Faculty of Health Science and Technology

Chemistry

Hoger Najem

An investigation for a method for measuring microbial degradation for

oxochlorates in waste water sludge

Master of Science in Chemistry

30 credit points biochemistry

Supervisor: Anna Smedja Bäcklund

Examiner: Thomas Nilsson

2

Abstract

The oxochlorates, such as ClO4-, ClO3

- and ClO2- are toxic compounds and therefore they must be

removed from effluents. In general they are synthetic compounds from industries and naturally also

presents in Chilean caliche. Salts of these compounds are used in many purposes for examples, NH4

ClO4 uses as a solid propellant in rocket, explosive and fireworks. It is difficult to removing this salt

from environmental pollutions because it is soluble in water.

Today, bioremediation process which is most used method to removing these toxic compounds in

wastewater treatment from industries. There are many species which have capability to using

perchlorate and/or chlorate as a sole electron acceptor in respiration pathway under anaerobic

conditions, for examples, Ideonella dechloratans, strain GR-1, CKB and perc1ace.

However, besides a perchlorate and chlorate, nitrate can also be used as an electron acceptor by

PRB. The using of nitrate instead ammonia is saving both cost and energy, but nitrate may be

interfere with the chlorate reduction. The reduction of (per) chlorate is catalyzed by two enzymes,

(per)chlorate reductase and chlorite dismutase(Cld). Chlorite dismutase (Cld) is a heme based

enzyme and has an important function in the pathway of reduction of (per)chlorate, which converted

chlorite into oxygen and chloride (ClO2 - Cl-+O2).

The main object of this work was to investigate and develop a method to measuring the potential

effect of nitrate on chlorate reduction in activated sludge from the paper and mill industry. The result

of this work is that no effect of nitrate concentration on chlorate reduction from the sludge was

found.

3

Sammanfattning

Oxoklorater t.ex. perklorat (Clo4-), klorat (ClO3

-) och klorit (ClO3-) är giftiga föreningar och därför

måste de tas bort från avloppsströmmar för att förhindra spridning i miljön. De bildas främst vid

blekning av pappersmassa inom pappers- och massaindustrin, men man har även hittat naturlig

förekomst i Chile. Deras salter används i flera ändamål t.ex. ammoniumperklorat kan användas som

raketbränsle, sprängämnen och fyrverkeripjäser. Den är vattenlöslig och därför är den svår att ta bort

från miljöföroreningar. Biologisk rening är den mest användbara metoden idag för att ta bort de

giftiga ämnena i avloppsrening från bruk. Bakteriestammar som deltar i processen är många, till

exempel, ideonella dechloratans,GR-1, CKB och perc1ace. De kan använda de giftiga ämnena i sina

andningskedjor och på så vis avlägsna dem från miljön. En del (per)kloratreducerande bakterier kan

också använda nitrat som elektronacceptor. Användning av nitrat kan spara både energi och

kostnader i reningsprocesser men nitrat kan störa kloratreduktionen. (Per)kloratreduktion

katalyseras av två enzymer. (per)kloratreduktas kan reducera både perklorat och klorat till klorit. Cld

är ett heminnehållande enzym som krävs för att klorit ska sönderdelas till syrgas och kloridjoner.

Syftet med det här arbetet var att undersöka och hitta en metod för att mäta eventuella nitrat

effekter på nedbrytning av klorat. Resultatet av studien har inte visat någon effekt av nitrat på klorat

nedbrytning i slammet.

4

Contents

Abstract................................................................................................................................................... 2

Sammanfattning………………………………………………………………………………………………………………………………….3

Contains ……………………………………………………………………………………………………………………………………………..4

Abbreviations...........................................................................................................................................5

