decolourization of azo dyes in textile wastewater by...

Decolourization of azo dyes in textile wastewater by microbial processes

Växjö februari 2010 Examensarbete nr: TEK 004/2010

Orçun Türgay Avdelningen för Bioenergiteknik

LINNÉUNIVERSITETET Författare Institutionen för teknik Orçun TÜRGAY Linnaeus University School of Engineering Dokumenttyp/Type of document Handledare/tutor Examinator/examiner Examensarbete/ Diploma work Ulrika Welander Ulrika Welander Titel och undertitel/Title and subtitle Decolourization of azo dyes in textile wastewater by microbial processes Keywords: Azo dyes, microbial, lignocellulosic material, textile wastewater Abstract (in English) Decolorization of Azo dyes in synthetic wastewater composition which is similar to real textile wastewater was carried out by microbial process. Experiments were performed in two continuous systems. Experiments were performed under anaerobic conditions in order to break the nitrogen bond of the azo group (-N=N-). A synthetic dye solution which contained 200 mg/L Reactive Black 5, 200 mg/L Procion Red MX-5B and 1 g/L yeast extract was prepared. In this study, living microorganisms were used to degrade the dyes in wastewater. Rice husks which contain bacteria and fungi were used in the reactors of continuous systems. The parameters tested on continuous system were wastewater composition, the number of reactors, the amount of yeast extract in wastewater composition, the wastewater flowrate, washing the system with wood chips solution, addition of yeast extract solution. Results have shown that increasing the number of reactors, the retention time, the amount of yeast extract and washing the system with wood chips solution had positive effects for degradation of the dyes from wastewater. When the flowrate was increased the retention time has decreased so degradation of dyes has decreased but although the flowrate increased twice, % degradation hasn’t decreased as the same ratio. Therefore this result showed that this process can be worked for faster flowrates. Microbial process is a promising technology which might be used to treat wastewater containing azo dyes with good performance. Key Words Utgivningsår/Year of issue Språk/Language Antal sidor/Number of pages 2010 English 30 Internet/WWW http://www.lnu.se

