3rd AMIREG International Conference (2009): Assessing the Footprint of 146

Resource Utilization and Hazardous Waste Management, Athens, Greece

Assessing biosorption on anaerobic dyes removal

I.C. Gonçalves, S. Abrantes and M.I. Ferra

University of Beira Interior, Covilhã, Portugal

H.M. Pinheiro
Technical University of Lisbon, Portugal

ABSTRACT region and the main characteristic peak re-

mained in HPLC chromatograms. This indicated
An improvement of color removal was achieved
that adsorption was prevalent during the initial
(96-100%) in an anaerobic bioreactor in which a
stages of dye removal in anaerobic bioreactors,
natural adsorbent, spent brewery grains (SBG)
bearing in mind that, for less soluble dyes, pre-
was incorporated. SBG could work as adsorbent
cipitation could also contribute to decoloriza-
as well as an electron shuttle (lignin frac-
tion.
tion/humic acids) in the dye removal mecha-
nism. Anaerobic biomass also can undergo ad-
sorption and reductive mechanisms. However, 1. INTRODUCTION
little is known about which dye removal mecha-
nism, degradation/adsorption, predominates and Azo dyes represent 60-70% of the more than
to what extent. The aim of the present work was 10000 dyes currently manufactured (Carliell
to evaluate the adsorption susceptibility of dyes et al., 1996). Most dyes cause negative aesthetic
on SBG and anaerobic biomass adapted and non effects. In addition, they can be toxic to aquatic
adapted to SBG (active and autoclaved). Batch life and carcinogenic and mutagenic to humans
tests were performed for 60 minutes at 37±1ºC (Brown and Hamburguer, 1987). Azo dyes may
with SBG, and both active and autoclaved an- be decolorized by several mechanisms including
aerobic mesophilic biomass. The adsorb- adsorption and reductive cleavage of the azo
ent/solution ratio was of 1:4 (v/v) and the bio- bond, to which the color is associated, via an-
mass concentration was of 6-8 gSS/L. In pre- aerobic degradation (Brás et al., 2001).
liminary tests performed with 24 azo dyes at a Adsorption is probably the first step of the
initial concentration of 100 mgl-1 and active an- mechanisms associated to anaerobic azo dye
removal, and depends on the sorbate-sorbent in-
aerobic biomass, a color removal rate between 0
teraction and the system conditions. It is known
and ~99% was achieved. Depending on the dye,
that materials of biological organic compounds,
adsorption and precipitation seem to be associ-
such as vegetal and bacterial biomass, display a
ated to color removal. More soluble dyes pre-
high adsorption capacity and have been used as
sented lower values for decolorization. A dye
biosorbents in the removal of several pollutants
concentration of 30 and 50 mg/L was then
(Akzu, 2001; Silva et al., 2004).
tested, with 5 dyes, and different types of bio-
Brewers’ spent grain (BSG) is a lignocellu-
mass (bacterial and vegetal). AO8 was then se-
losic material with a high content of protein and
lected for further studies. It is a soluble dye and
fibre (ca 20 and 70% dry basis, respectively),
displayed a significant adsorption (the percent-
non-cellulosic polysaccharides, lipids and some
age adsorbed was of 71% for SBG, 36% for ac-
polyphenolic components. Such rich composi-
tive biomass and 62% for autoclaved biomass).
tion makes it viable to be used as a conditioning
Ultraviolet-visible spectra of dye solutions did
agent in anaerobic reactors along with favoring
not show marked alterations (in systems with
the biodiversity in anaerobic biomass and bio-
and without BSG). The absorbance decreased in
gas production (Mussato et al., 2006).
the whole range of the spectrum in UV-visible
3rd AMIREG International Conference (2009): Assessing the Footprint of 147
Resource Utilization and Hazardous Waste Management, Athens, Greece

