Decolourization of azo and anthraquinone dyes by mean of ...218482/FULLTEXT01.pdf ·...

Decolourization of azo and anthraquinone

dyes by mean of microorganisms growing

on wood chips

Växjö May 2009

Thesis no: TD 003/2009

Sara Palacios

Department of Bioenergy

School of Technology and Design,TD

Sara Palacios Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips

1 INTRODUCTION................................................................................................. 5

1.1 REACTIVE DYES.......................................................................................... 5

1.1.1 AZO DYES.............................................................................................. 6

1.1.2 ANTHRAQUINONE DYES...................................................................... 7

2 EXPERIMENT 1 (50 mg each dye / L) ................................................................ 8

2.1 OBJECTIVE .................................................................................................. 8

2.2 THEORY ....................................................................................................... 8

2.2.1 MICROORGANISMS .............................................................................. 9

2.3 MATERIAL AND METHOD ........................................................................... 9

2.4 RESULTS OF THE EXPERIMENT...............................................................16

2.5 CONCLUSION .............................................................................................21

3 EXPERIMENT 1 (PART II, Reactive Black 5 and Procion Red MX 5B, 200 mg

each dye / L)..............................................................................................................21

3.1 OBJECTIVE .................................................................................................21

3.2 MATERIAL AND METHOD ..........................................................................22

3.3 RESULTS OF THE EXPERIMENT...............................................................22

3.4 CONCLUSION .............................................................................................25

4 EXPERIMENT 2 & 3 (Reactive Blue 4 and Cibacron Orange P-2R GR)............26

4.1 OBJECTIVE .................................................................................................26

4.2 THEORY ......................................................................................................28

4.3 MATERIAL AND METHOD ..........................................................................29

4.4 REACTIVE BLUE 4 SOLUTION...................................................................30

4.4.1 RESULTS ..............................................................................................30

4.4.2 CONCLUSION.......................................................................................33

4.5 CIBACRON ORANGE P-2R GR SOLUTION ...............................................34

4.5.1 RESULTS ..............................................................................................34

4.5.2 CONCLUSION.......................................................................................39

4.6 MIXTURE BETWEEN REACTIVE BLUE 4 AND CIBACRON ORANGE P-2R

GR SOLUTION ......................................................................................................39

4.6.1 RESULTS ..............................................................................................39

4.6.2 CONCLUSION.......................................................................................41

5 REFERENCES...................................................................................................44

6 APPENDIX .........................................................................................................45


6.1 Directive 2002/61/EC of the European Parlament and of the Council of 19

July 2002................................................................................................................45


ABSTRACT Reactive Black 5 and Procion Red MX 5B, an azo and anthraquinone dye repectively

were decoulorized by mean of microorganisms growing on wood chips. The process

consisted of three reactors, two anaerobic reactors and one aerobic reactor. The

anaerobic process was used in order to make it possible to break the nitrogen bond

of the azo group, (-N=N-) and the aerobic one to increase the possibility for the

degradation of possible intermediates. After pumping wastewater through the system

it was shown that mixtures or Reactive Black 5 and Procion Red MX 5B were

efficiently decolourised at 50 mg/l as well as 200 mg/l of each of the dyes.


SUMMARY

The wastewater from textile industries often contains azo and anthraquinone dyes

which can come from benzoic groups which might be carcinogenic and can cause

problems for the photosynthesizing aquatic plants due to absorption of light.

The item of this report is textile wastewater decolourization by mean of

microorganisms growing on wood chips. The wood chips acts as a support for the

microbial growth and it provides the microorganisms with carbon and nutrient

sources.

To imitate real wastewater two different dyes were used, one of them from the azo

group (Procion Red MX 5B) and the other one of the anthraquinone group (Reactive

black 5). The same amounts of both dyes were dissolved in tap water after which

yeast extract was added.

The system consisted of three reactors which contained wood chips. They were

connected in series and the synthetic wastewater prepared was pumped through the

system by mean of a pump.

The microorganisms living on the wood chips were capable of decolourization of the

wastewater. In order to get a more efficient decolourization, three different stages

were used, the first two were anaerobic and the last one aerobic. The anaerobic

stage favor more the break down of the bond of the azo group (-N = N), which is the

most difficult part of the process, and the aerobic process finnish to degrade the dye

solution.

After more than one month pumping approximately 3,5 L of the wastewater prepared

through the three stages process, it was demonstrated that the dye solution was

decolourized by the microorganisms growing on the wood chips.


After the successful results obtained in this experiment, another one with higher

concentration, similar to the one of the waste water from the textile industry was done

with the same procedure obtaining as well good results.

A batch experiment was also performed during the study in order to evaluate the

decolourization of Reactive Blue 4 and Cibacron Orange P-2R GR separately as well

as in mixture. The results show that especially Cibacron P-2R GR is a relatively

refractory dye.

