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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
6. References
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18
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20
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