2011

International Summer Water Resources Research School

Dept. of Water Resources Engineering, Lund University

Decolorization of C.I. Reactive Red 180 by immobilized

Citrobacter sp. CK3

By

Embla J. Mýrdal

Embla J. Mýrdal

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Instructor: Dr. Xiaojing Xiong Research assistant: Hehua Fu Lab partner: Congru Li

Embla J. Mýrdal

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ABSTRACT ................................................................................................................................................ 4

Keywords: .............................................................................................................................................................. 4

INTRODUCTION...................................................................................................................................... 5

Aim of project ........................................................................................................................................................ 5

Description ............................................................................................................................................................ 5

Restrictions ............................................................................................................................................................ 5

BACKGROUND AND THEORY ............................................................................................................. 5

Immobilization....................................................................................................................................................... 6

Previous experiments ............................................................................................................................................ 7

EXPERIMENT ........................................................................................................................................... 7

Material and reagents ........................................................................................................................................... 7

Method .................................................................................................................................................................. 8

Preparation of cell-entrapped gel beads ................................................................................................................... 8

De-coloring experiments ........................................................................................................................................... 9

RESULTS AND DISCUSSION .............................................................................................................. 10

Results of pH experiment .................................................................................................................................... 10

Results of salinity experiment .............................................................................................................................. 12

Results of glucose experiment ............................................................................................................................. 13

Detection of gel bead’s absorptive capacity to RR-180; the four hour experiment .............................................. 15

Mini extra experiment ......................................................................................................................................... 16

CONCLUSION AND RECOMMENDED IMPROVEMENTS ............................................................ 18

Acknowledgments ............................................................................................................................................... 19

REFERENCES .......................................................................................................................................... 20

Pictures ................................................................................................................................................................ 20

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Abstract In this experiment Citrobacter sp. CK3 bacteria were immobilized in PVA and sodium alginate gel beads

and the bacterial capability to remove the azo dye C.I. Reactive Red 180 was tested for various

conditions such as pH, salinity and glucose concentration. The experiment was done by a series of batch

experiments repeated five times for each factor with each test cycle being 24 hours. Absorption values

were determined using a spectrophotometer and the de-coloration efficiency was calculated.

It was observed that cell-free gel beads can hold some amount of dye and this ability changes with time.

The pH results indicate that the de-coloration efficiency is higher at a high pH although this was though

hard to determine since the gel beads easily expanded and broke in high pH environments. The results

from the salinity tests imply that a higher saline concentration is better for the de-coloration process.

From the glucose test results it was hard to determine the concentration that would result in the highest

de-coloration efficiency. An expansion of the gel beads was also noticed in the saline and glucose tests,

but this was not found to be as sensitive to different concentrations as in the pH test.

Keywords: Immobilized cell gel beads, CK3 bacteria, azo dye, pH, salinity, glucose, absorption, de-coloration

efficiency

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Introduction Dyes are used in many industries such as textile, food and pharmaceutical industries. Many dyes are

poisonous and since many are water soluble they can be hard to remove from industrial waste water. If

these dyes are not removed from the waste water this could lead to damage of ecosystems and become

poisonous for humans and animals. To be able to remove unwanted dye surplus from waste water

immobilized bacteria can be utilized.

Aim of project The aim of this project is to investigate the bacterial strain, Citrobacter sp. CK3’s ability to degrade the

azo dye Reactive red 180 (RR-180). In this experiment the parameters pH, salinity and glucose

concentration are investigated in terms of how they affect the CK3’s ability to degrade the dyestuff RR-

180.

Description The experiments are performed by a series of batch experiments containing immobilized CK3 bacteria in

dye waste water at different conditions of pH, glucose and salinity content. Each experiment is repeated

for five cycles, each cycle being 24 hours. After each cycle the absorption of the waste water is

measured and compared with a reference value in order to obtain how the de-coloration efficiency

changes with time at the different conditions. Also a small four hour experiment was done to investigate

cell-free beads ability to contain and hold the dyestuff.