1. Introduction…………………………………………………………………………………………………………………………. 6

2. Literature survey ………………………………………………………………………………………………………………..…7

2.1 Background ………………………………………………………………………………………………………………………….7

2.1.1 Metabolism of chlorate and perchlorate………………………………………………………………………8

2.1.2 Survey of perchlorate reducing bacteria and chlorate reducing bacteria ……………………..9

2.2 Nitrate effects from the earlier studies………………………………………………………………………………..10

3. Materials and Methods………………………………………………………………………………………………………..12

3.1 Preparation of sludge…………………………………………………………………………………………………………..12

3.2 Enzyme assay ……………………………………………………………………………………………………………………..13

3.3 Determination of total protein …………………………………………………………………………………………..13

4. Results and Discussion……………………………………………………………………………………………………………………14

4.1 Chlorite Dismutase Activity…………………………………………………………………………………………………………..14 4.2 The effect of nitrate……………………………………………………………………………………………………………………..14

5.Conclusions………………………………………………………………………………………………………………………………………16

6. References……………………………………………………………………………………………………………………………………..17

Appendix……………………………………………………………………………………………………………………………………………20

5

Abbreviations

PRB Perchlorate reducing bacteria

CRB Chlorate reducing bacteria

Cld Chlorite dismutase

Clr Chlorate reductase

Pcr Perchlorate reductase

ClO2- Chlorite

ClO3- Chlorate

ClO4- Perchlorate

6

1. Introduction

Oxochlorates such as, perchlorate (ClO4 -), chlorate (ClO3

-), hypochlorite (ClO-) and chlorite (ClO2 -) are

oxyanion ions of chlorine which do not occur naturally, only in a few exceptions. Perchlorate salts

such as, ammonium and sodium are high soluble and stable in water and they cannot be removed

easily, therefore perchlorate ions are presences and continues in ground and surface water.

Ammonium perchlorate (NH4ClO4) has been used as a strong oxidizing agent, in rocket and missile

propellant. According to the research by the Environmental protection Agency (EPA), perchlorate is

presence in the natural Caliche deposits in the Atacama Desert region of Chile. The contamination

with perchlorate is a problem, for example in United States a report showed that 361 out of ̴ 6800

public drinking water source have been contaminated with perchlorate [1].

Perchlorate can damage the function of thyroid gland in humans because it can interfere with iodine

and affect hormone production [2]. In the last 100 years, perchlorate, chlorate and chlorine dioxide

have been introduce into the environment through anthropogenic source for example, chlorine

dioxide is used as bleaching agent in the pulp and paper industry or for water disinfection [ 4].

The pollution with chlorate affects the aquatic environment, for example, in Sweden the bladder

wrack has been disappeared from the large area by the effluent from pulp mill (Mönsterås).This is

because they take chlorate instead nitrate and this occurs because chlorate is similar to nitrate and

nitrate reductase can reduce chlorate into toxic chlorite which causes inhibition of nitrate reduction

[4, 5].

In general, there are three methods of wastewater treatment oxochlorates primary, secondary

(biological) and tertiary treatment. Biological treatment is used to removing oxochlorates by

microorganisms which can use ClO3 – and ClO4

– as electron acceptor in the respiration pathway of the

pulp and paper mill effluent. Aerobic biological treatment is most used in Sweden and usually

involved a simple technique, such as aerated lagoons. Microorganisms also need nutrients (nitrogen,

phosphorus) to sustain life and support growth. Both energy and purchase nutrients are cost

efficient. In order to save costs, we can use the residue from NOx scrubbers which contains nitrate

ions as nutrients instead ammonia. Nitrate consists of one central nitrogen atom and three oxygen

atoms. These oxygen atoms can be used by microorganism in the process of treatment and this leads

to reducing supplementary of oxygen from the aeration. But the using of nitrate may interfere and

inhibit the chlorate reduction [19].

The aim of this study was to investigate the potential effect of nitrate on chlorate degradation in

activated sludge. A method was developed to showing if nitrate has an effect on chlorate reduction

of the sludge.