1 INTRODUCTION Textile industries use many chemical compounds, including dyes. Considerable

amounts are discarded in the wastewater. Wastewater from textile industries is highly

polluted because of most of the dyes that are used contain benzoic groups, which

could give rise to carcinogenic degradation products due to e.g. microbial processes,

but this is not all, the dye pollution generate many problems in the photosynthetic

aquatic plants and algae because the dye absorbs most of the light the organisms

need to survive [1,2].

Most of the textile industries in the industrial countries have moved to developing countries in order to save money in the production and earn more money. Many of

these developing countries lack laws in order to protect environment and health.

So the aim of this study was to try to develop an efficient, simple and relatively cheap

microbiological treatment method for textile wastewater. Wood chips were used as a

source of microorganisms. The wood chips furthermore contain carbon source and

nutrients needed by the microorganisms.

1.1 REACTIVE DYES

Reactive dyes is a class of highly coloured organic substances, primarily utilised for

tinting textiles, these kind of dyes bind to their substrates by a chemical reaction that

forms a covalent bond between the molecule of dye and that of the fibre.


These dyes can be used to dye wool, nylon and cotton, in the latter case they are

applied under weakly acidic conditions [3].

Reactive dyes are not readily removed by typical wastewater treatment processes

due to their inherent properties, such as stability and water solubility [4].

1.1.1 AZO DYES

Azo dyes, Figure 1, is the largest class of dyes used in the textile industry.

Figure 1. General structure of an azo dye

Azo dyes are often used in the colouring process of several textiles and leather

products. Some azo dyes contain chemical groups that bind metal ions. Often, the

metal ion also unites with the fibre, improving the resistance of the dye to washing

and also this bond between the dye and the ion can produce important changes in

shade [5].

Relatively recently it has been recognised that some azo colouring agents may form

amines (R – NH2), during degradation Figure 2, due that this kind of dye contains

nitrogen in the form of the azo group −N=N− [6, 7].

Figure 2. Amine general structure


These amine compounds might be carcinogenic and mutagenic [8]

There are high levels of azo dyes in the environment due to is quite difficult to

breakdown this azo bonds (R – N = N – R). They are very stable in acidic and

alkaline conditions and resistant to high temperatures and light. In spite of this, they

might be degraded with bacteria under anaerobic and aerobic conditions [9].

Various bacteria strains reduce azo dyes under anaerobic conditions. The most

generally accepted hypothesis for this phenomenon is that many bacterial strains

possess rather unspecific cytoplasmic enzymes, which act as “azo reductases” and

under anaerobic conditions transfer electrons via soluble flavins to the azo dyes [10].

The European Union published a Directive (2002/61/EC) to restrict the marketing

anda use of certain dangerous substances and preparations (azocolourants) in textile

and leather products. The legislation is relevant for all these products which come

into direct and prolonged contact with the skin and mouth. These include producers

of textiles and garments, leather goods, shoes, toys, furniture, decorative articles,

jewellery and accessories [11].

1.1.2 ANTHRAQUINONE DYES

Anthraquinone dyes, Figure 3, constitute the second largest group of textile dyes,

after azo dyes and are used extensively in the textile industry due to their variety of

colour shades and easy of application [12].

Figure 3. General structure of an anthraquinone dye


Anthraquinone acid dyes contain sulfonic acid groups that render them soluble in

water [13].

Among the textile dyes, anthraquinone dyes is an important class used not only to

colour cellulosic fabric (mainly cotton), but also wool and polyamide fibres.

2 EXPERIMENT 1 (50 mg each dye / L)

2.1 OBJECTIVE

The aim of this study was to develop an efficient and relatively cheap treatment

method based on microbial processes to clean the wastewater polluted with dyes.

The method should not be too technically advanced.

The experiment was performed to evaluate the decolourization of a dye solution

prepared to simulate wastewater from the textile industry. The experiment was

performed in continuous mode using wood chips as a source of microorganisms and

carbon.

2.2 THEORY

The dye solution used in this experiment was composed of two azo dyes, Reactive

Black 5 (Figure 4) and Procion Red MX 5B (Figure 5) in equal concentrations.


Figure 4. Reactive Black 5

Figure 5. Procion Red MX 5B

In order to get degradation three different stages were used in the same continuous

process, first two anaerobic stages and after an aerobic one.

2.2.1 MICROORGANISMS

As source of microorganisms we use wood chips because they contain

lignocellulosic material which can be used as carbon source. Wood chips often

contain bacteria as well as fungi which might be an advantage when molecules with

complex structures should be degraded. The fungi might degrade structures which

are difficult for bacteria to handle while the bacteria might degrade intermediates

formed by the fungi.

There are also other similar materials which could be tested from the countries when

the textile industries are common, as can be the cotton waste or rice husks [14].

These materials are waste products, which mean that they are available at large

volumes at very low costs.