1 INTRODUCTION.................................................................................... 4

1.1 REACTIVE DYES……………………………………………….....5 1.1.1 AZO DYES………………………………………………….. 5

1.1.2 ANTHRAQUINONE DYES………………………………... 5

2 EXPERIMENT 1 (Part I- 200 mg each dye and 1 g yeast/L)………...6

2.1 OBJECTIVE……………………………………………………… 6

2.2 THEORY…………………………………………………………. 6

2.2.1 MICROORGANISMS……………………………………………..7 2.3 MATERIAL AND METHOD……………………………………. 7

2.4 RESULTS OF THE EXPERIMENT……………………………...10

3 EXPERIMENT 1 (Part II- New composition which is more similar to real textile wastewater)………………………………………………. 12

3.1 OBJECTIVE……………………………………………………....12

3.2 MATERIAL AND METHOD…………………………………… 12

3.3 RESULTS OF THE EXPERIMENT…………………………….. 12

4 EXPERIMENT 1 (Part III- Series connected four reactors)……......14

4.1 OBJECTIVE……………………………………………………... 14



5 EXPERIMENT 1 (Part IV- Increased the amount of yeast extract)..16

5.1 OBJECTIVE……………………………………………………... 16

5.2 MATERIAL AND METHOD……………………………………17

5.3 RESULTS OF THE EXPERIMENT……………………………. 17

6 EXPERIMENT 1 (Part V- Increased the flowrate)…………………18

6.1 OBJECTIVE……………………………………………………...18

6.2 MATERIAL AND METHOD……………………………………18

6.3 RESULTS OF THE EXPERIMENT……………………………. 18

7 EXPERIMENT 2 (Part I- Rice husks with wood chips and dye mixture)……………………………………………………………......20

7.1 OBJECTIVE……………………………………………………... 20

7.2 MATERIAL AND METHOD………………………………….... 20


8 EXPERIMENT 2 (Part II- Increased the flow rate)………………... 23

8.1 OBJECTIVE……………………………………………………... 23

8.2 MATERIAL AND METHOD………………………………….... 23


9 EXPERIMENT 2 (Part III- Increased the amount of yeast extract)..24

9.1 OBJECTIVE……………………………………………………... 24



10 EXPERIMENT 2 (Part IV- Increased the flowrate)……………….. 27

10.1 OBJECTIVE……………………………………………………... 27



11 BATCH EXPERIMENTS…………………………………………...................29

11.1 OBJECTIVE……………………………………………………... 29



12 REFERENCES……………………………………………… . 31

1 INTRODUCTION The amount of wastewater from the textile industries has been increasing together with the effects of the growing demand for textile products. The release of wastewater into the environment can be the reason for serious health and environmental problems. The dyestuffs that the textile wastewaters contain and their breakdown products might have toxic and/or mutagenic features to life. (Weisburger, 2002) Without any treatment, these dyes can be stable and remain in the environment. (Hao et al, 2000) Together with the environmental and health problems, it is also reality that the textile industry has been using the large amounts of potable water in the production process of the textile products. Due to this reason, the recycling of wastewater has been becoming important and recommended for the protection of environment and decreasing the usage of potable waters. (Talarposhti et al, 2001; Dos Santos et al, 2006a) In order to reduce the pollution effects of the dyes to environment, there are three kinds of treatment techniques for removing of the dyes from wastewaters. These are chemical, physical and biological treatment techniques. (Robinson et al, 2001; Pearce et al, 2003) Chemical treatment techniques consist of two main divisions as advanced oxidation and chemical reduction. Advance oxidation includes some treatment processes like ozonation, fenton oxidation and photo catalytic oxidation. Chemical treatments contain also chemical reduction reactions. They convert hazardous polluting to nonhazardous or less toxic compounds. (http://www.brownfieldstsc.org/glossary.cfm?lett=C) Second treatment technique is physical. It contains adsorption, ion exchange, irradation, membrane filtration, electrolysis, coagulation/ flocculation and ultrasonic mineralization methods. The last treatment technique is biological, which includes bacterial and fungal biodegradation and biosorption in aerobic, anaerobic or combined treatment processes. (Chang and Lin, 2000; Robinson et al., 2001; Pearce et al., 2003) If we make a comparison among these treatment techniques, the chemical treatment techniques have some disadvantages. First of all, it makes the operation expensive which might create financial problems for applying these techniques in developing countries. (Ozbelge et al., 2002; Pera- Titus et al., 2004) Physical treatment methods also have some disadvantages. They need high investment cost and the replacement of equipment is difficult. (Mishra and Tripathy, 1993) In addition, physical methods can include the generation of hazardous sludge. (Mattioli et al 2002; Van der Zee, 2002) Biological methods are relatively cheap and environmental friendly; because living organisms are used for decolorization of the dyes from textile effluents. This also makes these methods applicable in developing countries where the textile industries are common. (Knackmuss, 1996) Therefore, the biological treatment method has been implemented for this research by working on anaerobic conditions. The aim of this study was to develop an efficient, cheap and simple microbiological treatment method for removing the dyes from textile wastewaters. For this aim, rice husks and wood chips were used in the reactors because these materials are natural environment for living microorganisms. They might also be used as carbon source for the microbes. Yeast extract was used as a nutrient source for microorganisms. 1.1 REACTIVE DYES

Reactive dyes are an important group of dyes and widely used in textile industry. Reactive dyes have some advantages such as wide variety of colour shades, easy of application, good colour fastness and brilliant colour. Reactive dyes with reactive groups constitute covalent bonds with OH, NH or SH groups in fibers such as cotton and wool. (Epolito et al., 2005; Lewis and Loan, 2007) During dyeing with reactive dyes, approximately 50 % of the dye cannot be reacted with the fibers and stay put hydrolyzed in water phase. If it is released into the environment, it can cause some environmental problems. (O’Neill et al., 1999) 1.1.1 AZO DYES

Azo dyes are the largest group of dyes with large industrial application. Azo dyes stand for almost 70 % of the textile dyestuffs produced by weight. (Knacckmuss, 1996; Meyer, 1981) They are used in the colouring process of several textiles and leather products. Azo dyes include at least one or more azo (N=N) double bond, with one or more aromatic systems.(Wong and Yuen, 1998) Azo dyes are classified into two subgroups according to number of their double bond as mono-azo and poly azo types. (Zollinger, 1986)

Figure 1: General structure of azo dyes There are high amounts of azo dyes in the environment because of breakdown of azo bonds (R-N=N-R) is quite difficult and they can be stable in acidic and alkaline conditions. They are resistant to high temperatures and light. (Wong and Yuen, 1996) 1.1.2 ANTHRAQUINONE DYES Anthraquinone dyes represent the second most important class of textile dyes. They have a large range of colours. Anthraquinone dyes are used to colour cellulosic fabric, wool and polyamide fibres. (Baughman and Weber, 1994)

Figure 2: General structure of Anthraquınone dyes

2 EXPERİMENT 1 (Part I- 200 mg of each dye and 1 g yeastextract / L ) 2.1 OBJECTIVE

The aim of this study was to develop an efficient, cheap and simple treatment method based on microbial process. The experiment was performed to consider the decolourization of a synthetic solution prepared. The experiment was performed in continuous mode and under anaerobic conditions. 2.2 THEORY The synthetic dye solution used in this experiment was prepared by use of two azo dyes. They were Reactive Black 5, (CAS number is 17095-24-8) Figure 3 and Procion Red MX 5B, (CAS Number is 17804-49-8) Figure 4. They were used in equal concentrations.