7

2. Literature Survey

2.1 Background

Microorganisms present in the sludge are most bacteria 95%, and 5% are other high organisms, such

as protozoa, metozoa, algae and fungi. The most used process to removing (per) chlorate

contamination in wastewater treatment is biological treatment. Microorganisms can utilize

perchlorate or chlorate as an electron acceptor under anaerobic conditions in the respiration

pathway and this process is called bioremediation. Bacteria which can reduce perchlorate are called

perchlorate reducing bacteria (PRB). The most predominant genera are Dechloromonas and

Dechlorosoma (Azospira) [11]. However, chlorate reducing bacteria (CRB) reduce only chlorate into

chlorite and then chlorite into chloride and oxygen. CRB, such as, Ideonella dechloratans,

Pseudomonas chloritidismutans strain ASK-1 may cannot reduce perchlorate [12]. Many bacteria

have been isolated from different contaminated sites and wastewater treatment sludge which are

capable of (per) chlorate bioremediation in anaerobic environments, such as, Dechlorosoma sp. KJ

and PDX, Psedumonas chloritidismutans ASK-1, Ideonella dechloratans, Wolinella Succinogenes HAP-

1, strain GR1, strain CKB, Acinetobacter thermos toleranticus and Pseudomanas chloritidismutans

AW-1 have been isolated and utilized different electron acceptors[6-8]. Most of these bacteria are

Gram negative, facultative anaerobes .The 16s rRNA sequence indicated that all isolated PRB are

located in α, β, γ and ξ subclass of proteobacteria [11,27]. The characterization of (per) chlorate

reducing bacteria have been described by other researchers [32,35].

They can use organic and inorganic compounds as energy source (electron donor), such as acetate,

lactate, methanol, sulfur and iron. Chlorate, oxygen and nitrate can be used as an electron acceptors

in the process of perchlorate reducing bacteria [2,20].

8

2.1.1 Metabolism of chlorate and perchlorate

The metabolism of perchlorate takes place in three steps. The first and second steps can be reduced

with perchlorate reductase [8, 12]. But this is not the case with Proteus, Pseudomonas and

Rhodobacter, which can reduce chlorate but not perchlorate [36]. Figure 1 shows that reaction 1 and

2 are energy yielding because they consume electrons (4 electrons). Perchlorate reductase has been

isolated in strains, such as, GR-1 and perc1ace [10,12]. It is the key enzyme that converts perchlorate

to chlorate and chlorate to chlorite. It is oxygen sensitive enzyme and located in the periplasm.

The second enzyme is chlorite dismutase which is a red colored and heme-containing enzyme that

converts chlorite into chloride and oxygen molecule. The location of this enzyme like a perchlorate

reductase is located in periplasmic area [12].This reaction does not consume any electrons. However,

the oxygen produced can be utilized by a terminal oxidase [8,9].

H2O H2O

ClO4- ClO3

- ClO2- Cl- + O2

2H+, 2e- 2H+, 2e-

Figure 1: The (per) chlorate reduction pathway.

(per)chlorate

reductase

Chlorite

dismutase

Perchlorate

reductase

1 2 3

9

2.1.2 Survey of perchlorate reducing bacteria and chlorate reducing bacteria

There is a study which has been shown that not all chlorate reducing bacteria can reduce

perchlorate, but most perchlorate reducing bacteria can also reduce chlorate [28]. To date, there are

a few microorganisms which have been reported to grow and reduce (per) chlorate. These

microorganisms have been investigated well, including, strain GR-1, strain CKB, and Wolinella

succinogenes strain HAP-1. While Ideonella dechloratans can only reduce chlorate [6,7,31].

Van Ginkel et al (1995) [16] have found chlorate reducing bacteria in different environments

including soil, sediments, ditch water and river water. In many industrial applications, these microbes

are attractive because they transform perchlorate and chlorate, which are toxic compounds into

innocuous chloride and oxygen molecule.

Perchlorate and chlorate can also be reduced to chlorite by the nitrate reductase in denitrifying

bacteria. The accumulation of chlorite, which is toxic, causes to inhibition of the cell growing and

therefore denitrifying bacteria cannot growing on chlorate because they lack chlorite dismutase to

reducing chlorite in to harmless oxygen and chloride [24,25]. Vibrio dechloraticans Cuznesove B-1168

is one of the microbes which utilize both perchlorate and chlorate as electron acceptor and acetate

as electron donor in the respiration pathway [2].

Malmqvist and Welander (1992) [13] have isolated four strains CRB from Kraft bleach effluent , which

are Gram negative and all strains could use nitrate as electron acceptor under aerobic conditions.