Restrictions The project will focus on the parameters pH, salinity and glucose content one by one and not in

combinations. Other restrictions are that only one bacterial strains effect of degradation is investigated

on one kind of dyestuff, the RR-180. Also the experiment will only show how much RR-180 that is

degraded or absorbed by the gel beads and no investigations are conducted to detect possible by-

products.

Background and theory Azo dyes are synthetically made compounds containing a double bound nitrogen group and are the

most commonly used dyes in industries. There are about 3000 different kinds of azo dyes and they have

a wide variety of colors and shades and are easy to use for industrial purposes. Unfortunately many of

them are known to be toxic and even carcinogenic (Chen et al. 2003). Azo dyes are often highly water

soluble and are therefore hard to remove from waste water. Because of this it is important to

investigate possible treatment methods to minimize the amount released into ecosystems. Since many

azo dyes have similar chemical structures one could assume that the degrading effects would be

somewhat similar for other types of azo dyes1.

1 Mail discussion with Dr. Xiaojing Xiong (May 4 2011).

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Figure 1. Chemical structure of the azo dye Reactive Red 180.

CK3 is a gram negative facultative anaerobe rod which has in previous experiments showed to be

particularly good at degrading the azo dye RR-180 (Wang et al. 2009). There are many advantages of

utilizing bacteria to degrade unwanted chemical substances from industrial waste water. Other methods

such as adsorption and oxidation are often very expensive and could also generate poisonous by-

products. When using bacteria to degrade these substances there is a possibility of almost totally

degrading the substance and thus remove the hazardous substance and also unwanted, possibly

dangerous, by-products as well. Using bacterial degradation is also relatively cheap and effective

compared with many other methods (Wang et al. 2009). It is therefore important to investigate the

optimal conditions for the bacteria to get an as efficient degradation as possible. By providing the most

optimal conditions for the bacteria the risk for the hazardous dyestuff to reach the surrounding

environment can be minimized.

Immobilization When bacteria are immobilized they are stuck to or inside a material, often a gel matrix or bead, which

inhibits the bacteria from flowing freely in the bioreactor. In this experiment the bacteria are trapped

inside small PVA and sodium alginate beads. The beads are created by cross linking an embedded

medium made of dissolved PVA in an acid solution (the method is described later in the report). As the

PVA comes in contact with the cross linking solution the polymer gels hydrophilic hydroxyl groups will

turn inwards against each other creating a small bead (Li 2009).

Immobilizing bacteria will increase the density of bacteria within the bioreactor which in terms will

increase the rate of degradation within the bioreactor (Chen et al. 2005). In a bioreactor containing

immobilized bacteria the substrate will diffuse through the gel surface allowing the bacteria to degrade

the substance in laminar flowing conditions inside the gel bead. Having the bacteria stuck to or inside a

carrying surface network has other advantages as well, such as the possibility for bacteria to be washed

out of the system is reduced and therefore the bacteria can be reused. The carrier can also provide

anaerobic conditions for the bacteria which in degradation of azo dyes has been shown to be of great

importance. This because when azo dyes are degraded the degradation can be inhibited by enzymatic

reduction in presence of oxygen (Chen et al. 2003).

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An important thing to keep in mind is that the rigidity of the gel bed needs to be suitable for the

conditions or else it might be damaged and reduce the efficiency in the reactor Also, as is done in

previous experiments the gel beads structure, size, rigidity and proportion to waste water need to be

analyzed (Chen et al. 2007). The shape and size is needed to obtain the suitable density of bacteria and

diffusivity of media around the carrier surface within the reactor. Size and shape tests are not

performed in this experiment due to shortage of time.

Previous experiments Previous experiments of CK3 bacteria degradation of RR-180 have been conducted, for example by

Wang et al. (2008). In the experiment it was found that if the glucose concentrations were very low (0, 5

g/L) this would inhibit the growth of bacteria and could even stop the degradation of dye completely. On

the other hand if the glucose concentration was very high (>12 g/L) it would more preferable for the

bacteria as energy source which also reduced the degradation of dyestuff.

