begin immediately, butwill start slowly, reaching the maximum growth rate in a gradual manner. Thisperiod prior to the maximum growth rate is called the lag phase. The durationof this period depends on many considerations, including the organism, themedium, the conditions of previous cultivation, the degree of aeration, and theway in which bacterial growth is measured. At the end of the lag phase, thecells are in a physiological steady state, undergoing balanced growth aspreviously described. This state ceases more or less gradually when the cellsbegin to exhaust essential nutrients or when they accumulate toxic products(See Figure 1 ). The time at which the log phase ends is entirely dependent onthe species of organism and the actual conditions of cultivation. When either


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10the carbon or the nitrogen source is limiting in a synthetic medium, growthof E. coli ends very abruptly (Mandelstam and Mc Quillen, 1968).

Heavy metals such as Cd+2 , Cr+6 , Hg+2 , Pb +2 , and Zn +2 , produce a decreaseof E. coli growth rate, and reduced total biomass. All the concentrations oftoxicants tested in the work done by Mariscal et al. (from 0.3 uM to 5 uM)produced at least a partial inhibition of E. coli growth rate. This inhibition wasreflected in the decrease of the respective values of growth rate, in

comparison with those of controls. Comparison of the values revealed that,Cr+6 , Hg+2 , Cd+2 , Pb+2 , and Zn +2 in that order, were the most to least toxic to E.coli (Mariscal et al., 1995).

Studies have resulted from the reduction of population growth of severalmicroorganisms in bodies of water from industrial effluents containing heavymetals. Heavy metals probably bound with the cellular membrane of thebacteria by impairing certain physiological and biochemical parameters. Theresults suggested that the degree of growth population of E. coli varied amongmetal ions in the aquatic environment. Effects of different concentrations ofmetals on the population growth of E. coli after 7 and 28 days were analyzed inthe treated and untreated experimental sets. A gradual decline in populationgrowth of E. coli was noted with increasing incubation time and metalconcentration. The harmful effects of the metals were found to be highest inCd, then Pb, Cu, As, Hg, and lowest in Cr (Jana and Bhattacharya, 1987).

In a study by Pickett and Dean (1979), the effect of differentconcentrations of Cd +2 and Zn +2 on Bacillus subtilis subsp. niger and


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11Pseudonomas fluorescens were investigated. Organisms in this study thatwere cultured many generations in liquid medium at concentrations between0.12 and 0.20 mM of Cd +2 or Zn +2 all survived on agar plates containing metalions at that concentration, but at higher concentrations the viability decreasedsharply. The minimum Cd +2 concentrations completely inhibiting the growth ofBacillus subtilis subsp. niger and Pseudonomas fluorescens were 0.62 and 0.13mM respectively, suggesting that Pseudonomas sp. is a more sensitive strain.The same trend was seen for Zn

+2, which inhibited these strains at 1.53 and

0.12 mM respectively. As with many other toxic agents, the lag increased asthe inhibitor concentration increased. There was also a distinct anomaly in theform of a plateau, after the lag phase, which occurred at Cd +2 concentrationsbetween 0.12 and 0.2 mM. Evidence suggests that well-defined sites such asthe cell walls on the organisms were initially attacked by metal ions. Thedegree of inhibition of growth rates was proportional to the concentration ofmetal ions present in the medium. When the cell walls were saturated withmetal ions, small increases in concentration had no effect, and a relativelyhigh concentration was required before other less accessible sites wereaffected. In liquid medium the lag and the mean generation time of Bacillussubtilis supsp. niger increased with increasing Cd +2 or Zn+2 concentrations,whereas only the total biomass was affected in Pseudonomas fluorescens.Nevertheless, the responses of both species indicted a specific action at lowconcentration and a more general toxic action at high concentrations (Pickettand Dean, 1979).


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12In a study by Morozzi et al. (1982) the effect of Hg2+ , Cd2+ , Cu2+ , Zn 2+ ,

Cr2+ , and Pb2+ on E.coli were studied. The growth curves of E. coli in culturescontaining sub-lethal concentrations of metal ions (ranging from 0.01 uM to 100uM) show that there is no effect on the lag phase time until the thresholdconcentration is reached. The metals studied, and the corresponding values ofthreshold concentration that were derived from the graph, were as follow:0.005 uM for Hg2+ , 0.5 uM for Cd 2+ , 5 uM for Cu 2+ , 23 uM for Cu 2+ , 30 uM for Zn 2+

and 45 uM for Pb2+ . In order to evaluate the persistence of the tolerance tometal ions, E. coli previously exposed to metals were subcultured into freshmedia and then re-inoculated into media plus metal at different times (0-12hours). Previous exposure to the single metals diminished the toxicity of Hg2+ ,Cd2+ , Zn 2+ , Cu 2+ , and Cr2+ . The mid-log phase times of primary sub-cultures,after four, five, and six hours respectively, came near the values of thecontrols without metals. The acquired tolerance is lost in a very short time ifbacteria grow in the absence of metal. In this experiment, the lag phase beganto lengthen again when bacteria without the metal were sub-cultured intomedium containing the corresponding ion. Loss of viability was only observedfor microorganisms tested after the exposure to lead. The reduction ofmetallic ions indicated a physiological adaptation of microbes rather than theselection of mutants. In a study by Morozzi et al. (1982) cells that havebecome adapted to the presence of metal ions lost this adaptation aftergrowing in a metal-free medium. In conclusion, metal ions Hg2+ , Cd2+ , Cu 2+ ,


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, , 13Cr , Zn + did not cause selection of metal-resistant mutants of E. coli(Morozzietal., 1982).

OBJECTIVES:The initial objective of this study is to find out the growth rate of E. coli

and M. roseus in the presence of 1mM and 10 uM concentrations of cadmiumand lead. E. coli and M. roseus will be used due to their ability to grow in acontrolled environment and ease of usage. Growth rates of Escherichia coli(gram-negative bacteria) and Micrococus roseus (gram positive bacteria) will beobserved before and after exposure to lead or cadmium. The growth rates ofthe bacteria will be measured via light scattering, and viable counts will beobtained. Bacteria will be grown on rich media (LB broth) as well as mineral

M9 media supplemented with and without casamino acids and growth rates willbe compared.