Figure 3: The chemical structure of Reactive Black 5

Figure 4: The chemical structure of Procion Red MX-5B In order to obtain a more efficient degradation two reactors which have same volume were used in the same continuous process.

2.2.1 MICROORGANISMS

Rice husks were used in the reactors because they are natural environment for living microorganisms such as fungi and bacteria. This situation might be an advantage when molecules with complex structures should be degraded. The fungi can degrade structures which are difficult for bacteria. Therefore, the bacteria can degrade intermediates formed by the fungi (Rosen et al, 1998). 2.3 MATERIAL AND METHOD The experiment was performed in a continuous two stage process.

Two reactors were performed anaerobically. Each anaerobic reactor (reactor 1&2) had a volume of 449, 3 ml. Reactors were filled with rice husks: Reactor 1: 42, 6 g rice husks Reactor 2: 41, 1 g rice husks Before starting this experiment, we have washed the connect tubes which are between reactors and enter bottles with ethanol for sterilization. Synthetic dye mixture was prepared. Dye mixture contains 200 mg/L Reactive Black 5 and 200 mg/L Procion Red MX-5B and 1g/L yeast extract. The dyes and yeast extract were dissolved in tap water. After preparing this solution, putting the dye mixture in autoclave, Figure 5, was necessary in order to sterilize the dye solution. Dye solution was in autoclave at high temperature (121 οC) and high pressure conditions during 15 min.

Figure 5: Photo of the autoclave used during the experiment After this sterilization operation, dye solution was ready to pass through the system, Figure 6. The speed of the pump (C6-MIDI multi-channel pump, Watson Marlow SCI) was as slow as possible in order to implement the dye degradation. If the speed of pumping is too high, the retention time will be too low and the microorganisms cannot degrade the dyes. The flow rate at 5 rpm was 0,351 ml/min (21, 06 ml/h). The retention time in the reactor 1 and 2 was around 0, 88 day (21, 3 hours) for each reactor.

Figure 6: Photo of the experiment which gives the details of two reactors. This photo was taken the day after the start-up of the process. To measure the absorbance, samples (6-7ml) were taken directly from each reactor and also from the prepared dye mixture. The samples were taken every second day. They were taken always from the same place of each reactor in order to obtain comparable measurements. An optimum dilution (200μl sample + 1000μl distilled water) was prepared and then they were scanned with a spectrophotometer, Perking Elmer Lambda 35 UV/VIS and analyzed by Perking Elmer UV winlab ver: 2.85.04 software, Figure 7. The absorbance was measured between 190 and 750 nm wavelengths.

Figure 7: Photo of spectrophotometer used in the experiment. The diluted samples were analyzed in the spectrophotometer with quartz cuvettes. These cuvettes should be perfectly clean and dry.

Figure 8: Photo of degradation of azo dyes in synthetic dye mixture in experiment 1. This photo was taken on the 23rd day of the experiment. The dye solution in the inlet bottle is very dark while the shade is a bit brighter in reactor 1 and even brighter in the outlet from reactor2. 2.4 RESULTS OF THE EXPERİMENT

This experiment was performed during 31 days (27 April- 28 May). The flow rate was 0,351 ml/min. When the absorbance couldn’t decrease any more, the experiment was finished.

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Figure 9: Absorbance data of synthetic dye wastewater and the outlets of every reactor in the 5th day of the experiment. Figure 9 shows the absorbances of the synthetic dye solution and the outlet of the reactors. The maximum peak was at 550 nm of wavelength. Decreasing of the absorbances of R1 and R2 can be seen. Reactive Black dye can be degrade at around 597 nm and Reactive Red dye

can be degrade at around 538 nm. λmax is given at 550 nm for the mixture of reactive black and reactive red dye. There is also a hump at 625 nm for the mixture of the dyes. After 5 days there was 14, 2 % of degradation at 549, 94 nm wave length for R2.There was 75, 3 % degradation for R2 at 625, 47 nm.

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Figure 10: Absorbance data of the dye wastewater and the outlets of the reactors in the 3rd week (in the 21st day in the process) after start-up the system. Figure 10 shows the absorbance in the R1 and R2 have been decreasing. There were a big difference between absorbance of dye solution and absorbances of reactors. Percents of degradation were 59, 7 % for R1 and 78, 8 % for R2 at 549, 94 nm wave length. There were 76, 7 % of degradation for R1 and 78, 3 % of degradation for R2 at 625, 47 nm.

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Figure 11: Absorbance of the synthetic dye mixture and the outlets of the reactors in the 30th day of the experiment.

