The biosorption behavior of inactive

Aspergillus niger modified by

autoclaving in treating dye wastewater

LingFeng Undergraduate Student Research Program 2009 Author: Frida Skult, Lund University Date: 2009-07-10 Istructor: Associate Professor Dr. Xiao-jing Xiong, Xiamen University Research assistant: Xue-jiao Meng, Xiamen University

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ABSTRACT The effluents generated by the textile industry are very polluting and it is of great importance to be able to treat these waste waters. Today dye wastewater is usually treated with activated carbon, but the producing costs is very high so there is a need of an alternative method that is more cost efficient. The aim of this experiment was to study the biosorption behavior of inactive Aspergillus niger in treating the dye Direct Blue 199 (FBL). The experiment contained two parts; biosorbent preparation and biosorption experiments. The preparation part contained cultivation of the A. niger in solid and liquid mediums and inactivation by autoclaving. Finally the biosorbent was dried and powdered. Three parameters were studied in the biosorption experiments including the biosorbent amount, pH and the initial dye concentration. The absorbance was measured at absorbance maximum (λmax) for the dye, 608nm, and those values were converted into concentration units by using a standard curve. The optimum experimental conditions were obtained by calculating the decolorization and the biosorption capacity in different intervals for each experiment. This experiment showed that the optimal biosorbent dosage was 8 g/L, at that point the decolorization value was 97.7%. The biosorption capacity decreases with increasing biosorbent amount. The optimal pH was between 4 and 10, then the decolorization is between 93% and 97.8%. Since the natural pH is 5.89 it is not necessary to adjust the pH. Even at high initial dye concentration the decolorization is very high. Aspergillus niger maybe could be a good alternative to activated carbon in treating Direct Blue 199. Key words: Aspergillus niger, dye, biosorption, autoclaving

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TABLE OF CONTENTS

1 BACKGROUND 4

1.1 INTRODUCTION 4 1.1.1 HOW IS THE TEXTILE EFFLUENTS TREATED TODAY? 4 1.2 OTHER STUDIES 4 1.2.1 KUMARI AND ABRAHAM, 2006 4 1.2.1 KHALAF, 2008 5 1.3 EXPECTED RESULTS ON THIS STUDY 5

2 MATERIALS 6

2.1 ASPERGILLUS NIGER 6 2.2 DIRECT BLUE 199 (FBL) 7

3 METHOD 7

3.1 BIOSORBENT PREPARATION 7 3.2 BIOSORPTION EXPERIMENT 9 3.2.1 BIOSORBENT DOSAGE 9 3.2.2 PH 10 3.2.3 INITIAL DYE CONCENTRATION 10 3.2.4 DATA ANALYSIS 10

