Biodegradation (2015) 26:289–298

DOI 10.1007/s10532-015-9734-5

ORIGINAL PAPER

Efficient anaerobic treatment of synthetic textile wastewater

in a UASB reactor with granular sludge enriched
with humic acids supported on alumina nanoparticles
Francisco J. Cervantes . Rafael Gómez . Luis H. Alvarez .
Claudia M. Martinez . Virginia Hernandez-Montoya

Received: 16 February 2015 / Accepted: 19 May 2015 / Published online: 23 May 2015
Ó Springer Science+Business Media Dordrecht 2015

Abstract A novel technique to co-immobilize hu-
mus-reducing microorganisms and humic substances
(HS), supported on c-Al2O3 nanoparticles (NP), by a
granulation process in an upflow anaerobic sludge bed
(UASB) reactor is reported in the present work. Larger
granules (predominantly between 1 and 1.7 mm) were
produced using NP coated with HS compared to those
obtained with uncoated NP (mostly between 0.25 and
0.5 mm). The HS-enriched granular biomass was then
tested for its capacity to achieve the reductive
decolorization of the recalcitrant azo dye, reactive
red 2 (RR2), in the same UASB reactor operated with a
hydraulic residence time of 12 h and with glucose as
electron donor. HS-enriched granules achieved higher
decolorization and COD removal efficiencies, as
F. J. Cervantes (&)
C. M. Martinez
División de Ciencias Ambientales, Instituto Potosino de
Investigación Cientı́fica y Tecnológica (IPICyT), Camino
a la Presa San José 2055, Col. Lomas 4a. Sección,
C. P. 78216 San Luis Potosı́, San Luis Potosı́, Mexico
e-mail: fjcervantes@ipicyt.edu.mx
R. Gómez
V. Hernandez-Montoya
Departamento de Ingenierı́a Quı́mica, Instituto
Tecnológico de Aguascalientes, Av. Adolfo López
Mateos No. 1801 Ote. Fracc. Bona Gens,
20256 Aguascalientes, Aguascalientes, Mexico
L. H. Alvarez
Facultad de Ciencias Quı́micas, Universidad Autónoma
de Nuevo León (UANL), Av. Universidad S/N, Cd.
Universitaria, 66451 San Nicolás de los Garza,
Nuevo León, Mexico
compared to the control reactor operated in the
absence of HS, in long term operation and applying
high concentrations of RR2 (40–400 mg/L). This co-
immobilizing technique could be attractive for its
application in UASB reactors for the reductive
biotransformation of several contaminants, such as
nitroaromatics, poly-halogenated compounds, metal-
loids, among others.
Keywords Anaerobic microorganisms
Humus

Immobilization
Redox mediator
Textile wastewater
Introduction
Humic substances (HS) constitute the most abundant
organic fraction in the biosphere accumulating in
terrestrial and aquatic environments. Despite their
remarkable stability in the environment, conferring
them a millenary residence time (Stevenson 1994), HS
are highly active in redox reactions catalyzing the
microbial and abiotic conversion of several recalcitrant
pollutants such as azo dyes, nitroaromatics, metalloids,
as well as aliphatic and aromatic polyhalogenated
compounds, by serving as redox mediators (RM,
Martı́nez et al. 2013b). Nevertheless, one of the main
limitations for applying RM on the reductive conver-
sion of these pollutants in wastewater treatment
systems is that continuous addition of HS or alternative
RM should be provided to increase conversion rates,
which is economically and environmentally unviable.