In tandem with adsorption the following
mechanism that presumably occurs in anaerobic
reactors, is reduction of azo dyes via anaerobic
bacteria as an non-specific process, in which re-
ducing equivalents from an external electron
donor (biologically or chemically generated) are
transferred to the dye (Santos et al., 2006). This
results in the formation of aromatic amines that
are hardly metabolized anaerobically, with the
exception of a few examples bearing hydroxyl
and carboxyl groups, which can be fully de-
graded (Razo-Flores et al., 1996). So a higher
biodiversity of anaerobic biomass could also
improve the elimination of the persistent aro-
matic amines.
The present study was undertaken to assess
the contribution of biosorption on the color re-
moval process that occurs in the presence of an-
aerobic biomass (adapted and non adapted to
BSG), in active and autoclaved conditions. This
will help to clarify which mechanism (adsorp-
tion/reduction) is prevailing, or if both play a vi-
tal role.
2. MATERIALS AND METHODS
2.1 Raw material
The BSG used in the experiments had the fol-
lowing composition (% dry weight, w/w): 24%
protein, 60% fibre, 10% ashes and moisture and
6% lipids. It was gently supplied by a brewery
from Lisbon region.
2.2 Anaerobic biomass
60; (NH4)6MoO7.4H2O 180; NiCl2.6H2O 100;
Na2SeO3.5H2O 200 and HCl (37%) (1). Sodium
bicarbonate was used (6-9 g l-1) to neutralize the
medium fed to the anaerobic vessels. For the ad-
sorption tests with BSG a phosphate buffer solu-
tion was used (1.28 g l-1 NaH2PO4.2H2O and
0.42 g l-1 NaH2PO4). All solutions were pre-
pared with tap water.
2.4 Chemicals
All dyes, including selected azo dyes Acid Or-
ange 8 (AO8), Acid Red 151 (AR151), Acid
Red 114 (AR114), Acid Blue 120 (AB120) and
Acid Blue 113 (AB113) were purchased from
Sigma-Aldrich (Germany). Their chemical
structures are indicated in Table 1. Chemicals
Table 1: Dyes selected for adsorption studies with differ-
ent types of biomass (bacterial and vegetal).
Commercial
Dye structure
name
Acid Orange 8
(AO 8)
C.I. *: 15575
Acid Red 151
(AR 151)
C.I. *: 26900
Acid Red 114
(AR 114)
C.I. *: 23635

Anaerobic biomass was collected from two

mesophilic lab-scale continuous reactors, fed
with a synthetic wastewater containing a basal
medium and glucose as soluble carbon source.
In one of the reactors BSG was incorporated as Acid Blue 113
biosorbent/conditioning agent (BSG adapted (AB 113)
biomass). C.I. *: 26360

2.3Basal medium
The basal medium used in the anaerobic reac-
tors and adsorption tests contained (mg l-1)
Ca(OH)2 90; NH4Cl 170; NaH2PO4.2H2O 48; Acid Blue 120
MgCl2.6H2O 11; KCl 25; and 0,5 ml l-1 of a (AB 120)
trace elements solution with (mg l-1) H3BO3 C.I. *: 26400
100; FeCl2.4H2O 4000; EDTA (Ethylene Dia-
mine Tetracetic Acid, sodium salt) 2000;
ZnCl2.4H2O 100; MnCl2.4H2O 50; CuCl2.2H2O *C.I. – Colour Index
3rd AMIREG International Conference (2009): Assessing the Footprint of 148
Resource Utilization and Hazardous Waste Management, Athens, Greece

were dissolved in the basal medium in concen-

trations of 30 mg l-1, 40 mg l-1 and 50 mg l-1.
These concentrations are usually found in textile
effluents. Methanol and acetonitrile were pur-
chased from Sigma and Merck respectively.
All chemicals were of high purity and com-
mercially available.
2.5 Analyses
Color was measured spectrophotometrically
with Helios Alpha spectrophotometer (Unicam,
UK.) at the maximum visible absorbance wave-
length of the dye (487 nm for AO8, 513 nm for
AR151, 516 nm for AR114, 559 nm for AB120
and 536 nm for AB113). Absorbance at these
wavelengths was correlated with dye concentra-
tion and used to quantify decolorization.
Suspended solids (SS), volatile suspended
solids (VSS) and pH in liquid samples were de-
termined according to Standard procedures
(APHA, 1995).
2.6 Batch adsorption tests
Figure 1: Scheme of the adsorption tests performed with
A total of 24 azo dyes were studied with active several azo dyes.
mesophilic anaerobic biomass in batch condi-
tions. Preliminary decolorization tests were car- BSG with buffer solution was used as blank
ried out in the same conditions as indicated for (Fig. 1).
biosorption studies, with an initial dye concen- UV-visible spectra and HPLC chroma-
tration of 100 mg/L. From those, 5 dyes were tograms were run to follow the adsorption of
selected to proceed with decolorization tests. those compounds. The amount of dye adsorbed
These 5 dyes (Table 1) and two types of an- was calculated as the difference between the
aerobic mesophilic biomass, with and without amount of test substance initially present in so-
BSG (both active and autoclaved), and dried lution and the amount remaining at the end of
BSG were suspended in 50 ml of solution con- the experiment.
taining a known concentration of dye. Dye solu-
tions (30, 40 and 50 mg l-1) were prepared with 3. RESULTS AND DISCUSSION
basal medium for tests performed with the two
types of bacterial biomass and with the buffer As pH is an important parameter to control on
solution for tests with dried BSG. The experi- adsorption and biological systems, all experi-
ments were conducted in amber glass flasks ments were carried out at neutral pH (7.5±1). In
(100 ml capacity) for 60 minutes, equipped with preliminary tests performed with active meso-
magnetic stirring, at 37±1ºC. The ad- philic anaerobic biomass, significant differences
sorvent/solution ratio was of 1:4 (v/v) and the were observed on color removal rate, for the 24
biomass (bacterial and vegetal) concentration dyes studied (Table 2). The number of aromatic
was of 6-8 gSS l-1. Samples were taken before rings, azo bonds and sulphonic and others sub-
mixing the biosorbent and dye bearing solutions stituent groups in the dye molecule determine
and at pre-determined time intervals for the re- their hydrophilic/hydrophobic character and the
sidual dye concentration in solution. Before type of interactions to be established with other
analysis samples were centrifuged at 14000 rpm groups present in substrates to be linked (as tex-
and the supernatant fraction, diluted when nec- tile substrates). So the behaviour of the different
essary, was analyzed for the remaining dye. A dyes when in presence of bacterial biomass will
flask containing biomass and basal medium or be affected in a similar way. Despite of all dyes
3rd AMIREG International Conference (2009): Assessing the Footprint of 149
Resource Utilization and Hazardous Waste Management, Athens, Greece