2.3 MATERIAL AND METHOD

The experiment was carried out in a continuous three stage process, Figure 6. The

first two reactors were operated anaerobically while the third reactor was operated in


an aerobic mode. Both anaerobic reactors (reactor 1 and 2) had a volume of 1L and

the aerobic reactor (reactor 3) had a volume of 0,5L, so in total there were 2,5L.

Figure 6. Photo of the experiment assembly which gives the details of the three different

reactors

Each reactor was filled with wood chips:

− Reactor 1: 96,545 g of wet wood chips (80% of water)

− Reactor 2: 103,927 g of wet wood chips (77% of water)

− Reactor 3: 28, 280 g of wet wood chips (80% of water)

All the reactors were filled with old wood chips used in a previous experiment, which

lasted some more than four and a half months (146 days).

It is important to have an anaerobic process as a first stage in order to break the

bond - N = N – which is in the azo dyes, and changing the molecular structure of the

dyes in other different molecular structures which might be possible for the aerobic

microorganisms to degrade [15].


The third reactor worked under aerobic conditions, so it was necessary to aerate it by

compressed air in order to have an environment rich in oxygen. In this case, the

reactor was smaller than the other two; in this case the volume was of 0,5L.

Before starting this new experiment, it was necessary to make pass distilled water

through the system in order to clean the possible wastes from a previous experiment.

It is necessary make pass three times more distilled water through the system than

the volume of the assembly; it has a volume of 2,5 L, so the necessary distilled water

through the system is 7,5 L.

After that, in order to put more microorganisms in the system, water rich in

microorganisms was also put in each reactor, this water was prepared taking 100g of

wood residues chips, 200 ml of 0,9 % saline (NaCl) solution, shaking it for about 1/2

hour with mechanical shaking and filtering through filter paper 3 or ordinary paper.

The dye solution was prepared with 50 mg of each dye (Reactive Black 5 and

Procion Red MX 5B) in 1 litre of water from the tap, so there was a concentration of

50 mg of each dye/L.

Yeast extract was also added to the solution from day 10, in a concentration of 1 g/L.

This yeast extract is an excellent substrate for so many microorganisms [16].

After prepare this solution, it was necessary to put it in the autoclave in order to

sterilize the dye solution prepared to be sure that there were not any microorganisms

in it, Figure 7.

To get the sterilization, the autoclave works with high temperature (121 ºC) and high

pressure during 15 min.


Figure 7. Photo of the autoclave used in the experiment

After the pass of the dye solution for the autoclave, it was ready to pass through the

system, for it, the solution was impelled by a mechanical pumping as the diagram

shows, Figure 8.

Figure 8. Diagram of the experiment

The speed of the pump was as slow as the pump permitted in order to achieve the

dye degradation. If the speed of pumping is too high, the hydraulic retention time will

be to low and the microorganisms cannot degrade the dyes. The flow rate was 104

mL/day (or of 4,36 mL/h). The retention time in the reactor 1 and 2 is around 9 days


and 14 hours (9,6 days) in each reactor and 4 days and 19 hours (4,8 days) in the

reactor 3.

Something is happening in the three different stages due to the work of the bacteria

and fungi, so in order to see what kind and what amount of bacteria there are in each

reactor, I studied them in the microscope using a method called Gram Staining. [17]

Gram Staining is an empirical method of differentiating bacterial species into two

large groups (Gram-positive and Gram-negative) based on the chemical and physical

properties of their cell walls.

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-

90% of cell wall), which stain purple and Gram-negative bacteria have a thinner layer

(10% of cell wall), which stain pink.

Gram-negative bacteria also have an additional outer membrane which contains

lipids, and is separated from the cell wall by the periplasmic space [18].

After the application of this method, the results obtained about the bacteria present in

each reactor were the next:

Figure 9. Microorganisms present in the water of the reactor 1 at the end of the experiment


Figure 10. Microorganisms present in the wood of the reactor1 at the end of the experiment

In order to get the Figure 9, the sample of the liquid from the reactor 1 was taken

directly by mean a pipette and this solution was put on the glass to see the

microorganisms with the microscope; and the Figure 10 was achieved taking some

wood chips and shaking them in distilled water, and after this water was put on the

glass.

After to do all this, it is necessary to dye the cell walls in order to know what type or

types of microorganisms there are in the solution. So, after make the Gram Staining

procedure, I can say the type of microorganisms there are is gram-negative because

of the colorization in purple color. [19]

As the Figures 9 and 10 show there are a quite high amount of bacteria working in

the experiment, and also there are different kind of bacteria, coccus, diplococcic,

bacillus, corkscrew’s form, streptococci, etc.

It is not enough to know what kind of bacteria there are or even the amount of them,

it is also important to know how the molecular structure of the dye solution changes

with the pass through the three different reactors, for it is necessary to take measure

of the absorbance.