Quastel et al [30] found that the strains of Escherichia coli (Balantidium coli) cannot reduce chlorite

which was formed from reduction of chlorate. This is because it lacks chlorite dismutase. The

similarity of the reduction potential between nitrate and perchlorate is very close, E˚=1.25 v and

E˚=1.28 v, respectively. It makes nitrate an excellent competitor than perchlorate [17]. Based on this

reduction potential several of PRB have different responsible when they use two electron acceptor

and therefore they reduce both electron acceptor at the same time [26]. In table I, an overview is

shown of bacteria reducing (per) chlorate.

10

Table I: Examples of isolated (per) chlorate reducing bacteria in the different environment.

Bacteria strain Electron acceptor Reference

Ideonella dechloratans ClO3-, NO3

-, IO3-, BrO3

-, O2 6

Wolinella succinogense HAP-1 ClO4-, ClO3

-, NO3- 7

Dechloromonas agitata ClO4-, ClO3

-, O2 20

(Strain CKB)

Azospiraoryzae ClO4-, ClO3

-, NO3-, O2, Mn (IV) 8

(Strain GR-1)

Dechlorosoma suillium ClO4-, ClO3

-, NO3-, O2 11

Dechlorosoma sp.KJ ClO4-, ClO3

-, O2 29

Dechlorosoma sp. PDX ClO4-, ClO3

-, O2 29

Vibrio dechloraticans ClO4

-, ClO3-, NO3

- 18 Cuznesove B-1168

2.2 Nitrate effects from the earlier studies

The study of N03- effect on ClO4

- reduction is very important in the bioremediation of perchlorate

contaminated sites. It is also necessary to know if they interfere which each other. The effect of

nitrate on perchlorate is unclear or may be vary among strains. Nitrate is a potential electron

acceptor for many perchlorate reducing bacteria (PRB) (Table 1).

In the case when nitrate preferred prior perchlorate, there is a possible for perchlorate to be

inhibited by nitrate. In this case nitrate is not useful as supplemental nitrogen source in the

bioremediation process. Previous studies have showed that the effect of nitrate concentration (125

mg/l or 250 mg/l) on the perchlorate reduction by Dechlorosoma suillum JPLRND was showed to be

inhibited [34].

In early studies, it was believed that both perchlorate and nitrate can be reduced simultaneously by

one single enzyme (nitrate reductase) because perchlorate reduction was found to be inhibited [21-

23]. From the point view of thermodynamics, the similarity of reduction of potential between nitrate

and perchlorate are very close and therefore it makes nitrate as an excellent competitor of

perchlorate. It has been shown that nitrate and perchlorate are simultaneously reduced by several

11

PRB, such as D.agitata strain CKB, W.succinogenes HAP-1 and Perc1ace [15,10,7]. But, recent studies

have showed that there is a different pathway and enzyme system for simultaneously reducing of

nitrate and perchlorate. When cells for two PRB strains (Dechlorosoma sp.KJ and PDX) grown on the

different medium, chlorate, perchlorate and nitrate, respectively. In the case when they only grown

on the chlorate or perchlorate, showed that both cells were unable to degrade nitrate. When the

medium of growth contains both perchlorate and nitrate, the cells could reduce both perchlorate

and nitrate. This result was suggested that pathway of perchlorate and nitrate reductase were

separate for both species [33].

Attaway and Smith (1993) showed that nitrate is not able to inhibit perchlorate reduction in a mixed

anaerobic sludge culture [35]. In previous work, it was suggested that reaction for perchlorate and

nitrate can be catalyzed by the same enzyme, nitrate reductase [21, 37]. But the discovering of using

ClO4- but not nitrate by Dechlorosoma agitate CKB has changed this suggestion [20]. Later study

showed that nitrate reductase was not responsible for both perchlorate and nitrate reduction. This is

because the enzyme perchlorate reductase was found in the periplasmic fraction. In the cell, while

the nitrate reductase was found in the membrane, which indicate that these two enzymes are not

the same [10].