Another objective in this study is to identify genes in E. coli mutants thatwill be more resistant or sensitive to cadmium. E. coli will be mutagenizedwith a transposon to identify mutants with altered sensitivities to Cd. Thegrowth rates of the mutants and wild type will be obtained with and withoutCd. The null hypothesis in this study states, Mutagenesis will have no effectin mean growth rates between wild type and mutant .


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AAATERIAL AND METHODS

Chemicals and instruments utilized were available in the Departments ofBiology and Chemistry and Geology Department, College of Science, ColumbusState University. Rifamycin (Rf) and tetracycline (Tc) were purchased fromSigma.

Bacterial strains and growth conditionsE. coli was supplied from Bactrol discs (ATCC 25922), and M.roseus from

Microbank (29). For transposon mutagenesis E.coli S17 mini Tn5-Tc (Tc r , Rif s )(de Lorenzo et al., 1990) was the plasmid donor. E. coli (ATCC 25922) wasgrown on Rif plates and surviving colonies were used as the recipient (Tcs , Rifr). Selection for antibiotic resistance was performed on LA platessupplemented with Tc or Rf to final concentrations of 15|ag/ml and 50|ag/ml,respectively from sterile stock solutions. Antibiotic stock solutions wereprepared by adding 50 mg of Rif per ml of Dimethyl Formamide (DMF), and 15mg of Tc per ml of 70% ethanol followed by filter sterilization.

Escherichia coli and Micrococus roseus were initially grown in minimalmedium containing 4.85ug Na2HP04 , 3.0g NaCl, 1.0g NH4Cl, 0.27g MgS04 , and100ul_ of 1 .0M CaCl2 per Liter of distilled water at pH 7.0. When necessary,5.0g of casamino acids and 5g of glucose or lactose were added. Agar powder

was added to 15 g/L to prepare solid medium. Bacteria were also grown in LB


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15broth (Difco Laboratories, Detroit, Ml) prepared according to manufacturer'sinstructions, always supplemented with 2g/L of glucose. When necessary, Tcand Rif were added to LB. All media were sterilized before addition ofantibiotics, by autoclaving at 121C for 15 minutes.

Filter sterilized stock solutions of 0.1 M of CdCl2 , PbN0 3 , and EDTA wereprepared in de-ionized water and metal solutions were simultaneouslyintroduced to EDTA. Solutions were then introduced at final concentrations of10 3 and 10

5M.

Measurement of growth ratesGrowth rates were monitored using a Milton Roy Spectronic 20D

spectrophotometer (St. Petesburg, FL) set at a wavelength of 650 nm.Measurements were plotted against time to determine growth rate for eachspecies.

Viable count procedureViable cell counts were performed by pour plate technique of tenfold

serial dilutions of control and 1 mM Cd and Pb containing samples. 1 ml ofculture dilutions was pipetted into a sterile petri plate, adding 20 ml of 45CTryptic Soy Agar and gently swirling the plate on the tabletop. Plates thathave between 30 and 300 colonies were counted. Three replicate plates ofeach dilution were prepared.


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16Experimental protocol for preparing different media

Bacteria were inoculated into M9 broth media (with 5% glucose or lactose)with and without casamino acids and LB broth media (supplemented with 2 %glucose) and grown overnight. These bacteria were then used to inoculatestanding tubes for the experiment. The experimental tubes included M9 mediacontaining lactose only, glucose only, lactose with severely limited glucose, M9media without casamino acids and LB broth. Cultures were inoculated on iceto prevent growth of the bacteria, and then transferred to a 37C water bathfor standing incubation. Turbidity was measured every 30 minutes withspectrophotometer at 650 nm.

Testing for sensitivity to Cd and Pb

After control readings of the four media types above were completed, ten-milliliter test tubes of differently supplemented broths were inoculated withbacteria using a sterile, plastic inoculating loop. Identical procedures wereused as the control, but medium contained the following:

1 Glucose with casamino acid and cadmium2. Glucose with casamino acid and lead3. Lactose with casamino acid and cadmium4. Lactose with casamino acid and lead5. Lactose with casamino acid, limited glucose, and cadmium


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176. Lactose with casamino acid, limited glucose, and lead7. Glucose with cadmium8. Glucose with lead9. LB broth with glucose and cadmium10. LB broth with glucose and lead

Controls were repeated at this stage. All tubes were incubated in a waterbath at 37C, and the absorbance was read every 30 minutes until bacterialgrowth was well into stationary phase. Three independent repetitions of eachexperiment were performed in this study.

ANOVA and Duncan's multiple testAnalysis of Variance (ANOVA) was used for analyzing data in this study.

ANOVA is used to uncover the overall effects of independent variables on aninterval dependent variable. The key statistic in ANOVA is the F-test ofdifference of group means, testing if the means of the groups formed by valuesof the independent variable are different enough not to have occurred bychance. If the group means do not differ significantly then it is inferred thatthe independent variables did not have an effect on the dependent variable. Ifthe F test shows that overall the independent variables are related todependent variables, then multiple comparisons tests of significance are used(Krebs, 1999). In this study, once it was determined that a significantdifference exists within the level means of a variable, it was necessary todetect the location of significant differences. Duncan's Multiple Test was used


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18to distinguish exactly where the differences were significant for E.coli andM.roseus. Duncan's Multiple Test involves three steps consisting of calculatingstandard error (Sx ), arraying or ranking the means for the treatment orinteraction in question, and calculating confidence intervals and comparingthem (Steel and Torrie, 1960).