To measure the absorbance, the first thing to do is to take the samples of each

reactor and also of the original dye solution. The samples were taken every second

day, and to be sure that they were taken always from the same place of each reactor,

and in order to get more comparable measurements, the samples were taken directly

from the outlet tube of each reactor.


After this, and after a optimum dilution in distilled water of the samples (2mL of

sample and 4mL of distilled water, 1:2) they were scanned with a spectrophotometer,

Perking Elmer lambda 35 UV/VIS and analysed by Perking Elmer UV winlab ver:

2.85.04 software. The absorbance was measured at wavelengths between 190 and

750 nm.

The diluted samples were introduced in the spectrophotometer with quartz cuvettes

of 1 cm of edge perfectly clean and dry.

2.4 RESULTS OF THE EXPERIMENT

This experiment lasted 45 days and 3640 mL of dye solution was pumped through

the system. During all this time the absorbance should decrease until some time

when the absorbance cannot decrease any more, then it is when the experiment is

finish.

First of all, is important to know how the absorbance of the dye solution is, in order to

compare how it is changing during treatment in the process.

Figure 11. Original dye solution and every reactor in the 3rd day


As the Figure 11 shows the original dye solution had an absorbance of 0,75 at 550

nm of wavelength, which is the maximum peak. This absorbance data is the

absorbance in the diluted sample, but the real one is 2,25.

After three days there were very satisfactory results, because the dye solution

suffered a decolourization of 50% at 550 nm of wavelength just in the reactor 1 and

a degradation of the 93% after passage of the whole process, Figure 11.

Until this moment the flow rate was more or less quite regular, around 4,2 mL/h, so

this makes that the graphics are quite representative because the amount of dye

introduced every day is more or less the same.

The yeast extract was added day 10, so in this day of the experiment (day 3) the

extract was not added yet. It is important to show how the absorbance changes

depending on e.g. addition of yeast extract.

Figure 12. First ten days (before the addition of the yeast extract) in the reactor 1

As Figure 12 shows the absorbance in the reactor 1 has been decreasing each time,

from 0,375 to 0,1 in a wavelength of 550 nm; these values are in the diluted solution,

so the real absorbance goes from 1,125 to 0,3.



The absorbance in the reactor 2, as the Figure 13 shows, is quite similar every day

around the 550 nm, but before 300 nm, the absorbance is each time higher.

Also it is important to see how the evolution of the absorbance in the reactor 3.


In this case, Figure 14, it occurs more or less the same as in the reactor 2, the

absorbance below 300 nm is each time higher.The increase in absorbance at the

lower wavelengths might be due to formation of smaller intermediate compounds.


Figure 15. Reactor 1 after the addition of the yeast extract until the day 25 of the experiment

The Figure 15 and 16 show the changes of the absorbance of the solution in reactor

1 after the addition of the yeast extract.

Figure 16. Reactor 1 until the last day of the experiment

On this time in the experiment, every day there were the same results and the flow

rate was perfectly normal, so the graphics are representative.


Table 1 shows the percent of the degradation in the dye solution during the whole

experiment:

Tabla 1. Table with the percent of the degradation of the dye solution during the whole

experiment in the reactor 1.

REACTOR 1 (550 nm)

Laboratory

day AbsorbancePercent of degradation

1 2,25 0,00 3 1,13 50,00 5 0,75 66,67 8 0,38 83,33

Before the addition of the yeast extract

10 0,30 86,67 12 0,21 90,67 19 0,13 94,44 25 0,21 90,67 33 0,15 93,33 38 0,15 93,33

After the addition of the yeast extract

45 0,15 93,33

Continuing with the other reactors, I found that the results of the reactor 2 after the

addition of the yeast extract are the next:



The Figure 17 shows that at 550 nm of wavelength the absorbance is the same

during day 12 to day 25, but the absorbance before 300 nm each time is higher, as in

the reactor 1.


The absorbance is the same each day, and at the entire wavelength range, even

before 300 nm during the later part of the experiment, Figure 18.

About the reactor 3, the evolution of the degradation of the dye is the next, Figures

19 and 20:




The absorbance of the solution in the reactor 3 is the same in the last 12 days at the

300 nm and also at 550 nm, Figure 20.

It can be seen that the major part of the decolourization takes place in reactor 1.

2.5 CONCLUSION

The start-up of the experiment is a period when the microorganisms starts to become

adapted to the environment and start to produce necessary enzymes for degradation.

To degrade a concentration of 50 mg of each dye per litre a flow of 104 mL/day has

been used, so in the reactors 1 and 2 there is a retention time of 9, 61 days

(1L/(104mL/day)) and a retention time in the reactor 3 of 4,80 days

(0,5/(104mL/day)). These times are enough to decolourize the dyes present in the

waste water. Also is important to say that the most important reactor in the

experiment is the reactor 1, because is which makes most of the degradation work.