4 RESULTS & DISCUSSION 10

4.1 BIOSORBENT DOSAGE 12 4.2 PH 13 4.3 DYE CONCENTRATION 14

5 CONCLUSIONS 16

6 REFERENCES 17

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1 BACKGROUND 1.1 Introduction The effluent generated by the textile industry is one of the most polluting among all industrial effluents and it is an important issue to be able to treat these waste waters1. Due to changes in the consumers’ demand the content of the effluents has changed rapidly, today there is a great usage of synthetic reactive dyes such as azo dye2 and phthalocyanine (PC) groups3. The Azo dyes is the largest group used industrially and the PC group dyes consists essentially of blue and green dyes4. The dye effluents generated by for example textile, paper and printing industries affect the visibility and the photosynthesis in the water bodies and it may also be toxic to the aquatic life5. There are also growing concerns in the community regarding their toxicity and carcinogenicity6. 1.1.1 How is the textile effluents treated today? It is quite difficult to treat the effluents by the conventional biological and physico-chemical processes, e.g. light, wash, heat and oxidizing agents7, used in regular treatment plants. That is because of the complexicity of the dyes aromatic molecular structures8. Adsorption is the most effective physical process in the treating these dye waste waters. Today activated carbon is commonly used for adsorption in many treatment plants9. But the producing costs for activated carbon is very high, there is a need of an alternative material that is more cost efficient10. A low costs adsorbent is defined as one which is abundant in nature or one that is produces as a byproduct in another industry11. There have been studies on lots of different natural materials as adsorbents in treating textile effluents, for example saw dust and agricultural wastes like wheat straw and corn cob12. Now biosorption is investigated as a method to absorb the effluents and different organisms treating different kinds of dyes are tested13. 1.2 Other studies 1.2.1 Kumari and Abraham, 200614 Kumari and Abraham (2006) made a study on the biosorption behavior of nonviable biomass of four fungi (R. nigricans, R. arrhizus, A. niger and A. japonica) and one yeast (S. cerevisiae) treating five different reactive dyes. These organisms are all produced as 1 Kumari and Abraham, 2006 2 Kumari and Abraham, 2006 3 Shu and Chang, 2005 4 Shu and Chang, 2005 5 Kumari and Abraham, 2006 6 Fu and Viraraghavan, 2001 7 Kumari and Abraham, 2006 8 Khalaf, 2008 9 Kumari and Abraham, 2006 10 Kumari and Abraham, 2006 11 Khalaf, 2008 12 Kumari and Abraham, 2006 13 Kumari and Abraham, 2006 14 Kumari and Abraham, 2006

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byproducts in fermentation industries and other studies show that they are good biosorbents of heavy metals and radioactive compounds15. The result of Kumari and Abraham’s study shows that most of these organisms also are good biosorbents of reactive dyes. Only A. japonica shows quite low values, around 4-20% adsorption, for the five dyes tested. Aspergillus niger adsorbed between 58-68%. S. cerevisiae and R. nigricans was the best adsorbents (55-91%) and was for some dyes comparable to the values given by adsorption with activated carbon. These two organisms were then studied further. The pH of the dye solutions was around 5.9-6.1, but there was also an experiment done at pH 10. At pH 6 the adsorption was most efficient, at pH 10 S. cerevisiae didn’t adsorb any dye at all while R. nigricans only could adsorb smaller amounts of reactive green and reactive blue 38. 1.2.1 Khalaf, 200816 A study made by Khalaf (2008) focused on textile wastewater treatment by non-viable biomass of Aspergillus niger and the alga Spirogyra. The dye solution contained the commercial Synazol reactive dye, a mixture solution with one red and one yellow dye. The biosorption experiments were performed at different initial pH (1-8), different temperature (15-45 ºC) and different biomass loading (4-12g/L). The A.niger and Spirogyra biomass was inactivated by either gamma radiation or autoclaving. Autoclaving resulted in the highest biosorption values. Another study made by Fu and Viraraghavan (2000 and 2002) even showed that autoclaved biomass of A. niger even had higher biosorption capacity compared to living biomass 17 . The surface characteristics of the biomass are changed in a way that improves the dye biosorption capacity. A possible explanation to that is that the autoclaving disrupts the biomass structure and then exposes the adsorption sites even more18. The result of the study showed that the highest biosorption values were obtained at pH 3 and the lowest values occurred around pH 6-8. The optimal temperature and biomass loading for the biosorption was found to be at 30 ºC respectively at 8 g/L. 1.3 Expected results on this study The study made by Kumari and Abraham (2006) shows R. nigricans to be the best adsorbent of the ones tested in that study. R. nigricans worked optimal at pH 6, R. nigricans is a fungi just like Aspergillus niger and maybe they will work optimally during similar circumstances. However Khalaf’s study showed a quite different result. There the optimal conditions for the biosorption with A. niger were pH 3 and pH 6-8 was not favorable at all. The optimal biosorption temperature was found to be 30 ºC and the optimal biomass loading was 8 g/L for A. niger. The biomass surface of course differs depending on the organisms used, which makes the biosorption capacity different depending on the origin of the biomass. The studies above 15 Kumari and Abraham, 2006 16 Khalaf, 2008 17 Khalaf, 2008 18 Khalaf, 2008

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showed that the biosorption capacity also differs depending on what dye one is dealing with. Maybe the dye also affects at the optimal conditions for the biosorption. 2 MATERIALS