The pH is also a parameter important to study since too acidic or alkaline environments could inhibit

the bacterial growth. The CK3 bacteria were in Wang’s experiment found to grow and degrade the

dyestuff best at a range of pH 6-7. For pH-values between 8 and 10 the degradation took longer time. In

another experiment performed by Li (2009) the optimum pH was found to be weekly acid. Since many

coloring agents are bound to fibers at alkaline conditions the waste water from coloring industries is

often quite alkaline, about pH 8-9, and therefore the bacteria used at such facilities need to be able to

withstand such conditions (Wang et al. 2008).

Experiment

Material and reagents Electronic balance, autoclave, heating and drying oven, clean bench, constant temperature magnetic

stirrer, thermostatic water bath cauldron, low speed large capacity centrifuge and UV-Vis

spectrophotometer.

Table 1. Chemicals and their concentrations used in the experiment. All concentrations are in relation to preparation of 500 ml of embedding medium.

Substance Chemical formula Concentration

Disodium Hydrogen Phosphate Na2HPO4∙12 H2O 31.7 g/L

Potassium Dihydrogen Phosphate KH2PO4 3 g/L

Ammonium Chloride NH4Cl 0.5 g/L

Sodium Chloride NaCl Standard 0.5 g/L, but

varies in the salinity test

Calcium Chloride Dihydrate CaCl2∙2 H2O 4 mg/L (dye substrate),

90g (crosslinking reagent)

Magnesium Sulfate MgSO4 0.12 g/L

Vitamin B1 0.15 mg/L

Glucose C6H12O6 8 g/L

Dye RR-180 50 mg/L

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Sodium Hydroxide NaOH Varies in pH test

Hydrochloric Acid HCl Varies in pH test

Polyvinyl Alcohol: PVA-124 (CH2 CHOH)n 10% (w/v)

Sodium Alginate (C6H7O6Na)n 1% (w/v)

Boric Acid H3BO3 Added until crosslinking

reagent solution is

saturated

Method The experiment can be divided into two parts, one being the preparation of gel beads and the other the

de-colorization experiments.

Preparation of cell-entrapped gel beads

Intermediate cultivation of CK3

Sterilize clean bench for 20 min and blow for 15 min. Prepare dye substrate by mixing Na2HPO4, KH2PO4,

NH4CL, NaCl, CaCl2∙2H2O, MgSO4, vitamin B1 and RR-180 dye. Sterilize at 121°C for 20 min. Add glucose

by filtrating it through a 0,22 µm membrane. Transfer activated2 bacteria into dye substrate inoculum

size about 10% inside clean bench close to burner. The bacterium is cultivated at a constant

temperature of 37°C for about 2-3 days until the dye color has faded. This amplification is carried out

every third day in order to have enough bacteria for the different parts of the experiment.

Preparation of synthetic dye waste water

The dye waste water synthetic waste water made of tap water. Its components are KH2PO4, Na4Cl, NaCl,

CaCl2∙2H2O, MgSO4, glucose and RR-180 dye. The pH needs to be adjusted with HCl and NaOH before

the pH test. NaCl and glucose concentrations also need to be adjusted for the saline and glucose tests.

Preparation of cross linking reagent

Dissolve CaCl2 into 1 L of saturated boric acid solution and place in refrigerator until temperature

reaches 4°C.

Preparation of embedding medium

Open thermostat cauldron and heat to 92°C. Add 500 ml of de-ionized water into a beaker and place it

into the cauldron. Add PVA (10% w/v) and of sodium alginate (1% w/v). As the solid substances melt in

the hot water mix carefully with glass stick, avoid getting air bubbles in the gel. When all the solids are

dissolved let the gel cool to room temperature.

Preparation of bacterial culture3

Sterilize clean bench for 20 minutes and blow for 15 minutes. Also sterilize centrifuge tubes at 121°C for

20 min. Pour cultivated germ solution into centrifuge tubes close to burner inside the clean bench to

avoid contamination. Centrifuge tubes at 5000 rpm for 10 minutes. Remove supernatant and take

bacteria from the bottom of the tubes and add into the cold embedded medium. Mix carefully.

2 The activation was prepared by lab assistant and lab partner.

3 One series of gel beads are for investigation of the absorption of the gel beads and thus should no bacterial

culture be added.