So to sum up, this experiment demonstrates, over the point of view of absorbance,

that with an easy (three reactors connected one after the other and a pump) and

cheap (using wood chips as a biological support) assembly, the wastewater used can

be decolourized and it would be important to make more analyses for instance by

high performance liquid chromatography (HPLC) in order to evaluate if any


intermediates are formed or not. It would also interesting evaluate the process in a

pilot scale if there are resources to be found.

3 EXPERIMENT 1 (PART II, Reactive Black 5 and Procion Red MX 5B, 200 mg each dye / L)

After the satisfactory results obtained in the part I of the experiment, I decided to

continue the experiment with increased dye concentration.

3.1 OBJECTIVE

This time the objective is also the decolourization of the dye solution but in this case

the dye solution has a concentration more similar as there is in the real textile

wastewater. It is prepared using again the same reactive dyes as in the previous

experiment, Reactive Black 5 and Procion Red MX 5B, but with a higher

concentration.


The assembly used in this case is exactly the same as the used in the part I of the

experiment, including the same wood chips, so the total volume continues being 2,5L

and the amount of wood chips is the same as the indicated in the paragraph 2.3, the

only thing that changes is the concentration of dye, which is higher, 200 mg of each

dye in 1L of water from the tap, also in this case the yeast extract is included since

the beginning, so there is 1 g of this compound as well, and of course, it is necessary

to put the solution obtained in the autoclave like always in order to sterilize the

solution.

To introduce the new dye solution in the assembly used in the previous experiment, it

is necessary to make it pass through the system during at least one week, to be sure

that in the moment to take samples that the only thing there is the new dye solution

and not wastes from the old one.


3.3 RESULTS OF THE EXPERIMENT

During the whole experiment through the system were pumped 3290 mL during 44

days.

In the first 8 days the absorbance decrease really fast as the Figure 21:

Figure 21. 8th day of the experiment

Figure 22. 8th day of the experiment in big scale

Using as a optimum dilution in distilled water of the samples 1mL of sample and 5mL

of distilled water, 1:5, in the first week the degradation was quite high, the

absorbance decreased from 1,2, in the original dye solution in the diluted solution, so


in the real one the absorbance is of 7,2, until 0,72 in the reactor 1, an absorbance of

0,22 in the reactor 2 and 0,15 in the reactor 3 (all in the real absorbance), Figure 22

and 23.

In the day 18 the absorbance has changed in different way in each reactor.


As Figure 23 shows, in the reactor 1, the absorbance was stable, so this mean than

the absorbance at 550 nm of wavelength is 0,72 for the real solution, in the reactor 2

it has totally decreased until an absorbance of approximately 0 and in the other hand,

in the reactor 3 the absorbance has increase until 0,36 (all data are given in real

absorbances).

In the last days it was almost of 0,00 in each reactor at 550 nm. In spite of the low

absorbance in the reactor 3, its content looked in quite dark colour, which made to

think that polymerisation occurred in that reactor, so in order to solve this, on the 23th

day of the experiment, the water phase was exchanged for 300 mL of activated

sludge from a municipal wastewater treatment plant in order to add some new

species of bacteria.

After the addition of this water the results obtained were the next:


Figure 24. 36th day of the experiment (13 days after the addition of activated sludge)

In this case, Figure 24, the absorbance at 550 nm of wavelenght in the reactor 1 and

3 are still stable, so their real absorbances are 0,72 and 0,36 respectively; in the

other way the absorbance in the reactor 2 has increased until a real absorbance of

0,36 from an absorbance very close to 0,0 in the previous figure, day 18th.

In the last day of the experiment, in the 44 day, the results are higher than during the

week before, at 550 nm of wavelength the absorbance in the reactor 1 around 1,20,

in the reactor 2 is the 0,9 and in the reactor 3 1,5, Figure 25.



Since the addition of the activated sludge 1700 mL of solution has been run through

the reactor 3.

3.4 CONCLUSION

The conclusion of this experiment is exactly the same as in the previous case, both

dyes, Reactive Black 5 and Procion Red MX 5B are decolourized in the process even

with a higher concentration of each dye, 200 mg/L of each instead 50 mg/L of each

dye. I only can confirm that this experiment worked as well as the previous one, but I

can confirm that the wastewater is cleaned by means only the absorbance measure.

Only one thing was a different, and it is important to realize about that, and it is that in

the reactor 3, the aerobic one, can be polymerisation, which has to be avoided ,

otherwise it won´t work correctly.

It is really good that this technique is effective also with this higher concentration

because this one is the most similar to the real life, it means, that in real polluted

waste water, the concentration of dyes that can be found is around 200 mg/L.

4 EXPERIMENT 2 & 3 (Reactive Blue 4 and Cibacron Orange P-2R GR)

This experiment is totally different from the first two, now there are different dyes and

assembly.

4.1 OBJECTIVE

The objective of these experiments is to determinate how different dyes can be

biodegrade just adding a biological support in the wastewater and how long time it

takes. The experiment was performed in batch.