2.1 Aspergillus niger In this project the dye solution will be treated with inactivated Aspergillus niger. A. niger is a fungi which has already been used industrially in producing citric acid. Citric acid used to be produced by extraction from lemons and other citrus fruits, but today microbial fermentation is a widely spread method and almost all citric acid is produced this way 19 . In these fermentation industries A. niger also comes out as a waste product which make it suitable for investigations of the biosorption capacity. A. niger is a dark colored fungi (see Figure 1 and 2) that could be seen at moldering food20 and is then called black mold21. It is mostly fruits and vegetables that are affected by the mold, for example grape fruits, onions and peanuts22. One should not forget when dealing with the fungi that it could cause fungus diseases on both humans and animals 23 . Aspergillus niger is a common saprophytic fungus in terrestrial environments and it has already been used in other studies to adsorb heavy metals and dyes. If the cells of the fungi are active they are easily affected by toxic compounds and chemicals in the waste water and they may then pollute the environment by releasing toxins or propagules. Another problem when dealing with active biomass is that it could not be stored at room temperature for long time periods before it may putrefy. When the biomass is dead and dried it could be stored and transported easier. Earlier studies (see above) also showed that autoclaved biomass actually has higher biosorption capacity than living biomass. These facts are the reasons why inactivated biomass by autoclaving is chosen for this experiment. The A. niger was supplied by the Department of Life Sciences at Xiamen University.

Figure 1: Aspergillus niger growing on Czapek dox agar in a Petri dish. 19 Prescott, Harley and Klein, 2005, p. 976 20 http://www.ne.se/l%C3%A5ng/borstm%C3%B6gel?i_h_word=Aspergillus, 2009-04-29 21 Widodo, Sukandar and Adnyana, 2008 22 Widodo, Sukandar and Adnyana, 2008 23 http://www.ne.se/l%C3%A5ng/borstm%C3%B6gel?i_h_word=Aspergillus, 2009-04-29

Figure 2: Onion with black mold

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2.2 Direct Blue 199 (FBL) The dye that will be investigated in this experiment is C.I. Direct Blue (DB) 199, see molecular structure in Figure 3. DB 199 belongs to the copper phthalocyanine (CPC) dyes24 and is sometimes also called Direct Blue FBL25. DB 199 is a widely used dye in the textile industry when dyeing cotton fabric, but it is also common in the paint and varnishes industry and in printer cartridge26. DB 199’s molecular formula is C32H14CuN8O6S2Na*NH4 and the adsorption maximum (λmax) is 608 nm. DB 199 is obtained from a local textile processing company in China called Ju-tai Textile Mill.

Figure 3: Molecular structure of C.I. Direct Blue 199 3 METHOD

The experiments could be divided into two different parts. The first part is the biosorbent preparation and the second part is the biosorption experiments. 3.1 Biosorbent preparation The solid agar, Potato Dextrose Agar (PDA), is prepared by mixing a prepared powder with de-ionized water. The composition of the agar is 5.0g/L potato, 20.0g/L dextrose, 13.0g/L agar and 0.1g/L chloramphenicol. After preparation the PDA is autoclaved at 121ºC for 30 minutes. The tubes containing the PDA are then laid down at room temperature until the medium is turned into solid mass. When it is solid the tubes are incubated for 1 day. After incubation one could be excluded that other biomass is growing at the agar. The A. niger grown on PDA is stored in a fridge at 4ºC. By inoculating the cultures further onto fresh PDA the biomass is deactivated, see figure 4. The inoculation is done in a fume cupboard that was first prepared by being irradiated with UV light to kill all possible biomass there. After inoculation the tubes are incubated for 3 days. 24 Shu and Chang, 2005 25 http://chinacjchemicals.com/pigments.asp 26 Shu and Chang, 2005