Figure 11 shows the final absorbance data of reactors for part 1 of experiment 1. Percents of degradation were 53, 9 % for R1 and 78, 2 % for R2 at 549, 94 nm wave length. Because of the absorbance didn’t decrease any more, some changes were made on the process. 3 EXPERİMENT 1 (Part II- New composition which is more

similar to real textile wastewater) After the satisfying results obtained in the part I of experiment 1, a new wastewater composition which is more similar to real textile wastewater was tried for this process. 3.1 OBJECTIVE The aim was to evaluate decolourization of a wastewater with a more realistic chemical composition. 3.2 MATERIAL AND METHOD

Enter dye mixture of continuous system 1 was changed and a new mixture which is more similar to real textile wastewater was used. This formula was: 12 g NaCl, 0,049 g Na3PO4*12H2O, 0,024 g NaNO3, 0,255 g KHSO4, 200 mg Reactive Black 5 dye, 200 mg Procion Red MX 5B and 1 g yeast for 1L solution. This composition was chosen to be more similar to real wastewater (dos Santos et al, 2006a) To prepare this solution, firstly one package of natural cotton (approximately 50 g) which contains oil and waxes was used and then 0, 5 ml soap was added over the cotton. 1L tap water was poured over mixture which contains cotton oil and soap. After mixing, it is waited for 15 min. Dyes and yeast extract were prepared in the other flask as powders. Finally, the mixture (cotton oil, soap and 1 L tap water) was poured over dye mixture (200 mg black dye/L, 200 mg red dye/L and 1 g yeast/L). This mixture which is similar to real textile wastewater was passed through the system 1. Again the samples were taken every second day and they were scanned with spectrophotometer. 3.3 RESULTS OF THE EXPERIMENT

This experiment was performed during 26 days (28 May- 23 June). The flow rate of dye composition was again 0,351 ml/min. The same dyes (Reactive Black 5 and Procion Red MX 5B) were used. With the results of part II of the experiment, effects of this process on new wastewater composition which is more similar to real wastewater were investigated.

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Figure 12: Absorbance data of new wastewater composition and the outlets of the reactors in the 5th day of this experiment. Percent of degradation were 18, 4 % for R1 and 41, 9 % for R2 at 549, 94 nm wave length after 5 days, Figure 12. Percents of degradation was 37, 5 % for R1 and 72, 0 % for R2 at 625, 47 nm.

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Figure 13: Absorbance data of dye wastewater and the outlets of the reactors in 3rd week of this experiment after using the new composition. Figure 13 shows the absorbance data of the outlets from the reactors were high. Percents of degradation were 11, 1 % for R1 and 29, 5 % for R2 at 549, 94 nm wave length. Percents of degradation were 43, 1 % for R1 and 69, 0 % for R2 at 625, 47 nm. There weren’t satisfied absorbance results and treatment in this period of time. Because of this reasons, some changes were made on the continuous system 1. 4 EXPERIMENT 1 (Part III- Series connected four reactors)

In order to try to improve the results for new composition which is more similar to real wastewater some changes were made on the system. The number of reactors was increased from two to four. Rice husks were put into the new two reactors. It means addition of new microorganisms. And then the system was washed with wood chips solution. The wood chips solution was used in order to add new microorganisms which have different capability for degradation of organic compounds. 4.1 OBJECTIVE The aim was to obtain a more efficient decolourization of dye composition after doing changes which include increasing the number of reactors and additions of microorganisms originating from wood chips. 4.2 MATERIAL AND METHOD The experiment was carried out in a continuous four stage process, Figure 14.

Figure 14: Photo of the new system 1 with four reactors in series. This photo was taken on the 7th day after re-building of the system. First of all, two new reactors were connected to the other reactors. The names of reactors of system 1 were changed. The new names of reactors are S1, S2, S3 and S4. Volume of our new reactors (S3&S4) was 430 ml each. New reactors were filled with rice husks. Reactor S3: 39, 5 g rice husks Reactor S4: 41, 8 g rice husks A new solution was prepared by adding 400 ml 0, 9 % NaCl to 50, 5 g wood chips in a flask. The flask was than placed on a magnetic stirrer for 15 minutes. 100 ml of the solution was added into every reactor. I used the dye mixture which has same formula as the model real textile used in part II of experiment 1.

The flow rate was 0,351 ml/min. To measure the absorbance in UV/VIS, the samples were taken every second day from reactors. 4.3 RESULTS OF THE EXPERIMENT This experiment was performed during 22 days (23 June- 15 July). The flow rate was again 0,351 ml/min. The same dyes were used and the wastewater composition was same according to part II of the experiment.

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Figure 15: The absorbance data of dye wastewater and the outlets of every reactor in 5th day of this experiment after re-building of the system. Figure 15 shows the absorbance of the outlets from every reactor. Percent of degradation was 87, 9 % for S4 at 549, 94 nm wave length. There was 87, 0 % degradation at 625, 47 nm. After using four reactors, good results were obtained. Big difference between absorbance of dye composition and absorbance of reactor S4 can be seen. However, there were important differences between absorbance data of every reactor. With increasing the number of reactors, new microorganisms were added into the system and retention time has increased so % of degradation has increased. There was a good decolourization of dye the dye mixture.