4.2 THEORY

There are two different dyes; Reactive Blue 4 and Cibacron Orange P-2R GR, and

also a mixture of both of them.

Reactive Blue 4 is an anthraquinone-based chlorotriazine dye very important in

dyeing of cellulosic fabrics, Figure 26 [19].

One of its properties is that it has relative slow biodecolorization kinetics [20].

Recent research shows that although the level of the anthraquinone dye Reactive

Blue 4 in the environment is expected to be orders of magnitude lower than that

found in commercial, spent reactive dye baths, the effect of long-term, low-level dye

exposure needs to be evaluated [21]

Figure 26 Reactive Blue 4

Cibacron Orange P-2R GR belongs to the azo dye group and is a variant on dyes

already in use in e.g. Australia and it is claimed that using it instead of older dyes

should reduce the quantity of dye released into the environment. In spite of it is a

very common dye in the industry, the main problem is that this dye is not

biodegradable [22].

.



There are two parallel experiments; the experiment 2 the active one which works with

a microbiological support (wood chips non sterilized), and on the other hand is the

experiment 3 or control one which works in a sterile way, with sterilized wood chips in

order to evaluate adsorption and non biodegradable based decrease of absorption.

The sterile one is just to have a control and in order to make comparison with the

biodegradable experiment possible.

Both experiments are composed of six 500 mL e-flasks, three different dyes

compositions are given out, this means that each experiment is made in duplicate, so

there are two flasks per each type of dye or dye mixture.

All the flasks contain wood chips; and it is important to know the amount of wood

used in each flask.

− EXPERIMENT 2

Reactive Blue 4

Flask 1 66,230 g

Flask 2 66,365 g

Cibacron Orange P – 2R GR

Flask 1 66,465 g

Flask 2 66,368 g

Mixture between Reactive Blue 4 and Cibacron Orange P – 2R GR

Flask 1 66,509g

Flask 2 66,650 g

− EXPERIMENT 3

Reactive Blue 4

Flask 1 66,038 g

Flask 2 66,165 g

Cibacron Orange P – 2R GR


Flask 1 66,859 g

Flask 2 66,778 g

Mixture between Reactive Blue 4 and Cibacron Orange P – 2R GR

Flask 1 66,330 g

Flask 2 66,384 g

All the wood chips used in the different flasks have a water contain of approximately

60%.

The three different dye mixtures used in both experiments are prepared in the next

way:

− DYE 1: 400 mg of Reactive Blue 4 and water from the tap until 1L.

− DYE 2: 400 mg of Cibacron Orange P – 2R GR and water from the tap

until 1L.

− DYE 3: 400 mg of Reactive Blue 4, 400 mg of Cibacron Orange P – 2R

GR and water from the tap until 1L.

Of course it is necessary to put in the autoclave all the dyes prepared during 15

minutes with 121ºC of temperature in order to have an original dye totally sterilized.

In the biodegradable experiment (experiment 2) it is enough to sterilize only the dye

solution because the microorganisms present in the wood chips are responsible for

the degradation, but for the control experiment (experiment 3), there is a need for a

complete sterilized environment. In order to achieve that it is necessary to put the

flasks with the wood chips in the autoclave during 90 minutes in 121 ºC twice.

Finally, each flask contains the amount of wood chips previously mentioned and 150

mL of the corresponding dye solution. All of them are kept in the darkness because

light e.g. sunlight might degrade dyes. All flasks were sealed with cotton stoppers.

To see the evolution of the experiments it is necessary to take samples for

absorbance measurements from each flask.


The samples of the biodegradable experiment were taken twice per week, and in the

case of the control experiment, the samples were taken once per week, because its

change in absorbance was almost zero, so it wasn’t necessary to take samples very

often.

The samples were taken directly from each flask with a pipette and filtrated through

regular paper filter.

After this, and after a optimum dilution in distilled water of the samples (3mL of

sample and distilled water until 25mL, they were scanned with a spectrophotometer,

Unicam Helios γ, which worked in an wavelength range between 200 and 800 nm.

The diluted samples were introduced in the spectrophotometer with quartz cuvettes

of 1 cm of edge perfectly clean and dry.

4.4 REACTIVE BLUE 4 SOLUTION

4.4.1 RESULTS

First of all it is necessary to know how is the original dye solution in order to compare

how the evolution of the dye solution degradation was; for that the control experiment

is used.


Figure 26. Graphic of the absorbance of the Reactive Blue 4 solution in the 1st day in the control

experiment (experiment 3)

Dilution: 3mL sample and distilled water until 25mL

As the Figure 26 shows, the maximum absorbance between a wavelength of 550 and

650 nm is 0,10, so the real absorbance in an undiluted sample is 0,83.

With the pass of the time the absorbance of the dye solution in the control

experiment shouldn’t change, or at least it shouldn’t change too much, exactly the

opposite that it is supposed to happen in the biodegradable experiment, so the

absorbance in the experiment 2 shouldn’t be higher than 0,10 in a diluted way or 0,83

in an undiluted one.


