School of Technology and Design,TD
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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
1 INTRODUCTION ................................................................................................. 5 1.1 2 REACTIVE DYES.......................................................................................... 5 1.1.1 AZO DYES.............................................................................................. 6 1.1.2 ANTHRAQUINONE DYES...................................................................... 7 EXPERIMENT 1 (50 mg each dye / L) ................................................................ 8 2.1 OBJECTIVE .................................................................................................. 8 2.2 THEORY ....................................................................................................... 8 2.2.1 3 MICROORGANISMS .............................................................................. 9 2.3 MATERIAL AND METHOD ........................................................................... 9 2.4 RESULTS OF THE EXPERIMENT...............................................................16 2.5 CONCLUSION .............................................................................................21 EXPERIMENT 1 (PART II, Reactive Black 5 and Procion Red MX 5B, 200 mg
each dye / L)..............................................................................................................21 4 3.1 OBJECTIVE .................................................................................................21 3.2 MATERIAL AND METHOD ..........................................................................22 3.3 RESULTS OF THE EXPERIMENT...............................................................22 3.4 CONCLUSION .............................................................................................25 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 Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
6.1 Directive 2002/61/EC of the European Parlament and of the Council of 19
July 2002................................................................................................................45
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on 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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
Figure 4.
Figure 5.
Reactive Black 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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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].
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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 (5090% 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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
Figure 13. First ten days (before the addition of the yeast extract) in the reactor 2
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.
Figure 14. First ten days (before the addition of the yeast extract) 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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Before the addition of the yeast extract After the addition of the yeast extract REACTOR 1 (550 nm) Laboratory day 1 3 5 8 10 12 19 25 33 38 Absorbance
2,25 1,13 0,75 0,38 0,30 0,21 0,13 0,21 0,15 0,15 Percent of degradation 0,00 50,00 66,67 83,33 86,67 90,67 94,44 90,67 93,33 93,33 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:
Figure 17. Reactor 2 after the addition of the yeast extract until the day 25 of the experiment
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Figure 18. Reactor 2 until the last day of the experiment
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:
Figure 19. Reactor 3 after the addition of the yeast extract until the day 25 of the experiment
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
Figure 20. Reactor 3 until the last day of the experiment
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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
3.2 MATERIAL AND METHOD
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Figure 23. 18th day of the experiment
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:
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Figure 25. 44th day of the experiment
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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].
.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
4.3 MATERIAL AND METHOD
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
ƒ Reactive Blue 4
x
Flask 1 Æ 66,230 g
x
Flask 2 Æ 66,365 g
ƒ Cibacron Orange P – 2R GR
x
Flask 1 Æ 66,465 g
x
Flask 2 Æ 66,368 g
ƒ Mixture between Reactive Blue 4 and Cibacron Orange P – 2R GR
x
Flask 1 Æ 66,509g
x
Flask 2 Æ 66,650 g
− EXPERIMENT 3
ƒ Reactive Blue 4
x
Flask 1 Æ 66,038 g
x
Flask 2 Æ 66,165 g
ƒ Cibacron Orange P – 2R GR
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
x
Flask 1 Æ 66,859 g
x
Flask 2 Æ 66,778 g
ƒ Mixture between Reactive Blue 4 and Cibacron Orange P – 2R GR
x
Flask 1 Æ 66,330 g
x
Flask 2 Æ 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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
Figure 27. Graphic of the absorbance of the Reactive Blue 4 solution in the 7th day in the
biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
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.
Figure 28. Graphic of the absorbance of the Reactive Blue 4 solution in the 18th day in the
biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
Figure 29. Graphic of the absorbance of the Reactive Blue 4 solution in the 35th day in the
biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
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.
Figure 30. Graphic of the absorbance of the Reactive Blue 4 solution in the 47th day in the
biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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)
Dilution: 3mL sample and distilled water until 25mL
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
Figure 33. Graphic of the absorbance of the Cibacron Orange P-2R GR solution in the 12th day in
the biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
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.
Figure 34. Graphic of the absorbance of the Cibacron Orange P-2R GR solution in the 35th day in
the biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Figure 35. Graphic of the absorbance of the Cibacron Orange P-2R GR solution in the 47th day in
the biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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)
Dilution: 3mL sample and distilled water until 25mL
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.
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
Figure 38. Graphic of the absorbance of the mixture between reactive Blue 4 and Cibacron
Orange P-2R GR solution in the12th day in the biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
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).
Figure 39. Graphic of the absorbance of the mixture between reactive Blue 4 and Cibacron
Orange P-2R GR solution in the35th day in the biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
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).
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
Figure 40. Graphic of the absorbance of the mixture between reactive Blue 4 and Cibacron
Orange P-2R GR solution in the 47th day in the biodegradable experiment (experiment 2)
Dilution: 3mL sample and distilled water until 25mL
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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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-dyecontaining 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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
[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
Sara Palacios
Decolourization of azo and anthraquinone dyes by mean of microorganisms growing on wood chips
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