An experiment was conducted to determine the effect of glucose concentration on the decoloring performance of immobilized bacteria. The aim was to measure the amount of glucose being consumed in the decoloring process, the dissolution of gel beads, and whether the concentration of glucose influences the dissolution of gel beads. Citrobacter koseri CK-3 , a high-effective decoloring bacteria, was activated and enriched before being immobilized using an improved phosphorylated polyvinyl alcohol (PVA) gel method. Cell counting showed that before and after activation, the cell concentration increased. The concentrations of cells were 1.2
× 10 8 mL -1 and
2.2
× 10 13 mL -1 respectively. The dyestuff used in the experiment was the azo dye C.I. Reactive
Red 180. Decoloring ratio, COD and glucose was measured for synthetic wastewater with glucose concentrations 0, 0.5, 1, 2 and 4g·L -1 . Eight cycles were run in the experiment, each cycle lasting for 24 hours. The decoloring performance of the beads decreased during the first two cycles, and increased during the following cycles. At the end the decoloring ratio reached 90%. The results of the decoloring ratio from the control showed high values. COD in effluent showed an increasing trend during the first three cycles and a decreasing trend throughout the following cycles. Glucose measurements did not show a significant trend in the values.
Key words: immobilization high-effective decoloring bacteria azo dye

The discharge of synthetic dye effluents damages the water recipient by impeding light penetration, oxygen transfer and causing aestethic problems. Moreover, azo dyes and their degradation products are either toxic or mutagenic and carcinogenic. Therefore, the removal of color from textile effluents is a matter of big concern. The textile industry is the main consumer of synthetic dyes, where azo dyes constitute the largest group. Not all dyes bind to the fabric. The loss of dyes in wastewater range from 2-50% and leads to severe contamination of surface- and ground waters in the vicinity of dyeing industries. (Pandey et Al, 2007).
Azo dyes are difficultly degraded by bacteria because the reduction of azo groups is inhibited by dissolved oxygen under aerobic conditions. In order to provide a low-oxygen environment, immobilization techniques involving cell entrapment inside polymeric materials can be used. Due to diffusion resistance, the inside of the materials becomes anaerobic, constituting the perfect environment for the decoloring bacteria to degrade the azo dye. PVA immobilization is an easy and low-cost method to use at large scale. Polyvinyl alcohol (PVA) has the property of being non-toxic to organisms and can be cheaply produced at industrial scale. Furthermore, PVA beads have proven to have the elasticity and high mechanical strength that is theoretically adequate for high shear stresses (Chen et Al, 2003).
Azo dyes can be degraded by several microorganisms, including bacteria. Though, individual bacterial strain cannot degrade azo dyes completely, and the intermediate products are carcinogenic aromatic amines which need to be further decomposed (Fang et Al, 2004).
The main objective of this study was to determine the effect of glucose concentration on the decoloring performance of immobilized bacteria. The method involved measuring the amount of glucose being consumed in the decoloring process. Following questions were investigated.
Can bacteria utilize the organic compounds from the dissolution of gel beads as a carbon source?
Does the concentration of glucose influence the dissolution of gel beads?

(SUKUN SKY-2102C) Shaker Incubator
(SYQ, DSX-280) Autoclave
(inoLab pH720 pH meter (TWT Co., Germany)) PH Meter
(SW-CJ-2F) Clean Bench
(Rayleigh UV-9200) Spectrophotometer
C.I. Reactive Red 180 was the dyestuff used in the experiment. It belongs to the group of azo dyes which constitutes of one or more azo groups (R1-N=N-R2) having aromatic rings mostly substituted by sulfonate groups (Khehra et Al, 2006). The structural formula of C.I. Reactive Red
180 is:
O
NaO
3
SOH
2
CH
2
CO
2
S C
OH HN
N N
SO
3
Na
NaO
3
S SO
3
Na
The molecular formula is C
29
H
19
N
3
Na
4
O
17
S
5 and λ max
= 543 nm. C.I. Reactive Red 180 can be degraded by microorganisms. The first step in the degradation of azo dyes is the reductive cleavage of azo bond, which leads to the decoloring of dye. The cleavage of azo bond generates amines of the dye realated structures that are not degraded under anaerobic conditions and which tend to accumulate to toxic levels. Though, the amines are reported to be readily biotransformed under aerobic conditions (Khehra et Al, 2006).
The bacteria used in the experiments are Citrobacter koseri CK-3 which has proved to be highly efficient in the decoloring of water. The decoloring bacteria was obtained from a wastewater treatment plant of a dyeing factory. Test tube experiments have shown that Citrobacter koseri
CK-3 are facultative anaerobe, optimal pH for decoloring is 5~7 and optimal temperature scale ranges between 27 and 37°C. In order to degrade the dyes in anaerobic condition, the bacteria