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Several strategies have recently been explored to
develop engineered systems, integrating the redox
mediating capacity of immobilized RM, to promote
enhanced conversion rates of contaminants. RM have
been immobilized by entrapment in calcium alginate,
polyvinyl alcohol-H3BO3 and agar (Guo et al. 2007),
by covalent binding on ceramic material (Yuan et al.
2012), polyurethane foam (Zhou et al. 2015), activated
carbon fibers (Amezquita-Garcia et al. 2015) and
polyethylene terephthalate (Zhang et al. 2014a; Xu
et al. 2015), by adsorption on metal oxides nanopar-
ticles (Alvarez et al. 2010; Alvarez 2012), by electro-
static attraction on the surface of anion exchange resins
(Cervantes et al. 2011), and by electro-polymerization
on activated carbon felt (Li et al. 2008). Furthermore,
humic-mineral complexes have also been demonstrat-
ed as effective solid-phase RM during the reductive
transformation of poly-halogenated contaminants (Al-
varez et al. 2012; Zhang et al. 2014b, c).
Lately, carbon based materials, such as granular (Van
der Zee et al. 2003) and powder activated carbon (Baêta
et al. 2013), black carbon (Xu et al. 2013; Gong et al.
2014), biochar (Oh et al. 2013), activated carbon fibers
(Amezquita-Garcia et al. 2013; Rı́os Del Toro et al.
2013), carbon nano-tubes and carbon xerogels (Pereira
et al. 2014), as well as graphene oxide (Colunga et al.
2015) have also been applied as RM to increase the
redox conversion of different contaminants.
The aim of the present study was to evaluate the co-
immobilization of HS supported on gamma alumina (c-
Al2O3) nano-particles (NP) and humus-reducing mi-
croorganisms (HRM) present in an anaerobic consor-
tium by a granulation process developed in an upflow
anaerobic sludge bed (UASB) reactor. The HS-enriched
consortium obtained by the granulation process was
then evaluated for its capacity to achieve the reductive
decolorization of the recalcitrant azo dye, reactive red 2
(RR2, Fig. 1), a model azo compound representative of
textile wastewaters for its recalcitrance and toxicity
(Beydilli et al. 2000; Cervantes and Dos Santos 2011;
Cervantes et al. 2011; Martı́nez et al. 2013a).
Materials and methods
Chemicals
NP of c-Al2O3 (20–50 nm and [150 m2/g) were
purchased from Inframat Advanced Materials
123
Biodegradation (2015) 26:289–298
(Manchester, CT USA). The source of HS was
leonardite, which was obtained from the International
Humic Substances Society (IHSS, Catalogue No.
1BS104L). HS were sulfonated and adsorbed on c-
Al2O3 NPs as previously described (Cervantes et al.
2011). RR2 (Procion Red MX-5B, *50 % of purity)
was purchased from Sigma-Aldrich Company and
used without additional purification. All other chemi-
cals used in this study were of analytical grade.
Adsorption of HS on c-Al2O3 NP
Both the capacity of c-Al2O3 NP to adsorb HS and
their surface charge distribution were measured as
described by Alvarez and Cervantes (2012). HS were
previously sulfonated in order to enhance their
solubility and affinity to c-Al2O3 NP, and also to
allow that quinone groups present in HS remain
available after adsorption (Cervantes et al. 2011). The
capacity of c-Al2O3 NP to adsorb HS was determined
by adsorption isotherms using the batch equilibrium
technique. Different concentrations of HS (100–5800
mg of total organic carbon (TOC)/L) were prepared
using deionized water, and the pH was adjusted to 4
with HCl (0.1 M). Then, 0.2 g of c-Al2O3 and 10 mL
of HS solution (previously filtered with 0.22 lm) were
mixed in polypropylene vials. Vials were placed on a
shaker (160 rpm at 27 °C) until equilibrium was
reached (after *3 days). After centrifugation (3200
rpm during 10 min) supernatant was analyzed to
determine the equilibrium concentration of HS in
terms of TOC; and the adsorption capacity was
determined by a mass balance based on this parameter.
In order to determine the stability of immobilized HS
on c-Al2O3 NP, several washing cycles with the basal
medium described below (pH 7.2) were carried out.
After each washing cycle, the amount of desorbed HS
was quantified from the supernatant based on the
concentration of TOC and the final adsorption ca-
pacity was assessed by a TOC balance once desorption
of HS was no longer detected.
Surface charge distribution of c-Al2O3 NP
Batch experiments were conducted to assess the
surface charge distribution of c-Al2O3 NPs at different
pH values under a CO2-free atmosphere. N2 was
bubbled for 15 min into the solutions and also in the
headspace of vials before sealing. First, a portion of
Biodegradation (2015) 26:289–298 291

UASB reactor
Humus reducing and
others anaerobic
microorganisms
Effluent GRANULATION PROCESS

y-Al2O3 NP Cations
coated with HS (Ca2+)