Figure 27. Graphic of the absorbance of the Reactive Blue 4 solution in the 7th day in the

biodegradable experiment (experiment 2)


Seven days after start the experiment, the absorbance in the maximum peak

wavelength is at 600 nm and it is of 0,10 (0,83 in an undiluted sample), Figure 27, is

the same as in the control experiment, so during this time the degradation of the dye

solution wasn’t so obvious.




Eighteen days after the beginning of the experiment, the absorbance is lower, in this

case is 0,05, (in an undiluted way the absorbance is 0,415) the half of the original

one.





After 35 days the absorbance is a little bit lower than in the Figure 29, in this case is

the 0,04 (0,33 in an undiluted way) for the same wavelength.




After forty seven days, the absorbance is ten times lower, so between 500 and 600

nm of wavelength the absorbance is 0,01 (0,083 in an undiluted way), Figure 30.


The next graphics show the evolution of the absorbance changes during the whole

experiment.

Figure 31. Graphic of laboratory day vs. percent of degradation of the dye solution

About the control experiment, the absorbance didn´t change during the whole

experiment, so there wasn´t decolourization because it was a sterile environment.

4.4.2 CONCLUSION

According to the results obtained, the experiment with the Reactive Blue 4

demonstrates that this dye is decolourized and that in 47 day, Absorption at the peak

wavelength decreased by 90% (Figure 30). That is a quite successful result. In spite

of the experiment lasted 47 days optimization of the conditions might hopefully lead

to a decrease in the time required for degradation.


4.5 CIBACRON ORANGE P-2R GR SOLUTION

4.5.1 RESULTS

As in the previous case, it is necessary to know how is the curve of the absorbance

between 200 and 800 nm in the control experiment, in order to compare the results

obtained in the biodegradable one.

Figure 32. Graphic of the absorbance of the Cibacron Orange P-2R GR solution in the 1st day in

the control experiment (experiment 3)


In this case, there is a maximum absorbance peak wavelength at approximately 500

nm, which is of 1,10 as the Figure 32 shows, but it is more interesting to show the

real absorbance, so it is of 9,17. The absorbance of the experiment 3, the control

one, is almost the same during the whole experiment, is for that it is mentioned only

once.

There is also an other "peak" at around 280 nm of wavelength with a real absorbance

of 12,5.


Figure 33. Graphic of the absorbance of the Cibacron Orange P-2R GR solution in the 12th day in

the biodegradable experiment (experiment 2)


After twelve days since the beginning of the experiment the absorbance of the

solution has decreased to 0,5, (4,17 in an undiluted way) so the degradation started

being quite fast, at 280 nm the real absorbance is of 5,8, so in this case the

absorbance is also decreasing, Figure 33.





After 35 days, Figure 34, the degradation is not so fast because in the last twenty

three days, the absorbances changed from 0,5 to 0,48, so in an undiluted way it is

4,17 to 4. And at 280 nm the absorbance continues decreasing; so the real

absorbance is 5.




The absorbance is decreasing very slowely during the final part of the experiment, in

the 47th day of the experiment the absorbance is 0,42 at 500 nm (3,5 in an undiluted

way), Figure 35; exactly the same occurs at 280 nm, so the absorbance stop

decreasing.

All the changes of the dye solution during the experiment are shown in the next

graphic, which represent the laboratory day vs. the percent of degradation of the

solution.


Figure 36. Graphic of laboratory day vs. percent of degradation of the dye solution

About the control experiment is always the same absorbance, so it doesn’t

decolourise at all.

4.5.2 CONCLUSION

This dye was more difficult to decolorized, actually, it wasn’t totally decolorized.

The decolourzation started very fast, and after that, it stopped, so maybe this is a

refractory dye (it is probably degradable to some extent according to the results).

4.6 MIXTURE BETWEEN REACTIVE BLUE 4 AND CIBACRON

ORANGE P-2R GR SOLUTION

4.6.1 RESULTS

As always, first of all, it is necessary to know how the original solution is, for that the

control experiment is used. The control experiment is used to see non-biological

related decrease of absorbance as a function of time. The importance of a control

experiment is the chance to follow the absorbance as a function of time e.g. how

adsorption is influencing the results.


Figure 37. Graphic of the absorbance of the mixture between reactive Blue 4 and Cibacron

Orange P-2R GR solution in the 1st day in the control experiment (experiment 3)


As Figure 37 shows, the maximum peak of absorbance is approximately at 500 nm

wavelength, the same as in the Cibacron P-2R GR solution, actually, the absorbance

curve is quite similar to the Cibacron P-2R GR one, even there is the same

absorbance in the maximum peak, 1,10 (9,16 in an undiluted solution). The

absorbance at approximately 600 nm is due to the blue dye, which is of 0,20 (1,67 in

an undiluted solution). So the total curve is due to absorbance of the blue as well as

the orange dyes.