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Making of cell-entrapped gel beads

Put the mixed medium into injectors and fix injectors to an iron stand. Let the medium drop into cross

linking reagent from 10 cm height. Stir the cross linking reagent with magnetic stirrer to form beads of

the medium. The beads should be about 3 mm in diameter and not have a long tail. When all embedded

medium has dropped into the cross linking reagent keep the gel beads in the cross linking reagent and

let them harden in a refrigerator at 4°C for 24 h.

Before usage wash gel beads with physical saline 3 times.

De-coloring experiments

Detection of gel bead’s absorptive capacity to RR-180; the four hour experiment

Add 30 ml of beads without germs into 100 ml synthetic dye waste water into sixteen flasks, eight

samples with two parallel samples each and zeal with aluminum foil. The dye concentration should be

about 20 mg/L and the pH 7. Also prepare two blank samples of dye water without gel beads. Put the

bottles into a shaker with 50 rpm and temperature 30°C. Take a pair of sample out of the shaker after

10, 30, 60, 90, 120, 150, 180 and 240 minutes and centrifuge. Determine the absorbance of the

supernatant at wavelength 543 nm and draw the absorbance variation curve depending on time for the

bacteria free beads. At each time interval also determine the absorbance of the blank samples and draw

absorbance variation curve.

Procedure of determining effect of pH

Add 30 ml of cell-entrapped beads and 100 ml of synthetic waste water into twelve flasks, dye

concentration 50 mg/L. Set two parallel samples of pH at 3, 5, 7, 9 and 11 by adjusting the pH with HCl

or NaOH. Also prepare one set of blank samples. Zeal all flasks with aluminum foil and place into a

shaker at 50 rpm and 30°C. Set 24 h as one period. As one period ends take all samples and centrifuge

the liquid at 5000 rpm for 20 min and determine the absorption at wavelength 543 nm. Then clean the

gel beads with new waste waster of corresponding pH and place in shaker for next period. Repeat for

five periods.

Procedure of determining effect of salinity

Do as in determining of pH, but instead prepare ten flasks. Set two parallel samples of NaCl

concentration at 0, 2, 6 and 12 g/L and blank samples. Perform analysis as in pH test. Then clean the gel

beads with new waste waster of corresponding NaCl concentration and place in shaker for next period.

Repeat until five periods.

Procedure of determining effect of glucose concentration

Do as in determining of pH and salinity, but instead prepare twelve flasks. Set two parallel samples of

glucose concentration at 0, 2, 4, 6 and 8 g/L and blank samples. Perform analysis as in previous tests.

Then clean the gel beads with new waste waster of corresponding glucose concentration and place in

shaker for next period. Repeat until five periods.

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Results and discussion In order to investigate the reduction of dyestuff the absorbance is measured and the color removal is

calculated by the following equation.

removal % = (initial dye conc. absorption value – residual dye conc. absorption value)/(initial dye conc.

absorption value) x 100

This gives percentage values of dyestuff removed by bacterial degradation which gives a good overview

of the different condition effects. If the absorption is lower than the reference value this should give a

positive result of color removal efficiency. The blank samples are supposed to work as a reference, i.e.

the initial dye concentration, for the normal degradation rate of the dyestuff with no CK3 bacteria

present.

Results of pH experiment The different pH of the samples influenced the shape of the gel beads strongly. In all samples the gel

beads became obviously weaker and softer but in the high pH samples, such as pH 9 and 11, almost all

the gel beads were completely destroyed after only 3 days, see figure 2. This could explain the

decreasing de-coloration efficiency values of the high pH samples, see figure 3. The gel beads effect on

the results could be due to two reasons in the pH test; one is the obvious swelling that was noticed in all

parts of the experiment (also in the glucose and salinity tests). The swelling and softening of the gel

beads made the beds release particles which affected the absorbance measurements of the samples.

The other was that in the pH test, the high pH environments changed the inner structure of the gel

beads. In the high pH conditions, the gel beads were more affected by the swelling and broke more

easily creating spongy materials when released particles joined together. In the other tests, such as

salinity and glucose concentration tests, the gel beads softening and breaking would not make the

particles join together.