Influent HS on y-Al2O3 NP
NaO3 S SO3 Na
O
R N
Cl N N
NH OH
Glucose
N N
O Red Reactive 2
Oxidized Cl
Humus reducing
microorganism

OH
R NaO 3S SO3Na
CO2 + H2O H2N
+
Cl N NH2
NH
OH OH
N N
Reduced Aromatic amines
Cl

DECOLORIZATION OF RR2
Fig. 1 Mechanisms involved in the reductive decolorization of reactive red 2 (RR2) mediated by immobilized humic substances (HS)
on c-Al2O3 nano-particles (NP) in a UASB reactor

0.4 g of c-Al2O3 NP was dispensed in vials of 50 mL,
and aliquots from a 0.1 M NaCl solution were taken to
maintain a constant ionic strength. Initial pH values
(2–12) were obtained by adding NaOH or HCl 0.1 M,
for a total volume of 25 mL. After 4 days in stirring
(150 rpm at 25 °C), the final pH was measured and the
surface charge (expressed as the amount of ions
released) was obtained with a mass balance based on
pH change according to Alvarez et al. (2012).
Granulation process
Two glass UASB reactors (height 40 cm, internal
diameter 6.2 cm) with a total volume of 1.1 L were
operated at a hydraulic residence time (HRT) of 36 h
in a controlled temperature room (28 ± 1 °C). The
control reactor was amended with untreated c-Al2O3
NP (without HS) and operated in parallel to the reactor
supplemented with HS (supported on c-Al2O3 NP).
Reactor amended with HS was supplied with 30 g of
HS supported on c-Al2O3 NP, which represents
*2.4 g TOC/L, while the control reactor was adjusted
with the same amount of NP, but using untreated c-
Al2O3 NP. Both reactors were inoculated with 25 g
volatile suspended solids (VSS)/L of a methanogenic
consortium obtained from a full-scale UASB reactor
treating brewery effluents (Sonora, Mexico). The
granular sludge was previously reported to reduce
both suspended (Alvarez and Cervantes 2012) and
immobilized HS (Martı́nez et al. 2013a) evidencing
the presence of HRM in this consortium. To achieve
homogeneous distribution in both reactors, anaerobic
granular sludge was disintegrated using a sieve of
250 lm in an anaerobic chamber (N2/H2, 95/5 % v/v
atmosphere) prior inoculation. Both UASB reactors
were fed with glucose (2 g chemical oxygen demand