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Figure 16: The absorbance results of dye wastewater and the outlets from every reactor in 3rd week of the experiment after using four reactors. There was an important difference between 5th day of the experiment and 3rd weeks of the experiment as percent of degradation. Percent of degradation was 78, 6 % for S4 at 549, 94 nm wave length. There was 80, 1 % of degradation for S4 at 625, 47 nm. Percent of degradation has decreased from 87, 9 % to 78, 6 % at 549, 94 nm from 5th day of the experiment to 3 rd week of the experiment. Because of this reason, more yeast extract was added into the wastewater composition in order to obtain a better decolourization. 5 EXPERIMENT 1 (Part IV- increased the amount of yeast

extract) The amount of yeast extract was increased in the formula of wastewater composition. The flow rate and the dye composition were same with part III of experiment 1. 5.1 OBJECTIVE The aim of this change on the system was to obtain a more efficient treatment. Because yeast extract is a nutrient source for microorganisms. If the number of microorganisms increases, decolourization might increase. 5.2 MATERIAL AND METHOD The amount of yeast extract was increased from 1g/L to 2 g/L in the formula of enter dye mixture of system1 on 100th day of experiment 1. Increasing the treatment in system 1 was intended by this way. The flow rate was 0,351 ml/min. The wastewater composition and the

process were also the same as in part III of experiment 1, except for the yeast extract concentration. 5.3 RESULTS OF THE EXPERIMENT This change was applied on the system during 22 days. (15 July- 6 August)

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Figure 17: The absorbance results of dye wastewater and the outlets from each reactor in the 5th day of the experiment after increasing the yeast extract. Figure 17 shows the absorbance of every reactor decreased in a short time as 5 days. Decreasing of the absorbances of all reactors can be seen in the figure 17. Percent of degradation was 82, 3 % for S4 at 549, 94 nm wave length. Percent of degradation was 75, 3 % of degradation for S4 at 625, 47 nm.

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Figure 18: The absorbance results of dye wastewater and the outlets from each reactor in 3rd week of the experiment after adding the yeast extract.

Decreasing of the absorbance data of all reactors have continued in Figure 18. Differences of absorbance data between storage flask and every reactor can be seen easily. 89, 2 % of degradation was obtained for S4 at 549, 94 nm. There was 79, 0 % of degradation for S4 at 625, 47 nm. After obtaining the results presented above, the flowrate of the system was increased. 6 EXPERİMENT 1 (Part V- Increased the Flowrate) 6.1 OBJECTIVE The aim of this experiment was also to investigate the efficiency of the process at a higher flowrate. 6.2 MATERIAL AND METHOD The process and wastewater composition were same with part IV of experiment 1. Only the flowrate of the system was changed. The flowrate was increased from 0,351 ml/min to 0, 803 ml/min. 6.3 RESULTS OF THE EXPERIMENT This experiment was performed for 34 days (6 August-8 September). The samples were taken every second days and their absorbance values were measured in UV/VIS spectrometer.

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Figure 19: The absorbance results of the outlets of each reactor and the wastewater in the 5th day of the experiment after increasing the flowrate. Percent of degradation was 85, 2 % for S4 at 549, 94 nm. There was 75, 4 % degradation for S4 at 625, 47 nm. There was a decrease as 4,0 % for treatment for first 5 day after increasing

the flowrate. This result is normal because after increasing the flowrate, retention time in the reactors has decreased so degradation has decreased.

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Figure 20: The absorbance results of the outlets of each reactors and the wastewater in 3rd week of the experiment after increasing the flowrate. Percent of degradation was 82, 76 % for S4 at 549, 94 nm. And 75, 3 % degradation was obtained for S4 at 625, 47 nm. After increasing the flowrate, % of degradation has decreased from 89, 2 % to 82, 7 % at 549, 94 nm. Although the flowrate was increased approximately twice, % of degradation has decreased only ~ 7 %. This result indicates that this process can be worked for faster flowrates. 7 EXPERİMENT 2 (Part I-Rice husks with wood chips and dye mixture) 7.1 OBJECTIVE The aim of this experiment was the decolorization of the synthetic dye solution which was used as in part 1 of Experiment 1. The system was washed with wood chips solution so more different kinds of microorganisms were added into the system. Increasing decolorization of the dye solution was intended by this way. 7.2 MATERIAL AND METHOD The experiment was carried out in a continuous two stage process. Two reactors (R3&R4) which had a volume of 449,3 ml volume each were operated anaerobically mode. Each reactor was filled with rice husks. Reactor 3: 42, 8 g rice husks Reactor 4: 38, 2 g rice husks The wood chips (50 g) was washed with 300 ml 0,9 % NaCl solution for 30 minutes. I filtered the mixture of wood chips and NaCl solution. I put 100 ml mixture’s water (filtered solution) into reactor 3 and 100 ml into reactor 4. The purpose was to add more and different kind of

bacteria and fungi. Different microorganisms have different abilities concerning degradation of organic compounds therefore a comparison with the results from part I of experiment I which only contained microorganisms from the rice husks is of an interest. After addition of the new microbes synthetic dye mixture which has the formula 200 mg reactive black 5, 200 mg Procion Red MX 5B and 1 g yeast for 1L solution was passed through the system.