need an external carbon source since there is not enough carbon in the dye. Previous research has found that simple organic substances such as glucose, amylum, acetate and ethanol can be used as carbon source (Pandey et Al, 2007). In this experiment, the carbon source used was glucose
8g·L -1 .
The decoloring bacteria was stored in refrigerator at 4°C, and would not show the decoloring ability because of the low activity. In order to regain the ability to degrade the dyes, the bacteria need to undergo an activation process before use. This involves activation by LB medium, 100mL, in incubator at 37°C for one night. The activation medium consist of peptone 10g·L -1 , yeast extract 5g·L -1 , NaCl 10g·L -1 and Reactive Red 180 50mg·L -1 , high temperature sterilized in 121°C for 20 min before use.
Activation process was followed by the enrichment process, which aims to obtain large quantities of bacteria. The bacteria was enriched by dye medium in incubator at 32°C for 2-4 days. The inoculation rate was 10%. The dye medium consist of Na
2
HPO
4
·12H
2
O 31.7g·L -1 , KH
2
PO
4
3g·L -1 ,
NH
4
Cl 0.5g·L -1 , NaCl 0.5g·L -1 , MgSO
4
0.12 g·L -1 , CaCl
2
3mg·L -1 , thiamine(vitamin B1) 0.15 mg·L -1 and Reactive Red 180 50 mg·L -1 , high temperature sterilized in 121°C for 20min. Dye medium was then mixed with glucose 8g·L -1 disinfected by 0.22 μm microporous filtering film.
Enrichment process is finished when the red color in the dye medium has .
After completion of the enrichment process, the decoloring bacteria was centrifuged (4500 rpm,
15 min). The cells were then added into a solution of polyvinyl alcohol (10%) and sodium alginate
(1%). The solution must first dissolve in water bath > 80°C and cool to room temperature before the bacteria was added. After adding the bacteria, the mixture was dropped into saturated boric acid solution with calcium chloride (3%) using a syringe to form beads with the approximate diameter of 3mm. To complete gel formation, the PVA-Calcium alginate gel immobilized bacteria was stored in saturated boric solution with calcium chloride (3%) at 4°C for 20h.
The bacteria was counted before and after activation. A small amount of decoloring bacteria was mixed with LB medium. 1 ml of the mixture was shifted into a test tube and diluted to 50 ml with

distilled water. 1 ml of the mixture in the test tube was shifted into another test tube and diluted the same way. Preparing for the cell count before activation, the procedure was repeated three times, so that in the end there was four test tubes with different concentration of bacteria. This gives a dilution of 50 4 in the last test tube. For the cell count after activation, seven test tubes were used, which gives a dilution of 50 7 in the last test tube. 1 ml from the last test tube was shifted into three agar plates respectively. The agar plates were then filled with 20 ml LB medium on each plate. Cell count was done after 3 days.
The synthetic dye wastewater used in the experiment was prepared the following way.
Mineral salts medium (MSM) of following composition: KH
2
PO
4
3g·L -1 , NH
4
Cl 0.5g·L -1 , NaCl
0.5g·L -1 , MgSO
4
0.12 g·L -1 , CaCl
2
3mg·L -1 , deionized water. The MSM was supplemented with glucose 8 g·L -1 and dye 50 mg·L -1 . The final pH of the synthetic wastewater was adjusted to 9.0 by NaOH solution.
2.7.1 COD measurement
Potassium dichromate method was used to measure the COD in the synthetic dye wastewater. The following function was used to calculate the COD in the samples.
COD
Cr