123
292
(COD)/L), peptone (1 g COD/L), and a mixture of
propionate, butyrate, and acetate (1 g COD/L) as
substrates. The basal medium contained (g/L): NH4Cl
(0.28), K2HPO4 (0.25), MgSO4
7H2O (0.1), CaCl2-
2H2O (0.2), FeSO4 (0.032), NaHCO3 (5) and 1 mL/L
of trace elements solution with a composition previ-
ously reported (Martı́nez et al. 2013a). The influent pH
was adjusted to 7.0 ± 0.15 throughout the granulation
process. The reactors were firstly operated in batch
mode with recirculation for 1 week in order to prevent
an important wash-out of either c-Al2O3 NP or
immobilized HS. Then, operation in continuous mode
followed during the remaining experimental period.
Characterization of produced granules
Granules were visible in both reactors after 60 days of
continuous operation and the operational conditions
were maintained for approximately 1 year. After
380 days, *200 mL of produced granules were
withdrawn from both reactors and characterized based
on their size and content of c-Al2O3 NP. Size
distribution of granules was determined by sieving
representative sludge samples through different sieves
(from 0.25 to C2.0 mm). To measure the content of c-
Al2O3 NP in sludge samples, digestion with a mixture
of HCl:HNO3:HF (2:2:1) was performed as follows:
(1) in effluent samples from both reactors: 5 mL of
water were mixed with 5 mL of acids solution; (2) in
granules collected from both reactors: a defined
portion of sample (*0.5 g) was suspended in 10 mL
of acids solution. Both water and granular sludge
samples were maintained in a shaker until digestion
was completed. Finally, filtered samples were collect-
ed to determine the concentration of dissolved Al3? by
Inductively Coupled Plasma (ICP-AES) (Varian 730
series, Palo Alto, CA, USA) under the conditions
described by Pat-Espadas et al. (2014).
Reductive decolorization of RR2 in UASB
reactors
After the granulation process was completed, both
reactors (reactor amended with HS and control reactor
without HS) were tested for their capacity to achieve
the reductive decolorization of the model azo com-
pound, RR2. A HRT of 12 h was established in both
reactors throughout the experiments and different
concentrations of RR2 (40–400 mg/L) were supplied
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Biodegradation (2015) 26:289–298
in both reactors. Glucose (1 g COD/L) was used as
electron donor during these experiments with the same
basal medium described above, which was adjusted to
a pH value of 7.0 ± 0.1 throughout the whole
experimental period. Both reactors were operated at
room temperature (20–28 °C). The COD removal and
decolorization efficiency, as well as the production of
aniline (product derived from RR2 reduction), were
determined using previously established analytical
techniques (Martı́nez et al. 2013a).
Results
Adsorption of HS on c-Al2O3 NP
The capacity of c-Al2O3 NP to adsorb HS and the
surface charge distribution of this material were deter-
mined. Surface charge distribution of c-Al2O3 NP at
different pH values is given in Fig. 2. While increasing
the pH value, a concomitant decrease in surface charge
of c-Al2O3 NP occurred. The point of zero charge of c-
Al2O3 NP was 8.1, which implies that positive charges
prevail on their surface below this pH value and
negative charges predominated above this value. Thus,
according to surface charge distribution, an appropriate
pH value for the adsorption of HS on c-Al2O3 NP was 4,
since sulfonated leonardite is negatively charged at this
pH value due to the presence of sulfonated groups
(pKa1 = 1.76 and pKa2 = 7.2, Tartar and Garretson
1941). In this context, adsorption assays revealed an
adsorption capacity of HS on c-Al2O3 NP of *165 mg
TOC/g. After six desorption cycles with basal medium
(pH 7.0 ± 0.1), the adsorption capacity became stable
at 89.5 mg TOC/L (Fig. 3). The adsorbed amount is
higher than that obtained by Alvarez et al. (2012), who
immobilized fulvic acids on c-Al2O3 (12 mg TOC/g),
but in that study alumina particles had an average size
[63 lm, whereas in the present study the size of the
alumina NP was between 20 and 50 nm.
Some studies have reported different adsorption
mechanisms of natural organic matter on mineral
surfaces: anion sorption, ligand exchange, protona-
tion, hydrogen bonding, cation bridging, and physical
adsorption (Yang et al. 2009). In the present study,
immobilization was conducted at pH 4 at which c-
Al2O3 NP are positively charged while sulfonated
groups in HS are negatively charged. Thus, the
obtained results suggest that immobilization of HS
Biodegradation (2015) 26:289–298 293

0.9 amended with HS and control reactor lacking HS,

respectively (Fig. 4a). Figure 4 also shows that the
0.7 pHPZC = 8.1 effluent concentrations of VSS and c-Al2O3 (after
Ions released (mmol/g)

samples digestion with acids, representing NP wash-

0.5
out) decreased over time when continuous mode was
0.3
established. During the first 25 days of operation,
washout of VSS decreased from *275 to *100 mg/
0.1 L. Later, the effluent concentration of VSS was
stabilized at around 50 mg/L in both reactors
-0.1 (Fig. 4b). On the other hand, the initial content of c-
Al2O3 in the effluent for reactor amended with HS and
-0.3 for control reactor lacking HS was 39 and 35 mg/L,
respectively. The concentration of c-Al2O3 gradually
-0.5
decreased to B1.0 mg/L and it was undetectable after
0 2 4 6 8 10 12
60 days of continuous operation (Fig. 4c). In order to
pH
confirm that c-Al2O3 was no longer released from both
Fig. 2 Surface charge distribution of c-Al2O3 NP. Data UASB reactors, its concentration was determined after
obtained at constant ionic strength under a N2 atmosphere and 1 year of operation. Results confirmed that alumina
calculated with a mass balance based on pH change according to was not present in the effluent of both reactors. Thus,
Alvarez et al. (2012). pHPZC as point of zero charge
the quantified amount of c-Al2O3 washed out was
5.1 g (corresponding to *20 % of NP initially added).
180 Speciation analysis (Fig. 5) revealed that alumina NP
Adsorption capacity (mg TOC/g)