Figure 21: Photo from the 16th day of the experiment. A change in colour can be seen as the wastewater trickles through the system. The flow rate was 0,311 ml/min (403U/VM4 Watson Marlow pump). Again the samples were taken every second day and they were scanned with spectrophotometer. 7.3 RESULTS OF THE EXPERIMENT This experiment was performed for 43 days (14 May- 25 June).

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Figure 22: The absorbance results of dye wastewater and the outlets from each reactor in the 5th day of the experiment. Percent of degradation was 34, 2 % for R4 at 549, 94 nm wave length for first 5 days. Percent of degradation was 70, 2 % for R4 at 625, 47 nm. Percent of degradation of system 1 was 14,2 % for first 5 days but percent of degradation was 34,2 % in system 2 for first 5 day, Figure 22. This important difference might be the result of addition of new microorganisms from wood chips.

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Figure 23: The absorbance results of dye wastewater and the outlets from each reactor in the 3rd week of the experiment. There was an important difference between absorbance results of 5th day of the experiment and 3rd weeks of the experiment. Decreases in the absorbance can be seen easily. Percent of degradation was 77, 6 % for R4 at 549, 94 nm wave length. There was good treatment for first 3 weeks, Figure 23. 85, 4 % of degradation was obtained at 549, 94 nm and 72, 9 % of

degradation was obtained at 625,47 nm for R4 in the final day(43th day) of experiment. There was an important increase for percent of degradation. Degradation has increased from 77, 6 % to 85, 4 % between 3rd week of the experiment and final day of the experiment. Result was stabilized in final day (43th day) of experiment. A comparison with the results of experiment 1 part 1 shows that the degradation at 549,94 nm was increased from 78,2 to 85,4 % while the degradation at 625, 47 nm was a bit lower or approximately the same in both experiments. After obtaining stabilized results, flowrate was increased. 8 EXPERIMENT 2 (Part II- Increased the flow rate) 8.1 OBJECTIVE The aim of this experiment was to see if the same result could be achieved for a faster flow rate because of retention time is important for decolourization. 8.2 MATERIAL AND METHOD The process and dye solution were same with part I of experiment 2. The flow rate was increased from 0,311 ml/min to 0,637 ml/min (approximately double) on 43rd day of experiment 2. 8.3 RESULTS OF THE EXPERIMENT This experiment was performed for 20 days (25 June- 15 July).

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Figure 24: The absorbance results of dye wastewater and the outlet of each reactor in the 5th day of the experiment after increasing the flow rate of the system. Percent of degradation was 80, 1 % for R4 at 549, 94 nm wave length day 5, Figure 24. Percent of degradation was 85, 4% for R4 on 43th day of part I of experiment 2. So after increasing the flowrate, degradation has decreased from 85, 4% to 80, 1 %. There was 72, 4 % of degradation for R4 at 625, 47 nm.

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Figure 25: The absorbance results of dye wastewater and the outlets of each reactor in the 3rd week of the experiment after increasing the flow rate. Figure 25 shows decreases in the absorbance of each reactor. There was 82,8 % of degradation for R4 at 549, 94 nm. Percent of degradation was 76, 4 for R4 at 625, 47 nm. Important results were obtained after increasing the flowrate. 85, 4 % of degradation was obtained at 549, 94 nm for R4 and 72,9 % of degradation was obtained at 625,47 nm for R4 in the final day of part I of the experiment 2. And then flowrate was increased approximately twice but after 3 weeks 82, 8% of degradation was obtained at 549, 94 nm for R4. Difference between 85, 4% and 82, 8% was not big in proportion to flowrate change. However, after increasing the flowrate 76, 4 % of degradation was obtained at 625, 47 nm for R4. Although increasing the flowrate, % of degradation has increased at 625, 47 nm! These results showed that similar % of degradation results can be obtained for faster flowrates. 9 EXPERIMENT 2 (Part III -Increased the amount of yeast extract) 9.1 OBJECTIVE Absorbance results of R4 didn’t decrease anymore, because of this reason a yeast extract solution was connected to R4. Yeast is nutrient for microorganisms. The increasing of the degradation was intended by this way. 9.2 MATERIAL AND METHOD A yeast extract solution was connected to R4 in order to decreasing the absorbance data of R4 and increasing the decolorization. Because of yeast is nutrient, obtaining more decolourization was intended by this way. Formula of yeast extract solution was 2g yeast/ L. Yeast extract was diluted with tap water. Due to being yeast extract in dye mixture, the total amount of yeast extract was increased. Flow rate of the yeast extract solution was 0,429 ml/min (403U/L2 pump).