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Figure 4: Aspergillus niger growing on a PDA. After three days of incubation the spores from the A. niger cultures grown on the PDA is inoculated further into a liquid medium, Mineral Salts Medium (MSM). The MSM is prepared by mixing the following ingredients, in the following order, with de-ionized water; 10g/L sucrose, 2g/L KH2SO4, 0.1g/L CaCl2, 0.5g/L MgSO4*7H2O, 0.1g/L NH4CL and 0.002g/L thiamine. The MSM is then poured into Erlenmeyer flasks, 50 ml in each. The flasks was then covered with 8 layers of fabric and one layer of paper and then autoclaved in 121 ºC for 30 minutes. By adding 50 ml de-ionized water into each tube with PDA the biomass could be loosened up into the water by working with an inoculation loop. The biomass mixture is then filtered to get out the spores. The spore mixture is then poured into a prepared Erlenmeyer flask containing de-ionized water and small glass balls. This flask is then incubated during shaking for 30 minutes. The spores are loosened from each other with help from the glass balls and the shaking. This procedure is done to get the spores free and not stuck to each other, then the contact area becomes as large as possible. After this short incubation 2 ml of this mixture is added into each Erlenmeyer flask containing MSM. They were then incubated during shaking for 3 days, see figure 5.

Figure 5: Aspergillus niger growing in MSM. After the incubation the biosorbent is inactivated by being autoclaved at 121ºC for 30 minutes. The mixture is then filtered to get rid of the water solution. After filtration the biosorbent is dried in an oven at 80ºC for 24 hours. When the biomass is dry it is powdered and sieved to a size of 0.15mm, see figure 6 and 7. Now the biosorbent is ready for the biosorption experiment.

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Figure 6 & 7: Powdered biosorbent 3.2 Biosorption experiment Three different biosorption experiments is performed according to three different parameters; biosorbent dosage, pH and the initial dye concentration. Each experiment will be done in different intervals and in duplicate samples. The dye solutions used is prepared by mixing the FBL stock solution with a water solution at a NaCl concentration off 30g/L. The concentration of the FBL stock solution is 1g/L. The biosorption will take place in an incubator at temperature 30+/-1 ºC, a shaking of 150 rpm and during 16 hours. After the biosorption the samples will be filtrated to get the supernatants, see figure 8. The absorbance of that solution is measured by a Vis/UV Spectrophotometer and the absorbance units are then converted into concentration units by using a standard curve.

Figure 8: The supernatant given after filtration after the biosorption experiments. 3.2.1 Biosorbent dosage The biosorption experiments with different biosorbent dosage is performed at natural pH and an initial dye concentration of 50mg/L. The dye solution is prepared by taking 50ml of the stock solution and adding de-ionized water until the volume is 1 liter. Seven different

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biosorbent dosages is used; 0, 0.1, 0.2, 0.3, 0.4, 0.5 and 0.6g. The biosorbent is weighted and put into a small plastic bottle. Then 50 ml of the dye solution is added to every bottle. 3.2.2 pH Now the biosorption experiments will be performed at different pH. As in the experiments above the initial dye concentration will be 50mg/L. The proper biosorbent dosage to use in this experiment is got in the previous experiment. Six different initial pH will be tested; 2, 4, 6, 8, 10 and 12. The pH of the dye solutions is prepared by adding 1M HCl or NaOH solutions. 3.2.3 Initial dye concentration In this last biosorption experiment the initial dye concentration will vary. Five different concentrations will be tested; 25, 50, 100, 200 and 400mg/L. These are prepared by mixing the FBL stock solution with the 30g/L salt solution. The proper biosorbent dosage and pH to use in this experiment is got in the two previous experiments 3.2.4 Data analysis The standard curve is used to convert the absorbance values into concentrations. Then the decolorization and amount of biosorbed dye per unit A. niger, the biosorption capacity (Qe), could be calculated. C0 is the dye concentration in a blank sample with no biosorbent. C0 could be defined as the initial dye concentration but then it is not taken into account that the dye could also be taken up by for example the filter paper during the filtration or lost during some other process. When C0 is defined as the blank concentration we are only taking the biosorption into account when calculating the decolorization and biosorption capacity. Ce is the concentration at equilibrium after the biosorption.