MutagenesisOvernight cultures of donor and recipient strains (0.1 ml) were

transferred into 10 ml of LB liquid cultures with appropriate antibiotics, andgrown to log phase. 0.1 ml of each strain was centrifuged in separateEppendorf tubes for 1 minute. The cell pellets were washed twice in 0.1 ml ofLB and re-suspended in 0.1 ml of LB. The two strains were mixed together,centrifuged for 1 minute, the liquid discarded, and the cell pellets re-suspended in 20 \d of LB broth. The mating mixture was spotted on LA platesand incubated overnight at 30C. The overnight growth was scraped off theplate and suspended in 1 ml of LB, and 0.1 ml samples were then spread on LAplates with Tc and Rif . Controls of donor and recipient only were treatedidentically. Approximately 1000 colonies that acquired the transposonantibiotic resistance were obtained on these plates. Tc r , Rif colonies werepicked and replica-plated on LA/Tc/Rf, and LA/Tc/Rf/CdCl2 plates. Putativemutants were cultured in liquid LB + 1 mM CdCl2 to confirm Cd sensitivity.Mutants exhibiting abnormal growth were selected for further analysis.


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19Determination of minimal inhibitory concentrationSuspected mutants that didn't grow on plates in the presence of 1 mM of

Cd were tested to find their minimal inhibitory concentration. Mutants weregrown overnight at 37C in LB medium + Tc + Rf +10 uM Cd +2 . Growth wasobserved after 24 hours, suggesting that the minimal inhibitory concentrationwas higher than 10 uM. Concentrations were incrementally increased until nocell growth was observed.

E. coli plasmid mini-prep1-2 ml of cell culture was centrifuged for 1 minute in a microfuge, the

pellet was collected and the culture fluid was discarded. The cell pellet wasresuspended in 0.1 ml of TE buffer. Lysis solution (0.2 ml) was added andmixed thoroughly by inversion. Potassium acetate (3M) 0.15 ml was added andmixed thoroughly by inversion until a white precipitate formed. The solutionwas centrifuged for 5 minutes in a microfuge. The cleared lysate wastransferred to a new tube, avoiding the white precipitate that was on the sideand bottom of the tube. Cold 95-100% (0.8 ml) was added to the lysate andmixed. The solution was centrifuged for 5 minutes in microfuge. The liquidwas discarded and the pellet was saved. The liquid was completely removedwith a pipetteman. The pellet was washed with 0.5 ml of cold 70% ethanol,dried, and resuspended in 20-50 (il water or TE buffer. Instructions outlined inthe Laboratory Manual by Sambrook et al. (1989) were used to prepare alkalinelysis buffers.


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20

Preparation of DNA electrophoresis gelPlasmid preparations were made for potential mutants, wild type, and

donor cell (Tc r ). Pellets from the plasmid preps were dissolved in 30 uL of TEbuffer and divided into a control portion and a portion to be digested. Theseplasmid preps were then digested with restriction enzyme Pst I (RocheDiagnostic Corporation, Indianapolis, IN) according to manufacturer'sinstructions. Instructions for preparing 1% agarose gel and 10X loading bufferwere extracted from the Labaratory Manual by Sambrook et al. (1989). Prior toelectrophoresis, 2 \iL of 10X loading buffer was added to each tube. DNAMolecular Marker IV (Roche Diagnostic Corporation, Indianapolis, IN) was alsorun on the gel with all of the samples. After electrophoresis, the gel wasstained in 10ug/ml ethidium bromide solution for 10 minutes. The gel wastransferred to distilled water for five minutes, and then visualized byFoto//Phoreisis I apparatus by Fotodyne at wavelength of 312 nm.Photography of these gels was taken afterwards.

Selection of a Cd+2 resistant E . coli strainE. coli was grown on LA plates that were prepared with different

concentrations of Cd +2 . The minimum starting concentration was 500 uM.Flourishing colonies were selected and grown in LB with the sameconcentration of Cd+2 . Once the bacteria reached log phase, a loopful wasspread on LA plates prepared with a higher concentration of Cd+2 . LA plates


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21were started with very low cadmium concentration of 0.5mM, followed by theintermediate concentrations of 1mM, 1.5mM, 2mM, 2.5mM, 3mM, 3.5mM, and4mM. This process was replicated until suspected mutant reached its maximalgrowth with Cd+2 of 4.5 mM. At the end, wild type E. coti and mutant that wasCd+2 resistant were tested for Cd+2 sensitivity. They were grown in LB with andwithout maximum concentration of Cd+2 that mutant could grow on and resultswere recorded.

UV exposureWild type (Rif r recipient), Tc r donor, Rif r donor, and suspected mutant

were grown overnight in LB at 37 C. Once grown to same OD of 0.35, an equalvolume of culture was placed on LA plates along five lines. Each line wasexposed to UV light in five seconds increments ranging from to 20 seconds.Uv mineralized lamp (UVS-54) was set five inches above the plates at awavelength of 254 nm. The plates were incubated for 24 hours at 37 C, andthen examined for growth.

The survival of bacterial cells exposed to 10 seconds of UV-light was thencompared to controls that were not exposed to the UV-light. Wild type (Rif r

recipient), Rif r donor, Tc r donor, and suspected mutant were grown to mid-logphase, and samples of grown culture were diluted 10 1 to 107 times with sterilephosphate buffer (PBS). Spread plating on a series of LA plates was done by100 pi of diluted samples. After 24-hour incubation at 37C, the number of


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22colonies formed before and after the UV-light exposure was counted and %survival was determined. Only plates that contained between 30 to 300colonies were counted. Three replicate plates of each dilution were prepared.


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RESULTSE . coli and M. roseus growth rates with glucose and lactose at 1x10 3 Mcadmium and lead.

Doubling times for E. coli and M. roseus were initially derived fromabsorbance vs. time. Two-Factor ANOVA with replication showed that therewas a significant change in absorbance observed with increasing concentrationof cadmium and lead (P-value < 0.001). Duncan's Multiple Test was used todistinguish where the differences are significant between E. coli and M. roseuswhen supplemented with glucose and lactose and exposed to Cd and Pb.Therefore there is no significant difference in doubling times between M.roseus and E. coli grown in M9 mineral medium supplemented with glucose and

lactose. There were significant differences noted in M. roseus doubling timeswhen exposed to Cd and Pb, but not between the heavy metals alone. Allinteractions between the heavy metals and control were significant in E. colidoubling times.