Previous work has shown that the effect of nitrate on perchlorate reduction by transferring the

nitrate grown culture of Dechlorosoma suillum into anaerobic medium. It was observed that both

nitrate reduction and growth occurred directly. But in the case when nitrate culture of the bacterium

inoculated with equimolar of nitrate and perchlorate, it was shown that the growth of the strain was

inhibited up to 40h lag phase and then nitrate reduced prior perchlorate. In contrast of this case

when perchlorate grown culture was inoculated with medium containing equimolar of nitrate and

perchlorate, no lag phase was observed and nitrate reduced prior perchlorate [15]. In contrast to

D.suillum, nitrate has been found to have a little effect on perchlorate reducing bacteria, strain CKB

[20].

Van Ginkel et al (1995) [16] have showed that the addition of nitrate into the culture lead to decreasing of chlorate. The main objective of this study is to investigate how nitrate effects on chlorate degradation of the active sludge. In order to study this objective, we can develop a method to measure chlorite dismutase activity.

12

3. Materials and Methods

3.1 Preparation of sludge

12 liter activated sludge was obtained from the aerated lagoon, Stora Enso Skoghall mill AB. After

uniform mixing of the sludge in the plastic bucket and the sludge samples were prepared in 1 liter

flask and then different concentration of chlorate and nitrate were added in all samples. The flasks

were placed on magnetic stirrer at room temperature and were storage under different time (Table

II). The experiment started with centrifugation of 1 L sludge at 8000 x g for 15 minutes. Cell pellets

from the samples 6a-7b were washed once again with 0.1M sodium phosphate buffer at pH 7.2,

containing 5 mM EDTA. Then the supernatant was discarded and the pellet was resuspended in 5-6

ml of 25 mM Bis–Tris propane buffer, pH 7.2, and then broken with a Branson Sonifer 450 set at duty

cycle in 50 % and output of 3 in 3 minutes. The samples were placed in ice bath during the

sonification. The cell homogenate was centrifuged in 90 s at 14,000 rpm (Micro centrifuge Tubes) at

rums temperature and then the supernatant transferred into a new centrifuge tubes and kept on ice

until enzyme assay was measured.

Table II: Preparation of the sludge samples.

Experiments

(samples)

ClO-3( g/L) NO-

3 ( g/L) Storage time (h)

1a control 1 - 24

1b 1 1 24

2a control 1 - 48

2b 1 1 48

3a control 1 - 48

3b 1 0.5 48

4a control 1 - 72

4b 1 0.5 72

5a control 1 - 120

5b 1 1 120

6a control 2 - 120

6b 2 5 120

7a control 2 - 144

7b 2 7 144

13

3.2 Enzyme assay

A Clark type electrode (Hansatech Instrument) was used to measure chlorite dismutase activity and

three measurements were done from range 100 to 500 µl of cell extract. The total volume of the

reaction chamber contained cell extract and 0.1 M sodium phosphate buffer at pH 7.2 with 5 mM

EDTA in a total volume of 2 ml. The reaction was started by adding20 µl sodium chlorite (NaClO2)

from the prepared stock solution (25 mM) in to the reaction mixture (chamber) using a syringe, to

create a final concentration 0,25 mM.

3.3 Determination of total protein

The Kit of pierceTM BCA protein Assay (Thermo Scientific) was used according Microplate procedure.

The standard of diluted albumin which containing bovine serum albumin (BSA) and working reagent

were prepared from ranging 0-2000 (µg/ml). 25 µl of standard and unknowns sample were added to

each well of microplate and then 200 µl working reagent (50:1, reagent A: B) was added into each

well. The plate was shaken by hand for 30 seconds and then incubated at 37 °c for 30 minutes. The

plate was cooled down in room temperature. Absorbance was measured at 562 nm by using plate

reader (TECAN Infinite®M200 pro).

14

4. Results and Discussion

The aim of this project was first to development a method to measuring microbial degradation and

second, to investigate the potential effect of nitrate on chlorate reduction in activated sludge. The

development method was performed in different concentration of nitrate during a 24-144 h storage

time of the sludge.