100*(%)0

0

CCC

tiondecoloriza e−=

mVCC

Q ee

)( 0 −=

4 RESULTS & DISCUSSION The standard curve is done by measuring the absorption at different known concentrations, see figure 9 and table 1. The standard concentrations are made by mixing 30g/L NaCl-solution with the FBL stock solution.

Figure 9: The dye solutions with known concentrations that is used to create the Standard curve.

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Table 1: The absorbance at different known concentrations of DB 199 dye solutions. dye concentration (mg/L) 0 2.5 5 10 20 50 70

absorbance 0.003 0.045 0.102 0.193 0.368 0.938 1.385

y = 0.0195x - 0.005

R2 = 0.9987

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 10 20 30 40 50 60 70 80

dye concentration (mg/L)

absorbance

c

Figure 10: The Standard curve of DB 199 dye solutions.

0050.00195.0 −= xy y = absorbance x = dye concentration

9987.02 =R ≈ 1 which makes the relationship above reliable. The dye concentrations are then calculated by the following relationship:

0195.0005.0+

=yx

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4.1 Biosorbent dosage The results of the absorbance and the calculated dye concentrations, by using the standard curve, and the decolorization and biosorption capacity values are given by table 2 and 3. Table 2: The absorbance of sample 1 and 2 and the calculated values of the dye concentration, decolorization and biosorbent capacity at different biosorbent amounts.

amount biosorbent

(g/L)

absorbance sample 1

absorbance sample 2

mean value

dye concentration

(mg/L), Ce

Decolorization (%)

Qe (mg/g)

12 0.002 0.008 0.005 0.512821 98.41396 2.651709 10 0.013 0.007 0.01 0.769231 97.62094 3.15641 8 0.008 0.011 0.0095 0.74359 97.70024 3.948718 6 0.025 0.018 0.0215 1.358974 95.79699 5.162393 4 0.06 0.06 3.333333 89.69072 7.25 2 0.183 0.212 0.1975 10.38462 67.88263 10.97436

Table 3: The absorbance and the calculated dye concentration of the blank sample.

amount biosorbent

(g)

absorbance sample 1

absorbance sample 2

mean value

dye concentration

(mg/L), C0 0 0.619 0.632 0.6255 32.33333

The dye concentration of the blank sample is 32 mg/L. The start concentration in each bottle was 50 mg/L. How come the concentration has decreased in the blank sample? Maybe some of the dye gets stuck on the inner walls of the plastic bottles used for the biosorption processes and maybe some of the dye gets stuck on the filter paper during the filtration after the 16 hour incubation. Since we define C0 as the dye concentration in the blank sample and not the initial dye concentration it is only the loss of dye during biosorption that is taken into account when calculating the decolorization and biosorption capacity.

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14

biosorbent amount (g/L)

decolorization (%)

0

0.1

0.2

0.3

0.4

0.5

0.6

Qe(mg/g)

decolorization

Qe

Figure 11: Decolorization and biosorbent capacity (Qe) at different biosorbent amounts. The decolorization increases with increasing biosorbent amount until a certain limit when it becomes almost constant, see figure 11. This happens at biosorbent amount 8 g/L, from this

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point the decolorization lies around 98 %. Due to this almost constant value of the decolorization and due to economical reasons the proper biosorbent amount is set to 8 g/L. The fact that the decolorization moves to a constant value is confirmed by other studies, for example the one made by Lataye, Mishra and Mall in 200727. An increased biosorbent amount increases the amount of adsorption sites which increases the removal amount. Eventually the surface concentration and the bulk concentration of the dye will reach equilibrium, then the decolorization reaches its constant value. The biosorption capacity decreases with increased biosorbent amount. That could also be explained by the larger availability of adsorption sites. Due to that increased amount there is also an increased amount of unsaturated adsorption sites. That is explained by Aydin and Baysal in 200528. 4.2 pH Since the optimal biosorbent amount was found to be 8 g/L this experiment is performed with a biosorbent loading of that concentration. The natural pH of the dye solution (50mg/L) is measured to be 5.89. The results of the absorbance and the calculated dye concentrations, by using the standard curve, and the decolorization and biosorption capacity values are given by table 4 and 5. Table 4: The absorbance of sample 1 and 2 and the calculated values of the dye concentration at different pH.