Table 2: Color removal in preliminary batch tests per- of absorbance in the visible region, suggesting
formed with mesophilic anaerobic biomass, at the end of biosorption/precipitation as mechanisms respon-
60 min.
sible for decolorization, for the three biosor-
Dye Colour removal (%)
bents tested. In the UV region the absorbance is
Acid Orange 6 0
mainly due to the blank test (Fig. 2)
Acid Orange 7 24.1
Color removal ranged from 25.0% to 92.4%
Acid Orange 8 34.6
at the end of 60 minutes contact time (Table 3).
Acid Orange 10 18.0
Acid Orange 12 35.2
AO8 reached the highest value for BSG and the
Acid Red 8 10.6
lowest for active biomass. It is the smallest dye
Acid Red 14 13.9 tested and has the smallest number of aromatic
Acid Red 18 18.8 rings. As obtained from preliminary tests,
Acid Red 73 21.6 AR151 and AR113 have a very efficient color
Acid Yellow 9 25.8 removal rate, for the three types of biomass
Acid Yellow 17 6.4 tested (from 89.7 to 98.3%).
Acid Yelow 36 27.3 AB120 andAB113 display similar biosorp-
Acid Blue 29 33.8 tion of the three type of biosorbents, although
Acid Blue 113 96.6 with lower values than those obtained in the
Acid Blue 120 >99 preliminary experiments, which can be associ-
Acid Blue 161 46.9 ated to the lower concentrations essayed.
Acid Black 1 36.9 Also it can be inferred that adsorption occurs
Acid Violet 7 15.9 essentially in the first 30 minutes (Table 3a, b).
Brilliant Black BN 49.1 Dye AO8 was selected for further studies
Brilliant Yellow 8.4 since it displays a markable absorption peak
Reactive Orange 16 0.0 (high absortivity) in the visible range, even for
Acid Red 88 12.1 low concentrations, which renders it easier to be
Acid Red 114 94.6 measured by UV-visible spectrophotometry
Acid Red 151 >99
Acid Orange 8
contain azo bonds, as shown in Table 1, the
color removal rate has a broad range of values 4,000
AO8
(ranging from no removal to almost a complete
Absorbance

3,000 t30min
removal). Spectra of UV-visible run in the be- 2,000 t60min
ginning and at the end of the experiments indi- 1,000 BSG blank
cate that the main mechanism involved on color 0,000
removal, in the first 60 minutes, was adsorption, 200,0 400,0 600,0 800,0
since the absorbance decreased in the whole
w avelength(nm )
range of the spectra. Nevertheless, some dyes
seem to display low solubility (as for instance
a)
AR151, AB113 and AB120). So in tandem with
adsorption a precipitation mechanism, rendering Acid Blue 120

the increase of dye removal rate from the solu-

4,000
tion, should not be discharged.
Absorbance

3,000 AB120
From these, 5 dyes, 4 with a high color re- 2,000 t30min
moval rate (AR114 AR151, AB113 and AB120) 1,000 t60min
and 1 with ca 35% color removal (AO8) were 0,000
studied for lower initial concentrations (30- 200,0 400,0 600,0 800,0
50 mg l-1), in the presence of 3 types of biosor- w avelength (nm )
bents. Examples of some ultraviolet-visible
spectra of dye solutions collected at the begin- b)
ning and at 30 and 60 minutes contact time, for
Figure 2: Examples of spectra obtained for dyes Acid Or-
different types of biomass, are depicted in Fig- ange 8 (a) and Acid Blue 120 (b) in tests performed with
ures 2 and 3. Results indicate a sharp decrease BSG.
3rd AMIREG International Conference (2009): Assessing the Footprint of 150
Resource Utilization and Hazardous Waste Management, Athens, Greece