Figure 2. Gel beds on day 3 of pH test. As can be seen the beads from the pH 9 and 11 samples are almost completely destroyed. (Photo: Congru Li July 2011)

From figure 3 it can be seen that all lines increase from day 3 to day 4 in the pH test. This might

implicate that the de-coloration rate on the fourth day is higher than the other days. An uncertainty is

though that the blank sample also increased on day 4.

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Figure 2. De-coloration efficiency of the pH experiment.

What the de-coloration efficiency actually was is hard to detect because of all particles that would

appear in all samples as the gel beads swelled and became softer. As can be seen in figure 2 the pH 3

and 5 are almost always above the blank sample even though there are some fluctuations of these

curves. The reason for the fluctuations could be due to two reasons; one is the error that presents by

making new samples every day and the other is that the gel beads also expanded and became weaker in

the pH 3 and pH 5 samples influencing the results. The strange declining value of the pH 3 in day 2 is

thought to be a measuring error since all the other values of pH 3 and 5 are above the blank one.

Since all the pH samples show negative values another way of determining the de-coloration is to study

the color of the different samples, see figure 4. The pH 11 and pH 9 had much lighter color than the

other pH samples every day indicating that a high pH is good for the biodegradation. This correlates

somewhat with results from previous experiments such as Wang et al.’s study.

Figure 3 Day 4 of pH test. Ph values from left to right: blank, pH 3, 5, 7, 9 and 11. (Photo: Congru Li July 2011)

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Results of salinity experiment The salinity test did not affect the shape and strength of the gel beads as much as the pH test,

compare figures 3 and 5. The gel beads did though become softer and swelled therefore releasing

particles into the salinity samples which would affect the absorption values. Another reason which could

affect the result is that the blank samples also gave negative de-coloration values. One improvement is

to perform all preparations of the experiment in a sterile environment and not use tap water, but

instead use de-ionized and sterilized water. The tap water used in the preparation of synthetic waste

water containing microorganisms from the environment might affect the results. The external

microorganisms present might be able to magnify with the nutrients present in the samples affecting

the absorption.

Figure 4. Gel beads on day 3 of the salinity test. It can clearly be seen that the gel beads from all salinity samples have expanded. (Photo: Congru Li July 2011)

For low concentrations of saline, 0-6 g/L, the salinity concentrations do not seem to affect the de-

coloration differently, see figure 6. This might also be due to the softening of the gel beads which could

cover the differences in de-coloration efficiency. On average the high salinity concentration, 12 g/L,

seems to have a better affect than the lower concentrations. On the fifth day all of the salinity samples

de-coloration efficiency decline which is believed to be due to the expansion of the gel beads and the

loss of bacteria caused by the cleaning procedures between cycles.

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Figure 5. De-coloration efficiency of the salinity experiment.

To investigate the different de-coloration affects the color differences of the samples could be

investigated. As can be seen in figure 7 there are some color differences between the samples with the

higher salinity concentrations being somewhat lighter than the lower concentrations.

Figure 6. Day 4 of salinity test. Salinity concentrations from left to right: blank 0, 2, 6, 12 g/L NaCl.

(Photo: Congru Li July 2011)

A thing that also can be seen in figure 6 is that the de-coloration efficiency in the 12 g/L group shows a

higher efficiency than the other samples. This may imply that the CK3 bacteria have a high salinity

tolerance which is a good property for bacteria used in waste water treatment facilities4.

Results of glucose experiment On the third day of the glucose test the color of the samples was noticeable lighter the higher

the concentration of glucose they contained. In the sample with glucose concentration 8 g/L there was

also noticeable white suspended mater floating on the surface. This is believed to be multiplied bacteria

colonies or particles from the gel beads. Another possible source of the material could be

macromolecules of by-products created by the biodegradation of glucose. As the gel beads break and

release particles these particles could flocculate creating the white substance. As the PVA pieces from

the gel beads conjoin they create a network trapping other matter such as free-flowing bacteria5.

4 Discussion with Dr. Xiaojing Xiong (July 12 2011).

5 Discussion with Dr. Xiaojing Xiong (July 12 2011).

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On the fourth day this was also seen in the other glucose samples, see figure 8. More suspended matter

could be noticed the higher the glucose concentration of the sample. In the 8 g/L sample the amount of

suspended matter was greatly reduced from the third to the fourth day. This may be due to that the

bacteria are washed away during preparations for the fourth day experimental cycle. On the fifth day

the 2 g/L test still contained white suspended material but none of the other samples, see figure 10.