Doubling times were also found in E. coli and M. roseus from cell growthvs. time and cell growth vs. absorbance. Two-Factor ANOVA with replicationshowed that increasing concentration of cadmium and lead (P-value < 0.001)caused a significant reduction in growth rates. Duncan's Multiple Testreplicates previously mentioned results for both E. coli and M. roseus.Therefore, only media with glucose was used since there was no significant


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24difference observed between media supplemented with glucose or lactose at95% confidence level.

M. roseus growth rates in different media at 1x10 3 M and 1x10 5 M cadmiumand lead.

Graphs of absorbance vs. time for M. roseus illustrate growth rates in M9mineral and rich media (LB) when exposed to 1x10 3 M and 1x10 5 M of Cd andPb. Two-Factor ANOVA with replication showed that there was a significantdecrease in growth rates observed with increasing concentration of cadmiumand lead (P-value < 0.001). Duncan's Multiple Test indicated that thedifferences between 1x10 3M and 1x10' 5M Cd and Pb were significant, whileinteractions between 1x10 3M Pb and 1x10 5M Cd, and 1*10 5M Pb and control(bacteria that hasn't been exposed to either Pb or Cd) were not significant.Also, there is no significant difference between M. roseus in M9 mineral andrich media.

E. coli growth rates in different media at 1x10 3 M and 1x10 5 M cadmiumand lead.

Since M. roseus couldn't grow in M9 media without casamino acid, growthrates of E. coli were compared in M9 medium with and without casamino acidand LB, at 1x10 3 M and 1x10 5 M Cd and Pb. Two-Factor ANOVA withreplication showed that there was a significant decrease in growth ratesobserved with increasing concentration of cadmium and lead (P-value < 0.001).


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25It can be observed from Duncan's Multiple Test that there is no significantdifference between 1*10 5 M of Pb and control. All of the other interactions ofgrowth rates in between the heavy metals, and heavy metals and controls weresignificant, as well as all of the growth rates of E. coti that were grown in M9medium with and without casamino acid and LB at 95% confidence level.

FIGURES A-1 through A-125 in APPENDIX A, and FIGURES B-1 through B-99in APPENDIX B, as well as TABLES 1-1 through 5-4 present all of the above raw

data of E.coli and M.roseus and their complete analysis.

MutagenesisApproximately 1000 Tc r , Rfr colonies that acquired the transposon

antibiotic resistance were obtained on LA plates. Colonies were replica-platedon LA/Tc/Rf, and LA/Tc/Rf/CdCl2 plates. The effect of Tc r , Rfr colonies withand without Cd was different. Their appearance varied from small and dry tolarge and slimy in the presence of Cd. Eight different mutants were found thatdidn't grow at all on plates in the presence of Cd. Putative mutants, wild typeE. coli, Rif r recipient, Rif r donor, and Tc r donor were grown in LB, LB/Rif/Tc,and LB/Rif/Tc and 50uM, 100uM, and 300uM of Cd. Observed growth rates arepresented in APPENDIX C, FIGURES C-1 to C-32, and in summary TABLE C-1 . Rifr donor, Tc r donor, and suspected mutant demonstrated the same growthrates. Minimal Inhibitory concentration (MIC) was found to be 300 uM in allmutants. It was possible that this Rif, Tc r phenotype would arise from a Tc r

mutant or from Rif donor. If that were true suspected mutant would have Tc


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26donor plasmid that has never entered the chromosome. Therefore donor andrecipient strains and their Cd sensitivity were tested and compared to themutants . Cd sensitivities in wild type, recipient, donor, and mutantstrains in summary TABLE C-1 suggest the possibility of picking Rif resistantdonor that was naturally more Cd sensitive than the recipient strain. Wildtype, recipient, donor, and mutant strains were prepared and run on DNAelectrophoresis gel. Figures C-33 to C-36 show the presence of tranposons in

the mutant strains.To confirm our results, UV exposure was done on wild type E. coli, Rif r

recipient, Rif r donor, Tc r donor, and suspected mutant. UV-light exposurewas necessary to see if suspected mutant behaved like the donor or wild type.After identical incubation the LA plates were inspected. Wild type E. coli andRif r recipient demonstrated the same UV resistance. Suspected mutant wassensitive to 5 seconds exposure to UV light. Tc r donor was sensitive to longerexposure of 10 seconds to UV mineral light. Rif r donor behaved in the samemanner as suspected mutant illustrating the same 5 seconds sensitivity to UVlight.

Since Uv-light exposure couldn't tell us the exact number of cells thatwere affected, it was decided to validate the results by exposing bacteria toUV-light to determine % survival. Viable counts were compared to non-exposed samples of the same culture. The control invariably contained 100 10colonies. Percent survival of wild type (Rif r recipient), Tc r donor, Rif r donor,and mutant was 78.7%, 13.5%, 6.2%, and 4.2 %, respectively. Single Factor


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27ANOVA showed that there was a significant change in % survival amongtested bacteria (P-value < 0.001). The calculated F-value of 287 and F-criticalvalue of 4 indicated that means of % survival are significantly different for twodegrees of freedom. Analysis of Variance and Duncan's Multiple Test wereused to distinguish exactly where the differences are significant. Analysis ofVariance Test shows overlapping of confidence intervals between Rif r donorand mutant. Therefore, both tests presented and supported that there was no

significant difference in % survival between Rif r donor and mutant. All of theother interactions in between the wild type, Tc r donor, Rif r donor, and mutantwere significant at 95% confidence level. TABLES 5-5 through 6-1 and FIGURE3-7 present all of the above analysis.

Wild type E. coli was exposed to different concentrations of Cd and strainthat is cadmium resistant was discovered. E. coli strain that is cadmiumresistant could grow at the maximum Cd concentration of 4.5 mM. Wild type E.coli and Cd resistant mutant were tested for Cd sensitivity. They were grownin LB with and without 4 mM Cd. Wild type E. coli grew in LB with no Cd, butdemonstrated no growth in LB supplemented with 4 mM Cd. Cadmium resistantE. coli strain grew in both, LB with and without Cd.