Figure 26: Photo of continuous system 2 after connecting the yeast solution to R4. This photo was taken in second week of system 2 after addition of yeast solution to R4. 9.2 RESULTS OF THE EXPERIMENT This experiment was performed for 22 days (15 July- 6 August).

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Figure 27: The absorbance results of dye wastewater and the outlet of each reactor after the first 5 days of the experiment after connecting the yeast solution to R4.

The dye solution’s flowrate was 0,637 ml/min and yeast extract solution’s flowrate was

0,429ml/min. Total flowrate in R4 was 0,637ml/min + 0,429ml/min = 1,066ml/min. The dyes

were diluted by yeast extract solution in R4. Dilution factor was calculated as shown below:

0.637 / min 100 59.8%1.066 / min

mlml

× = 0.598 was a dilution factor. To calculate the degradation % the

inital absorbance value (0,97) of storage flask was multiplied with 0.598 to compare with

outlet of R4.

0,97 0,598 0,58× = . Absorbance value of R4 was ~ 0,082 at 549,94 nm in the 5th day of the

experiment. From equation 0

0

( (%) 100)tC CX

C−

= × % degradation was calculated as 85,9 %

for R4 at 549,94 nm 5 days after addition of the yeast extract to the system. 0C and tC in

equation are the inital and final absorbance values of dye. Same calculations were done for

absorbance values at 625,47 nm. % of degradation was calculated as 77,9 % for R4 at 625,47

nm.

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Figure 28: The absorbance results of the dye solution and outlet of each reactor in the 3rd week of the experiment after connecting the yeast solution to R4. Percent of degradation was found to be 79,0 % for R4 at 549,94 nm and 71,2 % for R4 at 625,47 nm after 3 weeks, Figure 28. 10 EXPERIMENT 2 (Part IV- Increased the flowrate) 10.1 OBJECTIVE The aim of this experiment was to evaluate the process at an increased flowrate.

10.2 MATERIAL AND METHOD The process and dye solution and yeast solution were same with part III of experiment 2. I increased the flowrate from 0, 637 ml/min to 1, 36 ml/min for R3 and I increased the flowrate from 1,07 (0,637+ 0,429) ml/min to 2,35 (1,36+0,986) ml/min for R4 (approximately twice). 10.3 RESULTS OF THE EXPERIMENT This experiment was performed for 34 days (6 August-8 September). The samples were taken every second day and their absorbance values were measured.

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Figure 29: The absorbance results of the outlet of each reactor and dye solution in the 5th day of this experiment after increasing the flowrate. The results of the absorbance measurements 5 days after increasing of the flow rate is shown in Figure 29.

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Figure 30: The absorbance results of the outlet of each reactor and dye solution in 3rd week of this experiment after increasing the flowrate. To find the dilution effect same calculations were done after incresing the flowrate on continuous system 2. Total flowrate was 1,36 + 0,986= 2,346 ml/min in the second reactor of

the system. Dilution factor was calculated: 1.36 / min 100 57.9%2.346 / min

mlml

× = . The initial

absorbance value of storage flask was multiplied with 0.579 to compare the outlet of R4. After calculations, 62,7 % of degradation was obtained at 549,94 nm and 68,3 % of degradation was obtained at 625,47 nm. The degradation has decreased quite substantially after the flowrate increase especially at 549,94 nm which indicates that the flowrate is a bit too high. 11 BATCH EXPERIMENTS 11.1 OBJECTIVE Batch experiments were performed to see if any substances were released from the rice husks and if the substances in that case were degraded by the microorganisms. 11.2 MATERIAL AND METHOD Batch experiments were used for background. The retention time was higher in the batch system so we could see if the microorganisms were capable of degrading the compounds which might be released. Three batch experiments were prepared in 250 ml Erlenmeyer flasks. Formula of these batch experiments were 8 g rice husks + 250 ml 0, 9 % NaCl solution. Batch experiments were performed under anaerobic conditions.

11.3 RESULTS OF THE EXPERIMENTS Samples (3 ml) were taken from Erlenmeyer flasks every second days. They were scanned with spectrophotometer.

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Figure 31: Absorbance results in 8th day of batch exp 1 and in 30th day of batch exp 2 and batch exp 3. Batch exp 1 was prepared on 09/04/23. Batch exp 2 and 3 were prepared on 09/04/01. Because of the difference of preparing dates of samples, absorbance line of batch exp 1 was different according to other samples. Absorbance lines of batch exp 2 and 3 were closer each other. Batch exp 2 and 3 were used to compare with batch exp 1.