4.1 Measuring chlorite dismutase activity in sludge microorganism

In the first part of the project, a Clark type electrode was used to assay chlorite dismutase activity of

CRB or PRB in the sludge. Figure 2 is an example of the Cld activity and the detection of O2 evolution

from chlorite indicated the enzyme activity. In order to obtain the suitable rate of oxygen production,

different concentration of chlorate was tested (see appendix). The result showed that chlorite

dismutase activity assay is possible for the detection of chlorate reducing bacteria activity in the

sludge.

Figure 2: production of O2 in the cell extract by Cld of the sludge (sample 1).

4.2 The effect of nitrate on chlorate reduction

The effect of nitrate on the chlorate reducing bacteria activity was studied by exposing the chlorite

dismutase activity of the sludge samples to nitrogen for different period of time. The assay method

described above was used for this purpose.

Table II shows all samples studied and the results are given in the Appendix. Normalized chlorite

dismutase activity was calculated from the chlorite dismutase and protein concentration in the

sludge samples (also in the Appendix). The results are summarized in figure 3.

15

Figure 3: The relationship between normalized Cld activities with storage time.

Figure 3 shows variation between all samples and this may be the properties of the sludge has been

changed during the storage time. In all samples, there was no clear effect of nitrate and the storage

time on the Cld activity and the enzyme was active in all samples.

In the previous study, it has been shown that nitrate can inhibited perchlorate reduction because a

number PRB are utilized nitrate instead perchlorate as a terminal electron acceptor [3,14]. To the

contrary, there are studies which showed that perchlorate and nitrate can be reduced

simultaneously [15]. It has also been shown that the expression of chlorite dismutase in the

Dechlorosoma suillum strain PS, was inhibited by nitrate [15].

The result in this study indicated that neither nitrate concentration nor storage time inhibited the

chlorate reduction. It was interesting if I had more time so I would try to increase the storage time

and the concentration of nitrate and tested whether these changing can inhibit enzyme activity or

not. In all cases, chlorite dismutase was active because they showed an increasing of O2 production in

both samples control and in the presence of nitrate. The use of nitrate as electron acceptors had no

effect on chlorate reduction.

0,00

5,00

10,00

15,00

20,00

25,00

30,00

35,00

Normalized Cld activity

(µmol.g-1.min-1)

Time(h)

Control

Nitrate

24 48 48 72 120 120 144

16

5. Conclusions

This project investigated the nitrate effect on the CRB of the sludge by using different concentration

of nitrate and storage time. Based on the result, the following conclusions were drawn as below:

1. Evolution of oxygen is possible to detect and quantitate by measuring chlorite dismutase

activity with a Clark type oxygen electrode.

2. There is no harmful effect of nitrate on the chlorite dismutase activity of the sludge were

detected. Additional work needs to be done in future work of using nitrate from the NOx

scrubber as a nitrogen source of the sludge microorganisms.

3. In future studies, the correlation between chlorite dismutase activity and the chlorate

degrading capacity of the sludge should also be investigated.

17

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18

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[27] Xu J, Song Y, Min B, Steinberg L, Logan BE. Microbial Degradation of Perchlorate: principles and applications. Environ Eng Sci 2003; 20:405–22.

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[29] LOGAN, B.E., ZHANG, H.S., MULVANEY, P., MILNER, M.G., HEAD, I.M., and UNZ, R.F. (2001c). Kinetics of perchlorate and chlorate-respiring bacteria.Appl. Environ. Microbiol.67, 2499–2506.

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Appendix

Figure3: production of O2 in the cell extract by Cldof the sludge (sample 6).

Figure 4: production of O2 in the cell extract by Cld of the sludge (sample 7).

21

Figure 5: production of O2 in the cell extract by Cld of the sludge (sample 2).

Figure 6: production of O2 in the cell extract by Cld of the sludge ( sample 3).

22

Figure 7: production of O2 in the cell extract by Cld of the sludge (sample 4).

Figure 8: production of O2 in the cell extract by Cld of the sludge ( sample 5).

23

Total protein concentration

The total protein concentration of the both cells, control and nitrate were 1,3930–1,7845 g/ml , for

all samples. The total protein concentrations in all samples (1-5) have closely to each other (table III

see appendix).