pH absorbance sample 1

absorbance sample 2

mean value

dye concentration (mg/L), Ce

absorption blank

sample

dye concentration (mg/L) blank

sample, C0 2.03 0.009 0 0.0045 0.487179 0.011 0.820513 4.03 0.012 0.001 0.0065 0.589744 0.159 8.410256 5.97 0.008 0.003 0.0055 0.538462 0.466 24.15385 8.02 0.012 0.002 0.007 0.615385 0.502 26 10.04 0.016 0.016 1.076923 0.504 26.10256

12 0.762 0.752 0.757 39.07692 1.027 52.92308 Table 5: The calculated decolorization and biosorption capacity at different pH.

pH Decolorization (%) Qe (mg/g) 2.03 40.625 0.041667 4.03 92.9878 0.977564 5.97 97.7707 2.951923 8.02 97.63314 3.173077 10.04 95.87426 3.128205

12 26.16279 1.730769 As can be seen on the concentrations in the blank samples it is a huge loss of dye at lower pH. In some way the uptake by the wall or the filter paper or whatever is causing the sink is favored by a low pH. At pH 12 the concentration actually gets higher. But that is not a logical 27 Lataye, Mishra and Mall, 2007 28 Aydın, Baysal; 2005

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result but must be a result of some errors during the process, for example during the absorbance measurements.

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14pH

decolorization (%)

0

0.5

1

1.5

2

2.5

3

3.5

Qe (mg/g)

decolorization

Qe

Figure 12: Decolorization and biosorbent capacity (Qe) at different pH values. The decolorization is highest between pH 4 and 10 and the biosorption capacity is highest between pH 6 and 10, see figure 12. But when deciding what pH is most favorable for the biosorption experiment the decolorization is the most important factor. Since that is higher than 90% between pH 4 and 10 and the natural pH of the dye solution is 5.89 it is not necessary to adjust the pH before the biosorption experiment. Some other studies, for example Khalaf in 200829, mention that there is an electrostatic attraction between the dye and the biosorbent during the biosorption process. That could explain this response at high and low pH. If there for example is a negative charge on the dye and a positive charge on the biosorbent they would attract each other. But at low pH, when there is a large amount of H+, that attraction could be disrupted because of an attraction between the H+ and the dye. A similar but opposite reaction would happen at high pH when there is a large amount of OH- in the solution. 4.3 Dye concentration This experiment was performed with a biomass loading of 8 g/L and the natural pH of 5.89. The results of the absorbance and the calculated dye concentrations, by using the standard curve, and the decolorization and biosorption capacity values are given by table 6 and 7. 29 Khalaf, 2008

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Table 6: The absorbance of sample 1 and 2 and the calculated values of the dye concentration at different initial dye concentrations.

initial dye concentration

(mg/L)

absorbance sample 1

absorbance sample 2

mean value

dye concentration

(mg/L), Ce

absorption blank

sample

dye concentration (mg/L) blank

sample, C0 25 0.009 0.012 0.0105 0.794872 0.218 11.4359 50 0.009 0.011 0.01 0.769231 0.517 26.76923 100 0.013 0.016 0.0145 1 1.405 72.30769 200 0.023 0.019 0.021 1.333333 3.245 166.6667 400 0.708 0.681 0.6945 35.87179 6.8 348.9744

Table 7: The calculated decolorization and biosorption capacity at different initial dye concentrations. dye concentration (mg/L) Decolorization (%) Qe (mg/g) 25 93.04933 1.330128 50 97.12644 3.25 100 98.61702 8.913462 200 99.2 20.66667 400 89.72079 39.13782

010

2030

4050

6070

8090

100

0 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425

initial dye concentration (mg/L)

decolorization (%)