Figure 7. Day 4 of glucose test. Glucose concentrations from left to right; blank, 0, 2, 4, 6 and 8 g/L. (Photo: Embla J. Myrdal July 2011)

The results from the glucose test show that all the samples follow a similar pattern, see figure 9. On the

second day the de-coloration efficiency declines for all samples, which is believed to be due to bacteria

and small particles released from the gel beads as they softened. These particles were perhaps not big

enough to deposit in the centrifuge and could thus affect the results. The de-coloration efficiency

obviously increases during the third and the fourth day for all samples. The decline for most samples on

the fifth day is believed to be the result of that the gel beads have expanded and softened releasing

many particles that would affect the results.

Figure 8. Decoloration efficiency of the glucose experiment.

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From the results of the glucose test it is hard to see which concentration results in the highest de-

coloration. It can easily be seen that the higher glucose concentrations result in better bacterial growth.

In figure 10 it can be seen that the color does not vary much between the different glucose

concentrations. Also the gel beads shape was not affected by the different glucose concentrations as

much as in the pH test.

Figure 9 Day 5 of glucose test. Glucose concentrations from left to right: blank, 0, 2, 4, 6 and 8 g/L. (Photo: Embla J. Myrdal July 2011)

Using absorption as a method to evaluate the de-coloration efficiency is obviously not enough for these

experiments. The de-coloration mechanism is a very complicated process involving many parameters

such as biodegradation, flocculation and the absorbance of dyestuff to the gel beads. The damage of gel

beads is responsible for the flocculation and high absorption and covers thus the effect of the

biodegradation.

Detection of gel bead’s absorptive capacity to RR-180; the four hour

experiment The de-coloration of the waste water is not only due to the biodegradation by the CK3 bacteria but also

by the physical absorption of the bead itself. This means that the gel bead can “hold” dyestuff within

itself. Therefore the main reason of de-coloration cannot be confirmed from day to day. A small four

hour experiment was performed in order to investigate the cell-free beads absorption ability.

The results from this small experiment suggest that there is no distinct pattern of the cell-free

absorption test, see figure 11. After 4 hours the gel beads had already become transparent and they had

already begun to expand. The low efficiency at 120 min might be caused by external factors.

Figure 10 Decoloration efficiency of cell-free gel beads.

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In the first two to three hours the de-coloration efficiency increases on average, despite fluctuations in

data. After about three hours the observed de-coloration efficiency started to decline. This is believed to

be due to that the gel beads already started to become soft and release particles into the samples. This

phenomenon might also explain the fluctuations of the data. The releasing of particles might conceal the

actual absorption effect of the samples.

Mini extra experiment

Part 1: Expansion test

Since it was observed in all samples that the gel beads swelled and released particles, which in turn

influenced the results of the entire experiment, two small extra experiments were made. In the first one

different volumes of bacterial liquid were mixed into the embedding medium during the preparation of

gel beads to see if this would affect their strength.

The different relations in the embedding medium were:

1. Embedding medium made by 80 ml of distilled water and 20 ml of concentrated bacterial liquid.

2. Embedding medium made by 90 ml of distilled water and 10 ml of concentrated bacterial liquid.

3. Embedding medium made by 100 ml of distilled water and no concentrated bacterial liquid.

The second (2) proportion is the one used during all other parts of the experiment.

As can be seen in figure 12 the gel beads with different mixing proportions have different appearance

after hardening in cross linking reagent. The 80 ml gel beads have almost no tail and are light yellow

with a rough and viscous surface. The 100 ml have the longest tail and are white and have a much

harder and smother surface than the other two kinds. The 90 ml gel beads are a mixture of the others

with long tails and a soft, but not viscous, surface.

Figure 11. Different shape of different prepared gel beads. (Photo: Congru Li July 2011)

After the first 24 hours of the experiment with the different gel beads in synthetic waste water no

obvious difference was seen between the different samples. Therefore the samples were left to stand

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for another 24 hours. After 48 hours obvious differences were seen between the samples, see figure 13

and 14.