O
2
, mg

L

1



V
0

V
1

 c

8

1000
V
Where c is the concentration of ferrous ammonium sulfate solution, and V
0
and V
1
are the volumes of ferrous ammonium sulfate solutions required for titration of the blank and the sample, respectively, in mL; V is the sample volume in mL; 8 is the 1/2O mole mass in g/mol.
2.7.2 Glucose measurement
The iodometric method is used to measure the glucose concentration in the synthetic dye wastewater. The glucose concentration was measured in five wastewater samples with glucose

concentrations 0, 0.5, 1, 2 and 4 g·L -1 during 5 different cycles. The flasks can be contaminated by bacteria from the air, which can degrade the Reactive Red 180. To examine the impact of the contaminating bacteria, a control group without bacteria in the beads is included in the experiment.
2.7.3 Color measurement
The colourity of the synthetic dye wastewater was detected using a spectrophotometer
(λ max
=543nm) The colourity was measured in five wastewater samples with glucose concentrations 0, 0.5, 1, 2 and 4g·L -1 from 8 different cycles respectively, each cycle lasting for 24 hours. The same measurement was made on blank samples. The following function was used for calculating the decoloring ratio.
 
A
0

A i
A
0

Where A
0
: influent colourity; Ai: effluent colourity.
The results from the cell counting (Table 1) show that the quantity of bacteria increased in the activation process. The number of bacteria was 1.17·10 8 mL -1 and 2.19·10 13 mL -1 before and after activation respectively.
Table 1. Results from the cell counting before and after activation, and after enrichment. before activation after activation
1
9
11
2
27
45
3
20 blank result
110 1.17
·10 8 mL -1
2.19
·10 13 mL -1

A method for dissolving the beads is desired for further experiments. The dissolving of the beads is necessary to be able to count the bacteria in the beads after several cycles.
The results from the COD measurement (Figure 1) show an increasing trend during the first three cycles and a decreasing trend during the following cycles. The increasing trend reflects the dissolving of beads. The decreasing trend can be explained by the increasing growth of bacteria, leading to a higher consumption of glucose. On the other hand, the dissolution of gel beads becomes slower. When the glucose concentration in the influent water is 0 mg·L -1 , it is only the dyestuff that contributes to the COD.
18000.00
16000.00
14000.00
12000.00
10000.00
8000.00
6000.00
1
2
4
0
0.5
4000.00
2000.00
0.00
0 1 3 c yc les
4 5 7 8
Figure 1. COD measurement of five wastewater samples with glucose concentrations 0, 0.5, 1, 2 and 4g·L -1 measured during 6 different cycles. Cycle number 0 indicates the influent water.

Results from the glucose measurement are presented in Figure 2 and 3.
0.05250
0.05150
0.05050
0.04950
0.04850
0.04750
1
2
0
0.5
4
0.04650
0.04550
3 4 c yc les
5 7 8
Figure 2. Glucose measurement of five wastewater samples with glucose concentrations 0, 0.5, 1, 2 and 4g·L -1 from 5 different cycles.
The quantity of decoloring bacteria will increase for each cycle. blank
0.05050
0.05000
0.04950
0.04900
0.04850
0.04800
0.04750
0.04700
1
2
4
0
0.5
0.04650
3 4 5 c yc les
7 8
Figure 3. Glucose measurement of blank wastewater samples with glucose concentrations 0, 0.5, 1, 2 and 4g·L from 5 different cycles.
Results from the glucose measurement (Figure 2) do not show a significant trend. The number of bacteria increases with each cycle. This would imply a higher consumption of glucose, which is not indicated by the results. The experiment aimed to discover any tendencies of glucose in effluent. Since no trend was detected the method needs to be refined.