Calculation of total protein concentration (BCA)

k M x (ug/ml) Conc. (g/ml) Conc.(g/µl) Conc. i 300µl

0,0012 0,0431 1392,9583 1,3930 0,0014 0,4179

0,0012 0,0431 1316,5417 1,3165 0,0013 0,3950

0,0012 0,0431 1079,8750 1,0799 0,0011 0,3240

0,0012 0,0431 1926,4166 1,9264 0,0019 0,5779

0,0012 0,0431 626,3750 0,6264 0,0006 0,1879

0,0012 0,0431 2192,0416 2,1920 0,0022 0,6576

0,0012 0,0431 2546,4167 2,5464 0,0025 0,7639

0,0012 0,0431 2084,8750 2,0849 0,0021 0,6255

0,0012 0,0431 1358,6250 1,3586 0,0014 0,4076

0,0012 0,0431 2006,0417 2,0060 0,0020 0,6018

0,0012 0,0431 2631,7500 2,6317 0,0026 0,7895

0,0012 0,0431 2448,6667 2,4487 0,0024 0,7346 0,0012 0,0431 1048,0000 1,0480 0,0010 0,3144

0,0012 0,0431 1784,4583 1,7845 0,0018 0,5353

Aborbance Average Average

A 2000 2,515 2,4130001 2,464 2,2386

B 1500 2,0512 2,1303999 2,0908 1,8654

C 1000 1,5613 1,5367 1,549 1,3236

D 750 1,3353 1,1673 1,2513 1,0259

E

F 250 0,599 0,5004 0,5497 0,3243

G 125 0,3726 0,3567 0,36465 0,13925

H 25 0,2568 0,2582 0,2575 0,0321

I 0 0,2351 0,2157 0,2254 0

Unknown 1 2,0263 1,8538001 1,94005 1,71465

2 1,8771 1,8196 1,84835 1,62295

3 1,5228 1,6059 1,56435 1,33895

4 2,622 2,5383999 2,5802 2,3548

5 1,0447 0,9956 1,02015 0,79475

6 2,8471 2,9507999 2,89895 2,67355

7 3,2492 3,3992 3,3242 3,0988

8 2,7473 2,7934 2,77035 2,54495

9 1,849 1,9487 1,89885 1,67345

10 2,671 2,6805 2,67575 2,45035

11 3,4205 3,4326999 3,4266 3,2012

12 3,1834 3,2304001 3,2069 2,9815

13 1,514 1,5382 1,5261 1,3007

14 2,391 2,4287 2,40985 2,18445

Conc.(µg/ml)

24

Figure 5: Absorbance vs. protein concentration

From the equation: Y=0,0012x+0,0431; X=(y-0,0431)/0,001

Table III: the relationship between chlorite dismutase and total protein concentration per

minute.

y = 0,0012x + 0,0431R² = 0,9898

0

0,5

1

1,5

2

2,5

0 500 1000 1500 2000 2500

Ab

sorb

ance

Conc.(μg/ml)

Samples Storage time (h) Cld activity Total protein conce. Total protein conce Normalized Cld activity

(O2 roduction (µmol/(min*L)) (g/µl) i g/ 300 µl (µmol/(min*g))

1a (control) 24 3,23 0,001393 0,4179 7,73

1b 24 3,18 0,0013165 0,39495 8,05

2a (control) 48 1,74 0,0010799 0,32397 5,37

2b 48 9,93 0,0019264 0,57792 17,18

3a (control) 48 0,72 0,0006264 0,18792 3,83

3b 48 0,11 0,002192 0,6576 0,17

4a (control) 72 12,07 0,0025464 0,76392 15,80

4b 72 9,34 0,0020849 0,62547 14,93

5a (control) 120 5,33 0,0013586 0,40758 13,08

5b 120 7,47 0,002006 0,6018 12,41

6a (control) 120 17,64 0,002617 0,7851 22,47

6b 120 3,17 0,0024487 0,73461 4,32

7a (control) 144 3,27 0,001048 0,3144 10,40

7b 144 17,94 0,0017845 0,53535 33,51