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28

Table 1-1. Doubling times for E. coli absorbancevs. time in M9 media with glucose or lactoseexposed to 1x10'

3

M Cd and Pb1x10 3MCd 1x10 3MPb Control Avg

Ec-glu 150 90 60 100150 90 60 100150 90 60 100

Ec-lac 150 90 30 90150 90 30 90150 90 60 100

Variable 1: Bacteria and nutrients (glucose/lactose)Ec- E.coli

Variable 2: Treatment


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29

Table 1-2. Anova: Two-Factor With Replication forfor E. coli absorbance vs. time in M9 media with glucose

-3or lactose exposed to 1x10 M Cd and Pb

SUMMARY 1x10-3M 1x10-3M Control TotalEc-slu

CountSumAverageVariance

3450150

327090

318060

9900100

1575

Ec-lacCountSumAverageVariance

3450150

327090

3 9120 84040 93.3333300 2350

TotalCountSumAverageVariance

6900150

654090

630050

240

ANOVASource of Variation SS df MS F P-value F critSample 200 1 200 4 0.0687 4.7472Columns 30400 2 15200 304 5E-11 3.8853Interaction 400 2 200 4 0.0467 3.8853Within 600 12 50

Total 31600 17


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30

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ro osO

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Q.*4-o C

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32

Table 1-5. Summary of doubling times for E. coliabsorbance vs. time in M9 media with glucose orlactose exposed to 1x10 3 M Cd and Pb

1x10 3MCd 1x10 3MPb Control150.0 90.0 50.0

Ec-glu 100Ec-lac 93.3

1x10 3MCd 1*xO 3MPb ControlEc-glu 150 90 60Ec-lac 150 90 40

Variable 1: Bacteria and nutrients (glucose/ lactose)Ec- E.coli



48/320

33

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35

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(uiw) S9UUL) Sui|qnoo


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36

Table 1-6. Doubling times for M. roseus absorbancevs. time in M9 media with glucose or lactoseexposed to 1x1 0~ 3 M Cd and Pb

1x10 3MCd 1x10 3MPb Control AvgMr-glu 240 210 150 200

240 210 150 200240 210 150 200

Mr- lac 240 240 120 200240 210 120 190270 210 150 210

Avg 245 215 140

Variable 1: Bacteria and nutrients (glucose/ lactose)Mr - M. roseus



52/320


53/320

38

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,

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39

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40

Table 2-1 . Summary of doubling times for M. roseusabsorbance vs. time in M9 media with glucoseor lactose exposed to 1x10 3 M Cd and Pb1x10 3MCd 1x10'3MPb Control

i 245.0 215.0 140.0

Mr-glu 200Mr-lac 200

1x10 3MCd 1x10 3MPb ControlMr-glu 240 210 150Mr-lac 250 200 130

Variable 1: Bacteria and nutrients (glucose/lactose)Mr- M. roseus



56/320

41

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on

CO

co

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COb


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42

> m ^BBBBVJBBBBBjHB^flRjH9&]&3&3K 1 l/>> BBBBBBBBBBBBBBBBBBSBBBBBi ou_f010 ^5 oe -e 0)ngti abso

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43

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44

CDF 5:m-M o> Xr oD 4->O ou oui uroo 1_on oCD CDb i/>o+-> uon =3c on_Q si4->

5O O A3CM o J=>CO nCM TJCD o c.Q ^ 4-J 03u CDro _CD 1-^ rsiCD o CDo-O JD(13 uj fT3L_ t. UJ >


60/320

45

Table 2-3. Two-Factor ANOVA With Replicationfor E. coli cell count vs. time in M9 media withglucose or lactose exposed to 1x10 3M Cd and Pb

Anova: Two-Factor With ReplicationSUMMARY Control Cd

LactosePb Total


Glucose

3 3 3 9120 600 360 108040 200 120 120300 300 4950

3 3 3 9210 810 540 156070 270 180 173.333300 2700 2700 8950

6 6 6330 1410 90055 235 150

510 2670 2160



Total

ANOVASource of Variation SS df MS F P-value F critSample 12800 1 12800 12.1905 0.00445 4.74722Columns 97300 2 48650 46.3333 2.3E-06 3.88529Interaction 1300 2 650 0.61905 0.5548 3.88529Within 12600 12 1050

Total 124000 17


61/320

46

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Uh- aCD con fOc CDa: ocd UDn Ol)+j^~ JZD +J^ uoC *0ro OU CDC EQ o

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62/320

Q)E4->

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48

o L.o ouj 00i_ Oo U*4 13OOQj on -OE -C Q_>-> Donc ft^ a

a;O.a O13O E 3>T3

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1 c CD(VI ou CD00On 4->3r- QJu u_f0

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4->fD CfD QJsz EQJ >->)-> fCu QJfO i_CO I-^ (viQJ ~o QJu-Q -QfC uj fDi_ 1 L.fC u f0> LU >


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49

i/i>*.OO O

>E c+- Donc Ou*f^m ^^-* ^M_Q CDD


65/320

50

to>CDEu +-

Uj

to4- >O 4-CO cCD DE Ou+J -v^aon CDc U pM-Q ED oOCD CDtoon Oro 4-L_ uCD re> ^^< L.

o00 CD1 to OCD uD Ofon

m

fO

CD

Utd

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t/>OU_Dono4-1TD


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51

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CD

on

co

-O

T3

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cou Coo 2(D +in CO CDU U3 Coh S(h ^t/1 CO ro*S.ouui

ooro o oin ocm rsi oin oo oin(uluj) dim) SuiiqnoQ


67/320

52

CDUCroi_Oin-Oroin>+->C=3OuCDUOui

to *in4-JcCDi_J-J=3coc03 4-J(T3 CDs_ Fa; +->4-> fOu CDro l_CO h-- rsiCD o CDuX5 -Q(X3 Uj ros_ i_ro u ro> LU >


68/320

53

Table 2-8. Two-Factor ANOVA With Replicationfor E. coli cell count vs. absorbance in M9 media

v3with glucose or lactose exposed to 1x10 M Cdand Pb

Anova: Two-Factor With ReplicationSUMMARY Control Cd

LactosePb Total

Count 3 3 3 9Sum 0.242 0.077 0.127 0.446Average 0.08067 0.02567 0.04233 0.04956Variance