An unexpected result is that of the samples with 0g∙L -1 glucose, which measures glucose concentrations around 0.05g∙L -1 . An explanation of this could be that NaIO reacts with other compounds than glucose, thus contributing to the measurement of higher concentrations of glucose in the samples. Since the PVA beads dissolve to a certain extent in solution, we do not know the exact compounds in the liquid. When α-carbons adsorb H they can react with an oxidant, like NaIO. The PVA might dissolve into alcohols such as ethanol which will react with NaOH to form NaIO and I
2
. The I
2
-solution used in the glucose measurement should be 0.05mol∙L -1 , but was 0.183mol∙L -1 when we measured it. The higher concentration of I
2
-solution contributes to higher values of glucose concentration. The control show a stable value (Figure 3), ranging between 0.48 and 0.5g∙L -1 .
When analyzing the results, the ideal would be to study the measured concentration of glucose in a sample minus the concentration of glucose in the control. This was not done in this experiment since the control was contaminated by decoloring bacteria, and thus did not reflect the true value of glucose concentration in the water. The color of the control with 1g∙L -1 glucose was observed turning transparent after some cycles. This can be due to the flasks being contaminated by bacteria from other flasks. The same thing is likely to have happened for the other control. An improvement for the next round of experiments would be to keep control and regular samples separately in different incubators, and to irradiate the blank samples with UV-light.

The results from the colourity measurements are shown in Figure 4 and 5.
100
80 1
2
4
0
0.5
60
40
20
0
1 2 3 4 c yc les
5 6 7 8
Figure 4. Decoloring ratio of five wastewater samples with glucose concentrations 0, 0.5, 1, 2 and 4g·L -1 measured during 8 different cycles
100 blank
80
60
1
2
0
0.5
4
40
20
0
1 2 3 4 c yc les
5 6 7 8
Figure5. Decoloring ratio of the blank wastewater samples with glucose concentrations 0, 0.5, 1, 2 and 4g·L -1 measured during 8 different cycles
The results (Figure 4 and 5) show that the performance of the decoloring bacteria decreases during the first two cycles. This is because the bacteria in the beads absorb the dye, but only to a certain extent before the beads are saturated. Another reason is that the activity of the bacteria still is low.
The decoloring ratio increases during the following cycles due to the growth of bacteria. The bacteria growth increases the biodegration, which becomes the main reason for decoloring.

The results of the decoloring ratio from the control (Figure 5) show high values. The decoloring ratio should be low for control. Normally, the correct value for the decoloring ratio will be given by subtracting control from the absorption. This will not be done in this experiment, though, since the control show high values. One possible explanation for the high values can be that the control flasks have been contaminated by bacteria from other flasks. For next time the experiment is run, control flasks and other flasks will be stored in different incubators. To further impede the contamination of bacteria, the control flasks will be illuminated by UV-light.
The gel beads dissolve to a certain extent in water after some period of use. Since the PVA gel consist of organic compounds, organic material will be released to the water when the beads dissolve. The released organic material will benefit the microorganisms as an extra carbon source.
This can be observed by the results from the samples with 0g∙L -1 glucose (Figure 4 and 5). A decoloring ratio above 0 indicates that the bacteria obtain their carbon source from the beads.
After several cycles, the beads grow bigger. This is because the beads absorb water and the bacteria inside the beads generate gas which can not escape the beads.
The decoloring performance of the beads decreased during the first two cycles and increased during the following cycles. At the end the decoloring ratio reached 90%. The results of the decoloring ratio from the control showed high values. Improvements for further experiments would be to keep regular samples and control in different incubators and to illuminate the control with UV-light. COD in effluent showed an increasing trend during the first three cycles and a decreasing trend throughout the following cycles. Glucose measurements did not show a significant trend in the values. Another method is needed for measuring the glucose concentration.
The gel beads dissolve to a certain extent in water. The released organic material from the dissolved beads is utilized by the bacteria as an external carbon source. The results from the cell counting showed that the quantity of bacteria increased in the activation process. The number of bacteria was 1.174·10 8 mL -1 before and 2.19·10 13 mL -1 after activation. A method for dissolving the beads is desired to be able to count the bacteria in the beads after several cycles.

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Fang H., Wenrong H., Yuezhong L. 2004. Investigation of isolation and immobilization of a microbial consortium for decoloring of azo dye 4BS . Water Research 38: 3596-3604
Khehra M.S. et Al. (2006). Biodegration of azo dye C.I. Acid Red 88 by an anoxic-aerobic sequential bioreactor . Dyes and Pigments 70: 1-7
Pandey A., Singh P., Iyengar L. (2007). Bacterial decolorization and degradation of azo dyes .
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