165.0

160 are in solid state at the pH of operation in both reactors,

140
which confirms that c-Al2O3 NP are stable once
114.7
immobilized by the granulation process. The mea-
120
104.2
99.7 96.8
sured pH value in the effluent of both reactors was
100 93.2
89.5
7.2 ± 0.15 throughout the whole granulation process.
80
60 Characterization of granules
40
20 Once operational parameters were stable after ap-
proximately 1 year of continuous operation, granular
0
Initial 1 2 3 4 5 6 biomass samples were extracted from each reactor for
adsorption Desorption cycles their characterization. Figure 6a shows size distribu-
value
tion (by weight) of produced granules from both
Fig. 3 Adsorption capacity of HS on c-Al2O3 NP after reactors, which was different between them. While
desorption cycles with basal medium (pH 7.2). Reported data 73 % of granules from reactor amended with HS were
represent average from triplicate determinations and the of C0.5 to B2.0 mm in average, only 45 % of granules
standard deviation was less than 5 % in all cases
from the control reactor operated in the absence of HS
were found in the same range of size. In the control
on c-Al2O3 is principally due to electrostatic interac- reactor, 45 % of granules were in the range of
tion. However, it is possible that other mechanisms 0.25–0.5 mm. Thus, these results indicated that gran-
might also be involved. ules formed in the presence of HS were larger than
those produced in the control reactor lacking HS.
Performance of UASB reactors during granulation As shown in Fig. 6b, c-Al2O3 content in produced
process granules was slightly different depending on size of
granules and on whether c-Al2O3 NP were coated with
COD removal efficiencies at steady state were HS or not. The average content was 15.07 and
*95.5 % ± 2.2 and *96.7 % ± 2.0 for reactor 15.93 mg of c-Al2O3/g in sludge samples from control

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294 Biodegradation (2015) 26:289–298

a b Fig. 4 Performance of UASB reactors during granulation

process with c-Al2O3 modified (filled circle) and unmodified
(unfilled square) with HS. a COD removal, b and c indicates the
100 concentration of VSS and c-Al2O3 NP, respectively, in the
effluent. Reactors were operated at a hydraulic residence time of
COD Removal (%)

36 h and were fed with an enriched basal medium (pH 7.2)

95 including glucose (2 g COD/L), peptone (1 g COD/L), and a
mixture of propionate, butyrate, and acetate (1 g COD/L) as
substrates
90

85

80
0 25 50 75 100 125 150

b
300

250
Effluent VSS (mg/L)

200

150

100

50

0
0 25 50 75 100 125 150

c
40
Effluent -Al2O3 (mg/L)

30
Fig. 5 Speciation diagram of c-Al2O3 at different pH values
(Takeno 2005)
20

(89.5 mg TOC/g), the average content of HS in

10 granules formed in reactor amended with HS was
1.42 mg TOC/g granules or 14.79 mg TOC/g VSS,
which corresponds to a concentration of *369.7 mg
0
0 20 40 60 80 TOC/L in the UASB reactor amended with HS after
Time (d) the granulation process was completed.

Decolorization of RR2 in UASB reactors

reactor without HS and in samples derived from
reactor amended with HS, respectively, suggesting
appropriate agglomeration of biomass and c-Al2O3
NP in both reactors. According to these data and
considering the adsorption capacity of c-Al2O3 NP
UASB reactors in which granulation processes were
carried out, in the presence and in the absence of HS,
were evaluated for their capacity to achieve the
reductive decolorization of RR2. Both reactors