Glucose

8.2E-05 1.2E-05 2.2E-05 0.00063

Count 3 3 3 9Sum 0.332 0.066 0.162 0.56Average 0.11067 0.022 0.054 0.06222Variance

Total

0.00016 3.1E-05 6.3E-05 0.00158

Count 6 6 6Sum 0.574 0.143 0.289Average 0.09567 0.02383 0.04817Variance 0.00037 2.1E-05 7.5E-05

ANOVASource of Variation SS df MS P-value F critSample 0.00072Columns 0.01602Interaction 0.00085Within 0.00074

1 0.00072 11.7292 0.00503 4.747222 0.00801 130.1 7.3E-09 3.885292 0.00043 6.92329 0.01002 3.88529

12 6.2E-05

Total 0.01833 17


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96

UHa.>>

2 So I^ Oro c> 03 pMM> mL. L_D oCO coaM and 1mM concentrations as wellas Pb+2 at 1mM concentration, had a significant effect on E. coli in all mediatypes. Results suggest that E. coli did not show any significant differencebetween control and growth rates exposed to 10|aM Pb. However, 10^iM and1mM concentrations of both metals had a major effect on growth rates in M.roseus medium and rich media. Insignificant effect was found in M. roseusgrowth rates when exposed to 10>M Cd and 1mM Pb, as well as 10>M Pb andthe control. Also, there was no significant difference discovered between M.roseus medium and rich media. The responses of E. coli and M. roseus tocadmium salts were, in general, increased in comparison to lead salts,suggesting slightly different biological effect of these heavy metals. The key to


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100understanding distinctions may lie in the analysis of different geneinduction by the two metals if they are significantly different.

Results by Mariscal et al. showed similar effects of Cd and Pb salts on E.coli growth rate (Mariscal et al., 1995). Cenci et al. (1985) previously discussedthe complexity of metal-bacteria interaction. Their results emphasize that thebinding of heavy metals to the bacterial envelope may play an important rolein removing metals from environments polluted with heavy metals (Cenci et

al., 1985). Higher concentrations of metals are toxic because of their ability tocause breakage in DNA (Shapiro and Keasling, 1996). Since metals play animportant role in bacterial metabolism and the concentration in naturalsettings limits growth, bacteria have developed numerous high affinitytransporters to import essential nutrients. However at high concentrationsmany metals ions can precipitate or bind inappropriately to macromoleculesleading to toxic effects (Gaballa and Helmann, 2003).

In a study by Pazirandeh (1996), expression of the Neurospora crassametallothionein gene (NCP) in E. coli was reported, and this recombinant E.coli (NCP) was able to sequester cadmium from solution rapidly and with highselectivity. It has been previously shown that NCP was capable of sequesteringmore Cd from solutions than control bacteria, which did not contain themetallothionein gene. The results in this study indicate that the NCP hasproperties that are desirable and necessary for a heavy metal biosorbent.These properties include affinity for a wide range of heavy metals, reusability


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101and durability, it's potential to be used as non-viable biomass, and itsability to be immobilized into a matrix (Pazirandeh, 1996).

Microbes can be used to assay the toxicity and mutagenicity of heavymetals. In this study E. coli strains and cadmium were used to some extent toshow effects in mutagenesis. The isolation of mutants resistant to specifictoxic agents such as Tc and Rif antibiotics was not a very useful geneticapproach in this study. It was unfortunate that it cannot be proven that real

mutants were created. Instead, the donor was already cadmium sensitive, andthe Rif donor appeared to be even more sensitive to cadmium and thereforebehaved like a mutant. UV-light exposure to wild type (Rif r recipient), Rif r

donor, Tc r donor, and suspected mutant, and % survival among abovementioned bacteria confirmed exactly the same behavior of Rif resistant donorand suspected mutant. Growth rates of Rif resistant donor and mutant alsosupported our theory. Minimal inhibitory Cd concentration of 300uM wasestablished by growing suspected mutants in LB. Cadmium sensitivity of 300uMwas then tested on Tc and Rif resistant donor strain and compared withsuspected mutant. Absence of growth was observed on all three strains.Therefore it seemed like our donor strain was already a mutant that has veryhigh cadmium sensitivity. Further mating of the donor and recipient will berequired to identify true mutant. PCR screening of these cadmium sensitivedonors will be necessary to assure that the suspected mutants are valid.

In order to identify E. coii genes involved with cadmium resistance, wildtype E. coli was exposed to different concentrations of Cd. Suspected mutant


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105The metal binding sites in ZntA and CadA are yet to be discovered. Itcan be hypothesized that certain residues confer specificity for certain metals.Identification of the metal binding sites on ZntA and CadA may lead tounderstanding the basis of metal recognition and perhaps engineering of theprotein to modify metal specificity (Sharma et al., 1999).

Future studies involving the identification of E. coli genes that are Cdresistant can provide better insight into Cd resistant mechanisms in bacteria.

Additional research is required where two different bacteria should be used asdonor and recipient instead of the two different strains of the same bacteria.Further potential improvements may be achieved by modification of thepresent study procedure for discovering mutants. Characterization of genes forwild type and donor may provide insight into the cellular targets, and themechanisms of sensitivity to metal exposure. Therefore, it is hoped that theresults described in this research thesis will be useful for further studies basedon the use of this environmentally important scientific approach.


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107Gadd, G. M. 1990. Heavy metal accumulation by bacteria and otherorganisms. Experientia 46: 834-839.

Gadd, G. M. and A. J. Griffiths. 1978. Microorganisms and heavy metaltoxicity. Microbial Ecology 4: 303-317.

Gelmi, M., P. Apostoli, E. Cabibbo, S. Porru, L. Alessio, and A. Turano. 1994.Resistance to cadmium salts and metal absorption by different microbialspecies. Current Microbiology 29: 335-341.