Table 3: Dye removal (%) in BSG, active and autoclaved 4. CONCLUSIONS

biomass tests at 30 (a) and 60min (b) contact time.
a) In order to clarify some aspects involved on azo
Biosorbent Active Autoclaved dyes decolorization in anaerobic bio-reactors, it
BSG
Dyes Biomass Biomass should be pointed out:
AO8 67.4 33.1 52.6
- Results from experiments performed with 24
AR151 92.0 95.3 97.0
azo dyes and active biomass show that, in the
AR114 90.0 91.2 92.9
first 60 min, only dyes Acid Orange 6 and
AB113 56.0 74.9 59.5
AB120 17.2 38.4 58.9
Reactive Orange 16 were not removed. For
b) the other 22 dyes decolorization ranged from
Biosorbent Active Autoclaved 6.4 to >99% (Table 2).
BSG - For the 5 dyes studied with active and auto-
Dyes Biomass Biomass
AO8 71.0 36.0 62.1 claved biomass and BSG, removal rate
AR151 92.0 96.0 98.3 ranged from 36 to 98.3%, being the highest
AR114 94.9 89.7 92.4 values achieved for dyes with the lowest
AB113 77.7 70.0 66.3 solubility. The most soluble dye (AO8) dis-
AB120 50.1 41.8 58.9 played lower color removal rates. In this case
dye precipitation should not be relevant.
(Fig. 3). - Depending on the dye tests also suggest that
From the results obtained with anaerobic adsorption and/or precipitation could be the
biomass incubated with BSG, adsorbed and non main mechanisms responsible for color re-
adsorbed dye was measured and the recovered moval in anaerobic bio-reactors, in the first
dye was calculated from extraction tests and 30-60 min.
analyzed by HPLC. Adsorption seems to be the - AO8 was also tested with active biomass in-
predominant mechanism of color removal. cubated in the presence of BSG leading to an
improvement of dye adsorption (higher de-
AO8
colorization).

5
Absorbance

4 ACKNOWLEDGEMENTS
3 AO8
2 t30min The authors are grateful to the project
1
t60min
PDCT/AMB/59388/2004 from Fundação para a
0
t7days
Ciência e Tecnologia (FCT), Portugal
200 400 600 800
w avelength(nm )
REFERENCES
a) Akzu, Z., 2001. Biosorption of reactive dyes by dried ac-
tivated sludge: equilibrium and kinetic modelling.
AB120
Biochemical Engineering Journal, 7: 79-84.
Brás, R., M.I.A. Ferra, H.M. Pinheiro and I.C. Gonçalves,
6,000 2001. Batch tests for assessing decolourisation of azo
A b so rb an ce

dyes by methanogenic and mixed cultures. Journal of

4,000 AB120 Biotechnology, 89, 155-162.
2,000 t30min Brown, D. and B. Hamburguer, 1987. The degradation of
dyestuffs: part III - investigations of their ultimate de-
0,000 t60min gradability. Chemosphere, 16, 1539-1553.
200 400 600 800 t7days Carliell, C.M., S.J. Barclay and C.A. Buckley, 1996.
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w avelength (nm) ing anaerobic digestion: laboratory and full-scale tri-
als. Water SA, 22, 3, 275-233.
b) Mussato, S.I., G. Dragone and I.C. Roberto, 2006.
Figure 3: Examples of spectra obtained for dyes Acid Or- Brewer´s spent grain: generation, characteristics and
ange 8 (a) and Acid Blue 120 (b) in tests performed with potential applications. Journal of Cereal Science, 43:
active biomass. 1-14.
3rd AMIREG International Conference (2009): Assessing the Footprint of 151
Resource Utilization and Hazardous Waste Management, Athens, Greece

Santos, A.B., M.P. de Madrid, F.A.M. de Bok, A.J.M.

Stams, J.B. van Lier and F.J. Cervantes, 2006. The
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Silva, J.P., S. Sousa, J. Rodrigues, H. Antunes, J.J. Porter,
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