123
Biodegradation (2015) 26:289–298
Fig. 6 Size (a) and content of c-Al2O3 (b) in granules collected
from UASB reactors after one year of operation under the
experimental conditions described in Fig. 4. Data represent
average from triplicate determinations and the standard
deviation was \7 % in all cases
showed high COD removal efficiencies ([95 %)
under steady state conditions, before addition of the
azo dye, indicating that the presence of c-Al2O3 NP
(regardless if coated with HS or not) did not affect the
methanogenic activity of the anaerobic sludge. Nev-
ertheless, increase in the concentration of RR2 caused
inhibitory effects on the methanogenic consortia as
evidenced in a gradual drop in COD removal in both
reactors, although bioreactor including granules en-
riched with HS always showed a better performance as
compared to the control reactor operated in the
absence of HS (Table 1). The pH value in the effluent
of both reactors was 7.2 ± 0.16 throughout the whole
experimental period.
Regarding the reduction of RR2, it was observed
that the bioreactor enriched with HS achieved more
stable reductive decolorization of the azo dye and
showed higher decolorization capacity as compared to
295
the control bioreactor (Fig. 7; Table 1). A more
pronounced difference between both reactors was
observed during the first two periods of operation in
which concentrations of RR2 of 40 and 100 mg/L were
tested. Statistical analysis confirmed a significant
difference in the performance of both reactors during
the first two operational periods (Table 1). The biore-
actor operated in the absence of HS eventually became
stable after perturbations caused by increased concen-
trations of RR2 and reached almost the same color
removal efficiencies as the bioreactor enriched with
HS. The reduction of RR2 was confirmed by quanti-
fying aniline (one of the aromatic amines derived from
RR2 azo cleavage) in both reactors (data not shown).
Adsorption isotherms of RR2 on c-Al2O3 NP
(uncoated and coated with HS) were also assessed to
further elucidate the removal mechanisms of RR2
removal in both reactors. These adsorption isotherms
revealed a maximum adsorption capacity of 345.3 and
1079.9 mg RR2/g for uncoated and HS-coated c-
Al2O3 NP, respectively. Considering the amount of c-
Al2O3 NP (with and without HS) supplied in both
reactors (30 g), saturation of these materials with RR2
would have occurred during the first 12 days of
operation at a RR2 concentration of 40 mg/L. There-
fore, reduction rather than adsorption is postulated as
the main mechanism of RR2 removal in both reactors.
Discussion
The aim of the present study was to evaluate the co-
immobilization of HS supported on c-Al2O3 NP and
HRM present in an anaerobic consortium by a
granulation process developed in a UASB reactor.
The HS-enriched consortium obtained by the granula-
tion process was then evaluated for its capacity to
achieve the reductive decolorization of the recalcitrant
azo dye RR2. Several strategies have previously been
reported documenting the immobilization of HS or
model RM to increase the reductive biotransformation
of different contaminants (for a review see Martı́nez
et al. 2013b). However, only a few attempts have been
reported on the simultaneous immobilization of RM
and microorganisms responsible for their reduction.
For instance, co-immobilization of anthraquinone
moieties and quinone-reducing microorganisms could
be accomplished by entrapment in calcium alginate.
The co-immobilized beads exhibited good catalytic
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296 Biodegradation (2015) 26:289–298

Table 1 Performance of UASB reactors during the reductive decolorization of reactive red 2 (RR2)
RR2 concentration (mg/L) COD removal efficiency (%) Decolorization efficiency (%)
With HS Without HS With HS Without HS

40 73.7 ± 8.5a 70.5 ± 4.9a 67.6 ± 17a 54.3 ± 16a

a
100 65.9 ± 9.9 57.6 ± 12 81.6 ± 11.7 60.6 ± 14.9a
200 66.9 ± 8.3a 54.4 ± 6.7a 98.3 ± 1.5 94.9 ± 1.5
400 36.3 ± 4.9 32.3 ± 6.9 97.8 ± 1.3 94.5 ± 5.4
Reactors operated at a hydraulic residence time of 12 h with glucose (1 g/L) as electron donor. Influent pH was 7.2 for both reactors,
which were incubated at room temperature (20–28 °C)
COD chemical oxygen demand, HS humic substances
a
Statistical difference (p = 0.05) was found between both reactors under these experimental conditions

recently, a composite integrated by reduced graphene

RR2 concentration (mg/L)
oxide (RGO) and activated sludge was synthesized
40 100 200 400
and applied for the reduction of nitrobenzene. The
Decolorization efficiency (%)