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batch 1batch 2

batch 3

Figure 32: The absorbance results in 15th day of batch exp 1 and in 37th day of batch exp 2 and batch exp 3. Figure 32 shows that the absorbance data of batch 1 has decreased. This shows that microorganisms could degrade the compounds which were released from rise husks.

12 REFERENCES [1] Rita Alex, 2009,Characterization of dye decolourizing microorganisms from textile effluents in dar es sallam. Environmental Biotechnology: 9-17. [2] Andre B. Dos Santos, Francisco J. Cervantes, Jules B. Van Lier; 2006, Review paper on current technologies for decolourisation of textile wastewaters: Perspectives for anaerobic biotechnology. Biosource Technology 98: 2369-2385 [3] Weisburger, J.H ;2002, Comments on the history and importance of aromatic and heterocyclic amines in public health. Mutation Research 506: 9-20 [4] Hao, O.J., Kim,H and Chiang, P.C ;2000, Decolorization of wastewater. Critical Reviews in Environmental Science and Technology 30: 449-505 [5] Talarposhti,A.M., Donnely,T., Anderson,G.K., 2001, Colour removal from a simulated dye wastewater using a two-phase anaerobic packed bed reactor. Water Res. 35, 425-432. [6] Robinson,T., McMullan, G., Marchant,R., Nigam,P., 2001. Remediation of dyes in textile effluent: a critical review on current treatment technologies with a proposed alternative. Biores. Technology 77, 247-255. [7] Pearce,C.I., Lloyd,J.R., Guthrie,J.T, 2003. The removal of colour from textile wastewater using whole bacterial cells: a review. Dyes Pigments 58, 179-196. [8] Chang, J.S and Lin, Y.C 2000. Fed-batch bioreactor strategies for microbial decolorisation of azo dye using a Pseudomonas luteola strain. Biotechnology Progress 16: 979-985. [9] Ozbelge, T.A, Ozbelge, O.H and Baskaya, S.Z; 2002. Removal of phenolic compounds from rubber-textile wastewaters by physical-chemical methods. Chemical Engineering and Procecessing 41: 479-491. [10] Pera-Titus, M., Garcia-Monalina, V., Banos, M.A., Gimenez, J and Esplugas, S, 2004. Degradation of chlorophenols by means of advanced oxidation process: a general review. Applied Catalysis Environmental 47: 219-256. [11] Mishra, G and Tripathy, M ;1993, A critical review of the Treatment for decolorisation of textile effluent. Colourage 40: 35-38. [12] Mattioli, D., Malpei, F., Bortone, G and Rozzi, A; 2002. Water minimization and reuse in textile industry. Analysis, technologies and implementation. IWA Publishing, Cornwall,UK. p. 677. [13] Van der Zee, F.P., 2002, Azo dye decolorization by anaerobic granular sludge. Chemosphere 44: 1169-1176. [14] Knackmuss, H.J; 1996, Basic knowledge and perspectives of biolimination of xenobiotic compounds. Biotechnology 51: 287-295 [15] Epolito, W.J., Lee, Y.H., Bottomley, L.A and Paulostathis, G.G; 2005, Characterization of the textile anthraquinone dyes, Reactive blue 4. Dyes and Pigments 67: 35-46.

[16] Lewis, D and Loan, T ; 2007. Dyeing cotton with reactive dyes under neutral conditions. Colouration Technology 123: 306-311. [17] O’Neill, C., Hawkes, F.R., Hawkes, D.L., Lourenço, N.D., Pinheiro, H.M., Delee, W., 1999. Colour in textile effluents-sources,measurement,discharge consents and simulation: a review.J. Chem. Technol. Biotechnol. 74, 10009-10018. [18] Meyer, U, 1981. Biodegradation of synthetic organic colorants. In: Leisinger T, Hutter R, Cook, A.M, Nuesh, J (eds). Microbial degradation of xenobiotics and recalcitrant compounds. Academic press,London. [19] Wong, P.K and Yuen, P.Y (1998). Decolorisation and Biodegradation of N, N-dimethly-p-phenylenediamine by Klebisiella pneumoniae RS-13 and Acetobactobar liquefaciens. S-I. Applied Microbiology 85: 99-87. [20] Zollinger, H (1987). Color Chemistry- Syntheseses,properties and application of organic dyes pigment. VCH, New York. [21] Wong and Yuen, 1996. Metabolism of azo dyes by lactobacillus casei TISTR 1500 and effect s of various factors on decolorization. [22] Baughman, G.L., Weber, E.J., 1994. Transformation of dyes and related compounds in anoxic sediment: kinetics and products. Environ. Sci. Technol. 28, 267-276 [23] Rosen M., Welander T., Löfqvist A., Holmgren J., 1998 Development of a new process for treatment of pharmaceutical wastewater. Wat. Sci.Tech. 37, 251-258. [24] http://www.brownfieldstsc.org/glossary.cfm?lett=C

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