Orange P-2R GR solution in the12th day in the biodegradable experiment (experiment 2)


The absorbance got in twelve days after the beginning of the experiment is 0,50

(4,16 in an undiluted solution) at 500 nm, so for the moment this solution has a

behaviour quite similar as the Cibacron P-2R GR, but also it has a behaviour similar

as Reactive Blue 4 at 600 nm, which is of 0,10 (0,83 in an undiluted solution), also

the half of the original absorbance (Figure 38).


Orange P-2R GR solution in the35th day in the biodegradable experiment (experiment 2)


In the 35th day of experiment, Figure 39, the absorbance is 0,45 (3,75 in an undiluted

solution), a little bit lower than in the case of the Cibacron P-2R GR, which was of

0,48 (4,00 in an undiluted solution) with the same days of experiment, and the

absorbance due to the Reactive Blue 4 is a little bit lower than in the previous

measure, which is of 0,08 ( 0,67 in an undiluted solution).



Orange P-2R GR solution in the 47th day in the biodegradable experiment (experiment 2)


The absorbance continues decreasing, but very slowly, in the last day of the

experiment, day 47, the absorbance at 500 nm is 0,35 (2,91 in an undiluted solution)

and at 600 nm 0,075 (0,625 in an undiluted solution), Figure 40.

So in order to see the evolution of the degradation at 500 nm and 600 nm of

wavelength there is the next graphics, Figures 41 and 42.

Figure 41. Graphic of laboratory day vs. percent of degradation of the dye solution at 500 nm


Figure 42. Graphic of laboratory days vs. Percent of degradation of the dye solution at 600 nm

4.6.2 CONCLUSION

For the study of this solution it is convenient to compare with the other two dye

solution (Reactive Blue 4 and Cibacron P-2R GR) because is made with both of

them.

Figure 43. Graphic of laboratory day vs. percent of degradation of each dye solution


After comparison of the data obtained I can say that the behaviour of the mixture

solution is more similar to Cibacron P-2R GR one. During the beginning they were

nearly the same, but in the end, the mixture solution degradation is between blue and

orange solution, so the mixture solution has a behaviour intermediate between blue

and orange solution, Figure 43.

5 REFERENCES [1] Banat IM, Nigam p, Singh D, Marchant R. Microbial decolorization of textile-dye-

containing effluents: a review. Bioresour Technol 1996;58:217-27.

[2] Das SS, Dey S, Bhattacharyya BC. Dye decolorization in a column bioreactor

using wood-degrading fungus Phanerochaete chrisosporioum.Indiand Chem Eng

sect A 1995;37:176-80.

[3] Philips (1996).

[4] Poots et al., 196;1979; Tincher and Rrobertson, 1982; Banat et al., 1996; Lee,

2003; Lee et al., 2005.

[5 ]www.britannica.com/eb/article-9011550/azo-dye

[6] www.britannica.com/eb/article-9011550/azo-dye and Wikipedia.

[7] http://en.wikipedia.org/wiki/Amine

[8] Allinger, Cava, De Jongh, Johnson, Lebel, Stevens; Química Orgánica

[9] Wong and Yuen, 1996)(Metabolism of azo dyes by Lactobacillus casei TISTR

1500 and effects of various factors on decolorization.

[10] Walker R. The metabolism of azo compounds; a review of the literature. Food

Cosmet Toxicol.

[11]

http://www.cbi.eu/marketinfo/cbi/docs/eu_legislation_azo_dyes_in_textile_and_leathe

r_articles

[12] Baughman and Weber, 1994; Aspland, 1997.

[13] http://www.britannica.com/eb/article-9007791/anthraquinone-dye

[14] Carin Zander. Evaluation of the released thermal power in wood pellets

http://es.wikipedia.org/wiki/Microorganismo

[15] http://www.britannica.com/eb/article-9011550/azo-dye

http://es.wikipedia.org/wiki/ADN


[16] http:// www.vinotec.com/pdf/T154.pdf

[17], [18] http://en.wikipedia.org/wiki/Gram_stain

[19] Lehninger Princeples of Biochemestry

[20] H. Zollinger, Color in Chemestry, 2 ed, V.C.H. Publisher: New York, 1991

[21] Lee, 2003; Lee et al ., 2005

[22], Epolito et al,.2005

[23] http://www.nicnas.gov.au/search/cache.cgi?collection=nicnas-

web&doc=http/www.nicnas.gov.au/publications/car/new/na/nasummr/na0100sr/na16

8.asp

6 APPENDIX

6.1 Directive 2002/61/EC of the European Parlament and of the

Council of 19 July 2002

School of Technology and Design

SE- 351 95 Växjö

Sweden

tel +46 470-70 80 00, fax +46 470-76 85 40

www.vxu.se

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