Jana, S. and D. N. Bhattacharya. 1987. Effect of heavy metals on growthpopulation of a fecal coliform bacterium Escherichia coli in aquaticenvironment. Water, Air, and Soil Pollution 38: 251-254.Krebs, C. J. 1999. Ecological Methodology. Addison-Welsey EducationalPublishers, Menlo Park, CA. 349-357 pp.LaRossa, R. A., D. R. Smulski, and T. K. Van Dyk. 1995. Interaction of leadnitrate and cadmium chloride with Escherichia coli K-12 and Salmonellatyphimurium global regulatory mutants. Journal of Industrial Microbiology14: 252-258.Mietz, J. A., and R. E. Sjogren. 1982. Incidence of plasmid-linked antibiotic-heavy metal resistant enterics in water-sediment from agricultural andharbor sites. Water, Air, and Soil Pollution 20: 147-159.Mandelstam, J. and K. McQuillen. 1968. Biochemistry of bacterial growth.John Wilson & Sons, New York, NY. 137-153 pp.Mariscal, A., A. Garcia, M. Carnero, J. Gomez, A. Pinedo, and J. Fernandez-Crehuet. 1995. Evaluation of the toxicity of several heavy metals by afluorescent bacterial bioassay. Journal of Applied Toxicology 15(2): 103-107.

Morozzi, G., G. Cenci, and G. Caldini. 1982. The tolerance of anenvironmental strain of Escherichia coli to some heavy metals. Zbl. Bakt.Hyg., I. Abt. Orig. B 176: 55-62.Mullen, M. D., D. C. Wolf, F. G. Ferris, T. J. Beveridge, C. A. Flemming, and G.W.Bailey. 1989. Bacterial sorption of heavy metals. Appplied andEnvironmental Microbiology 55(12): 3143-3149.Okkeri, J., and T. Haltia. 1999. Expression and mutagenesis of ZntA, a zinc-transporting P-type ATPase from Escherichia coli. Biochemistry 38: 14109-14116.


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APPENDIX A

All of the doubling times for E.coli are presented in APPENDIX A

Data for E.coli from absorbance vs. timeLactosecontrolTime Run1 Run2 Run 3 Average

306090120150180210240 0.09 0.11 0.10 0.10270 0.11 0.10 0.12 0.11300 0.14 0.12 0.14 0.13330 0.14 0.11 0.15 0.13360 0.29 0.22 0.29 0.27390 0.31 0.26 0.30 0.29420 0.33 0.29 0.31 0.31450 0.35 0.40 0.34 0.36480 0.47 0.39 0.45 0.44510 0.42 0.38 0.46 0.42540 0.41 0.42 0.47 0.43570 0.47 0.49 0.47 0.48600 0.54 0.41 0.54 0.50630 0.50 0.37 0.54 0.47660 0.52 0.52 0.55 0.53690 0.52 0.41 0.55 0.49720 0.46 0.47 0.54 0.49750 0.55 0.52 0.55 0.54780 0.48 0.54 0.53 0.52810 0.48 0.54 0.54 0.52840 0.56 0.54 0.54 0.55870 0.52 0.54 0.53 0.53900 0.51 0.50 0.52 0.51930 0.51 0.52 0.52 0.52


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APPENDIX A (cont.) 119

Data for E.coli from absorbance vs. time in M9 media

Glucose controlTime(min) Run1 Run2 Run3 Average

306090120150180210240270300330360390420450480510540570600630660690720750

0.0510.0920.0910.1330.2110.2670.2780.3110.3160.3520.5960.6440.7020.7180.7220.7160.7120.7540.8450.8210.7200.7580.7200.7920.8200.820

0.0490.0830.0850.1170.2160.2340.2780.2900.5560.5440.6460.6980.7260.7600.7860.8300.8450.8300.8700.8450.8400.8450.8450.8650.8400.845

0.0510.0890.090.1290.2130.2590.2720.3080.4110.4820.5460.6120.6980.7120.7360.7580.7630.7820.8430.8410.8360.8410.8380.8350.8380.837

0.0500.0880.0880.1250.2140.2510.2780.3010.4360.4480.6210.6710.7140.7390.7540.7730.7790.7920.8580.8330.7800.8020.7830.8290.8300.833


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Data for E.coli from absorbance vs. time in M9 mediaGlucose and Cd at 1*10 JMTime (min) Run 1 Run 2 Run 3 Run 4 Average

306090120150180210240270300330360390420450480510540570600630660690720750780810840

0.0630.0710.0610.0890.1010.1040.1460.2140.2830.3510.4560.5620.6280.6960.7280.7420.7360.7560.7020.7120.7180.7200.7020.7120.722

0.0630.0660.0590.0840.0960.0930.1350.1720.2310.3120.3780.4680.5420.5840.6280.6660.6620.6500.6300.6440.6720.6520.6380.6680.640

0.0360.0790.0630.0750.0860.0820.0890.1360.1690.2750.2800.3650.4240.4660.5490.5720.6000.6060.5840.5760.5640.5600.5540.5500.546

0.0750.0790.0950.0830.0940.0940.1200.1590.2210.2910.3400.4000.4740.5040.5460.5800.5750.5720.5620.5560.5200.5600.5650.5430.546

0.0590.0740.0700.0830.0940.0930.1230.1700.2260.3070.3640.4490.5170.5630.6130.6400.6430.6460.6200.6220.6190.6230.6150.6180.614


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Figure A-22. Average Glucose At Higher Cd Concentration

c- 0.1o ,> ^\ ,> > yO ,l /(V /^ /^ %>Time (min)Absorbance increases from 0.283 to 0.568

Actual growth rate = (600 min -510 min) = 90 min

C10-g 0.1o & $> #f f to& tovP toP^ A$> A#^^Time (min)

Absorbance increases from 0.303 to 0.608



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Figure A-27. Average Glucose At Higher Pb Concentration

c(T3.Qi_o1/1.Q C$ -^ l O^ ^


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0.01

Figure A-34. Glucose With Pb 3

~i 1 r~

o &> # & 4 $P ? jP to^ ^ ApTime (min)Absorbance increases from 0.118 to 0.237


Figure A-35. Average Glucose And Pb At Lower Concentration1.000

0.01030 60 90 120 150 180 210 240 270 300 330 360 390 420 450 480 510 540 570 600 630 660 690 720 750

Time (min)

Absorbance increases from 0.119 to .0.239Actual growth rate = (1 50 min -90 min) = 60 min