Figure 12. Size and shape differences between different gel beads after 48 hours in synthetic dye waste water. (Photo: Congru Li July 2011)

Figure 13 Close-up of gel beads after 48 hours. (Photo: Congru Li July 2011)

After 48 hours the 80 ml gel beads were sticky, fragile and had lost most of their original shape. They

had also lost most of their color and were almost transparent. The 90 ml gel beads had also expanded

and lost most of their color but were not as transparent as the 80 ml gel beads. The 90 ml gel beads

were not as fragile as the 80 ml beads, but would also break easily. The 100 ml beads kept most of their

original shape and were much harder than the other two.

For further experiments it is recommended that different proportions are tested in order to reduce the

bacteria liquid to gain stronger gel beads without reducing the de-coloring efficiency. Also more

experiments are needed to research the bacterial content within the beads since the number and

activity of bacteria may not have been enough for the entire experiment6.

6 Discussion with Dr. Xiaojing Xiong (July 12 2011).

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Part 2: Filtration test

Since all results were strongly influenced by gel bead particles a small filtration experiment was

conducted to try to remove some particles. This experiment was done by filtering all glucose samples on

day 2 of the glucose experiment with common filter paper. As can be seen in figure 6 the lines are

almost parallel indicating that the filtration works equally for all samples. It can also be seen in the figure

15 that this filtration method was not efficient enough to remove all particles since most values are still

negative. So unfortunately this method could not be used to improve the experiment.

Figure 14 Effect of filtration on glucose test day 2.

Another filtering method with vacuum filtration, membrane size 0, 45 nm, was also conducted. But this

method could not be used since dye particles were suspected to stick to the membrane which would

give incorrect results. The observed dye particles in the membrane could also be dye stuck to larger

particles released from the gel beads. More filtering methods should be tested in order to remove

particles without threaten to remove dye.

Conclusion and recommended improvements The main conclusion from the experiment is that in order to get more accurate and well-founded results

of the different parameters effect on the de-coloration more testing of the gel beads are needed. Since

the gel beads swelling and releasing of particles strongly affected all results the results are inconclusive

and hard to draw any conclusions from.

This experiment can be seen as a pre-experiment for de-colorization tests. In order for future

experiments to become more successful the main improvement that needs to be done is to make the

gel beads stronger. Therefore should a much larger experiment than the mini extra experiment be

performed in order to investigate a better relation between the embedding medium and bacterial liquid

content without minimizing the de-coloring ability.

Also a more efficient experimental method should also be investigated. The many steps in each part of

the experiment might increase the source of error since small differences could occur between the

sample preparations. Some errors in our experiment are also due to human error and apparatus failure.

In order to improve the experiment further test would be needed on various combinations of the

parameters and also new parameters such as temperature and dye concentration could be tested. It is

hard to imitate a real waste water treatment facility in the lab since so many parameters have to be

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taken in to account which would lead to difficulties analyzing data. Though are experiments like this

valuable for the understanding of the importance of certain parameters.

Acknowledgments Thanks to Dr. Xiaojing Xiong for valuable thoughts and discussion about the results, Hehua Fu for all help

with the experimental work, preparations and translations and a special thanks to Congru Li for being a

great and patient group member. I would also like to thank the sponsors ITT, Sweco and Tyréns for the

financial support making this experience a possibility.

Embla J. Mýrdal

20

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Citrobacter sp. CK3, International Biodeterioration & Biodegradation 63: 395–399

Chen et al. (2003), Decolorization of azo dye using PVA-immobilized microorganisms, Journal of

Biotechnology 101: 241-252

Chen et al. (2007), Decolorization of azo dye by immobilized Pseudomonas luteola entrapped in

alginate–silicate sol–gel beads, Process Biochemistry 42: 934–942

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Bo Li (2009), Decolorization of Reactive Red Azo Dyes by Immobilized Citrobacter sp.CK, Xiamen

University (Chinese)

Pictures Figure 1: http://www.lookchem.com/cas-728/72828-03-6.html (July 10 2011)