0

5

10

15

20

25

30

35

40

45

Qe (mg/g)

decolorization

Qe

Figure 13: Decolorization and biosorbent capacity (Qe) at different initial dye concentrations. As can be seen in figure 13 there is a high decolorization value even at the high initial dye concentrations. A study made by Lataye, Mishra and Mall in 2007 30 says that the decolorization probably would be at a constant value at the lowest concentration but that it then would decrease with higher concentrations. In this case the decolorization actually increases at the lower concentrations and is decreasing at only the highest concentration. Maybe this could be due to the NaCl solution that the dye was mixed with. In this other study they used double-distilled water when preparing the adsorbate solution. There is no other study found with the same result as this study. The biosorption capacity is increasing with increasing initial dye concentration. That is also the result in the study made by Lataye, Mishra and Mall31. This is explained as a decreasing resistance to mass transfer from the bulk to the biosorbent when the dye concentration is increasing. That increases the amount of dye that could be adsorbed per unit adsorbent. 30 Lataye, Mishra and Mall, 2007 31 Lataye, Mishra and Mall, 2007

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5 CONCLUSIONS This experiment showed that at an initial dye concentration of 50 mg/L the proper biosorbent amount to use is 8 g/L and the proper pH to use is the natural pH of the dye solution, in this case 5.89. During the experiments with increasing initial dye concentration one could see that the biosorption process work very well even at high concentrations. The decolorization is high, above or around 90 % until a dye concentration of 400 mg/L. In this report there is no experiments done on activated carbon that is the commonly used method today to deal with these dye waste waters. Since there are no such results found for Direct Blue 199 it is not possible to evaluate if Aspergillus niger could be used as an alternative method to activated carbon in treatment plants. Since A. niger would be a cheaper alternative than activated carbon when it is produced as a byproduct in fermentation industries it would be a better alternative than activated carbon as soon as it is proved that the decolorization level is at least as good as with activated carbon.

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6 REFERENCES Lansing M. Prescott, John P. Harley, Donald A. Klein; 2005, Microbiology (Sixth Edition). ISBN: 0-07-255678-1 Mahmoud A. Khalaf; 2008, Biosorption of reactive dye from textile wastewater by non-viable biomass of Aspergillus niger and Spirogyra sp., Science Direct, Bioresource Technology 99 (2008) 6631-6634. Kumad Kumari, Emilia Abraham; 2006, Biosorption of anionic textile dyes by nonviable biomass of fungi and yeast, Science Direct, Bioresource Technology 98 (2007) 1704-1710 Hung-Yee Shu, Ming-Chin Chang; 2005, Decolorization and mineralization of a phthalocyanine dye C.I. Direct Blue 199 using UV/H2O2 process, Journal of Hazardous Materials B125 (2005) 96–101 Gunawan Pamudji Widodo, Elin Yulinah Sukandar, Sukrasno and I. Ketut Adnyana; 2008, A Coumarin from Ageratum Leaves (Ageratum conyzoides L.), ISSN 1811-7775, International Journal of Pharmacology 4 (1): 56-59, 2008 Homepage of Nationalencyklopedin, http://www.ne.se/l%C3%A5ng/borstm%C3%B6gel?i_h_word=Aspergillus Dilip Hiradram Lataye, Indra Mani Mishra, Indra Deo Mall; 2007, Adsorption of 2-picoline onto bagasse fly ash from aqueous solution, Chemical Engineering Journal 138 (2008) 35-46 Haluk Aydın, Gülay Baysal; 2005,Adsorption of acid dyes in aqueous solutions by shells of bittim (Pistacia khinjuk Stocks), Desalination 196 (2006) 248–259 Figure 1: http://www.mycology.adelaide.edu.au/gallery/photos/aspergillus12.html Figure 2: http://ipmnet.org/plant-disease/disease.cfm?RecordID=749 Figure 3: Hung-Yee Shu; 2005, Degradation of dyehouse effluent containing C.I. Direct Blue 199 by processes of ozonation, UV/H2O2 and in sequence of ozonation with UV/H2O2, Journal of Hazardous Materials B133 (2006) 92–98