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Figure A-37. Glucose Control Run 2

0)uc(0 0.1o A> & so A> rfP ^> A


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Time(min)AverageCell Count Absorbance Antilog Transmitance

300 167 0.070 1.18 0.850360 225 0.101 1.26 0.793420 283 0.119 1.32 0.760480 467 0.210 1.62 0.617540 867 0.317 2.07 0.482600 950 0.356 2.27 0.440660 1033 0.418 2.62 0.382720 1133 0.448 2.81 0.356750 1583 0.466 2.92 0.342780 1750 0.474 2.98 0.336840 2467 0.491 3.10 0.323900 4367 0.511 3 25 0.308960 8750 0.551 3.56 0.2811020 12300 0.564 3.66 0.2731080 13733 0.579 3.79 0.2641140 17500 0.589 3.88 0.2571170 23333 0.609 4.06 0.2461200 24167 0.612 4.10 0.2441260 25833 0.619 4.16 0.2411320 29167 0.621 4.18 0.2401380 45000 0.636 4.32 0.2311440 48333 0.642 4.38 0.2281500 33333 0.622 4.19 0.239

Cell Count Absorbance167 0.070225 0.101283 0.119467 0.210867 0.317950 0.3561033 0.4181133 0.4481583 0.4661750 0.4742467 0.4914367 0.5118750 0.55112300 0.56413733 0.57917500 0.58923333 0.60924167 0.61225833 0.61929167 0.62145000 0.63648333 0.64233333 0.622

1.000

0.010

Figure A-1 10. Average Lactose With Cd Absorbance vs. Cell Count

N* ^ # * # $ ^ ^ g, ^ ^ ^ tf^^jP^j ?^^ $>>,>Cell Count

Cell Count increases from 8750 to 17500

Difference in absorbance = (0.589 -0.551) = 0.038


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APPENDIX A (cont.)

Time(min) Cell Count 2 Absorbance Antilog Transmitance300 250 0.456 2.86 0.350360 250 0.489 3.08 0.324420 500 0.596 3.94 0.254480 750 0.698 4.99 0.200540 1000 0.716 5.20 0.192600 1000 0.729 5.36 0.187660 1000 0.759 5.74 0.174720 1500 0.764 5.81 0.172750 2000 0.796 6.25 0.160780 3000 0.873 7.46 0.134840 3000 0.882 7.62 0.131900 9000 0.896 7.87 0.127960 22500 0.903 8.00 0.1251020 67500 0.912 8.17 0.1221080 132500 0.933 8.57 0.1171140 100000 0.938 8.67 0.1151170 125000 0.941 8.73 0.1151200 225000 0.945 8.81 0.1141260 250000 0.935 8.61 0.1161320 300000 0.931 8.53 0.1171380 325000 0.928 8.47 0.1181440 350000 0.930 8.51 0.1171500 300000 0.931 8.53 0.117


UcID

A> ^ C? * rfP -# s$> -# rfP .o jS>Cell Count

Cell Count increases from 1500 to 3000

Difference in absorbance = (0.873-0.764) = 0.109


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Time(min)AverageCell Count

AverageAbsorbance Antilog Transmitance

300 208 0.417 2.61 0.383360 267 0.458 2.87 0.349420 592 0.613 4.10 0.244480 833 0.690 4.89 0.204540 1017 0.717 5.21 0.192600 1200 0.737 5.46 0.183660 1283 0.758 5.73 0.175720 1567 0.767 5.85 0.171750 2050 0.785 6.10 0.164780 2517 0.832 6.80 0.147840 3767 0.860 7.25 0.138900 9383 0.895 7.85 0.127960 22667 0.908 8.10 r 0.1231020 69500 0.914 8.20 0.1221080 128833 0.925 8.42 0.1191140 117500 0.925 8.42 0.1191170 126667 0.930 8.52 0.1171200 240000 0.938 8.66 0.1151260 254167 0.935 8.62 0.1161320 282500 0.933 8.58 0.1171380 335833 0.936 8.62 0.1161440 359167 0.932 8.56 0.1171500 281667 0.906 8.06 0.124


1.000

uc

v v v v &


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Time(min)AverageCell Count

AverageAbsorbance Ant i log Transmitance

300 133 0.108 1.28 0.780360 200 0.204 1.60 0.626420 208 0.292 1.96 0.510480 217 0.356 2.27 0.440540 225 0.393 2.47 0.405600 467 0.481 3.02 0.331660 533 0.510 3.23 0.309720 733 0.603 4.01 0.249750 950 0.647 4.44 0.225780 1117 0.684 4.83 0.207840 1933 0.704 5.05 0.198900 4133 0.717 5.21 0.192960 4933 0.736 5.45 0.1841020 5250 0.744 5.55 0.1801080 5400 0.751 5.64 0.1771140 5833 0.756 5.70 0.1751170 6667 0.760 5.76 0.1741200 7500 0.764 5.81 0.1721260 7500 0.764 5.81 0.1721320 8333 0.769 5.87 0.1701380 11667 0.781 6.04 0.1651440 13333 0.782 6.06 0.1651500 12500 0.778 6.00 0.167


Figure A- 122. Average Glucose With Cd Absorbance vs. Cell Count1.000

o0)

0.100A rb A .


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GLUCOSE WITH Pb CELL COUNTTime(min) Cell Count 1 Absorbance Antilog Transmitance

300 200 0.387 2.44 0.410360 275 0.412 2.58 0.387420 325 0.456 2.86 0.350480 400 0.489 3.08 0.324540 425 0.511 3.24 0.308600 650 0.535 3.43 0.292660 850 0.559 3.62 0.276720 900 0.581 3.81 0.262750 1000 0.589 3.88 0.258780 1000 0.594 3.93 0.255840 2500 0.620 4.17 0.240900 3900 0.702 5.04 0.198960 5650 0.719 5.24 0.1911020 7150 0.730 5.37 0.1861080 7450 0.742 5.52 0.1811140 7500 0.751 5.63 0.1781170 10000 0.754 5.68 0.1761200 10000 0.755 5.6

effect of heavy metals

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