100 presence of RGO resulted in enhanced nitrobenzene

80
60
40
20
0 quinone-reducing (Cervantes et al. 2003), have been
0 20 40 60
Time (d)
Fig. 7 Decolorization efficiencies by UASB reactors contain-
ing granules produced with c-Al2O3 NP modified (filled circle)
and unmodified (unfilled square) with HS at different concen-
trations of RR2. Reactors were operated at a hydraulic residence
time of 12 h with glucose (1 g/L) as electron donor. Influent pH
was 7.2 for both reactors, which were incubated at room
temperature (20–28 °C)
activity and increased the decolorization rate of
different azo dyes (Su et al. 2009). Terylene fiber also
served both as supporting material for different redox
mediators and as a bio-carrier. Decolorization rate of
10 azo dyes was enhanced up to threefold by the
functionalized bio-carrier (Guo et al. 2010). Ceramic
material was also used to co-immobilize an-
thraquinone-2-sulphonate and quinone-reducing mi-
croorganisms and the results revealed that the
synthesized composite increased up to 6.4-fold the
decolorization rate of different azo dyes under high
salinity concentrations (Yuan et al. 2012). More
biotransformation by activated sludge, and the highest
removal efficiency of nitrobenzene was observed with
initial RGO (3–5 lm) to sludge ratio of 0.075 (w/w)
(Wang et al. 2014).
Microbial consortia with different metabolic ca-
pacities, such as methanogenic (Lettinga et al. 1980),
sulfate-reducing, halorespiring (Guerrero-Barajas
et al. 2014), denitrifying (Cervantes et al. 1998), and
80 100 120 140
enriched by granulation processes in UASB reactors.
However, to our knowledge no previous studies have
been performed to co-immobilize HS and HRM by
this technique. This-co-immobilization strategy is
attractive due to a number of reasons: (1) granulation
allows the enrichment of several distinct types of
microorganisms, which boosts the metabolic capacity
of the consortium for degrading recalcitrant pollu-
tants; (2) it is a simple and cheap immobilization
technique, which allows a relatively fast co-immobi-
lization of HS and HRM; (3) the supporting material
employed to immobilized HS (c-Al2O3 NP) is an
abundant and cheaply available source, which did not
show negative effects on the methanogenic activity of
the consortium evaluated; (4) once immobilized, HS
appeared to remain in the UASB system, which
warrant the long term applicability of the redox-
mediating material; (5) compared to synthetic RM
previously immobilized in different studies, HS rep-
resent the most abundant and cheaply available redox-
mediating material.

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Biodegradation (2015) 26:289–298
Previous studies documented that c-Al2O3 NP
coated with HS can interact better with HRM as
compared to uncoated c-Al2O3 NP (Alvarez and
Cervantes 2012). This could explain why NP modified
with HS in their surface promoted a faster granulation
and production of granules with a bigger size
(Fig. 6a). The HS-enriched granules produced by this
technique were stable during the long-term ex-
periments, as evidenced by the absence of aluminum
in the effluent of the UASB reactors. Furthermore, the
immobilized HS effectively enhanced the reductive
decolorization of RR2, which promoted a faster
adaptation of the consortium to the high azo dye
loading rates applied, as compared to the control
reactor operated in the absence of HS. However, the
control UASB reactor could also reach similar decol-
orization efficiency at the end of the experiments
probably due to gradual adaptation to the recalcitrant
azo dye evaluated, although with lower COD removal.
Several lines of evidence indicate that immobilized
HS remained in the amended UASB reactor once the
granulation process was completed. Firstly, aluminum
was not detected in the effluent of both reactors
suggesting that c-Al2O3 NP (the supporting material)
was stable during the long-term operational period.
Furthermore, Fig. 5 clearly indicates that alumina NP
are in solid state at the pH of operation in both reactors
(7.2), which confirms that c-Al2O3 NP are stable once
immobilized by the granulation process. Moreover,
adsorption isotherms of RR2 in the supporting mate-
rial (uncoated and HS-coated c-Al2O3 NP) revealed
that the limited adsorption capacity would have been
overcome during the first 12 days of operation at a
RR2 concentration of 40 mg/L. Additionally, aniline
was confirmed as one of the products derived from
RR2 cleavage. The collected evidence strongly sug-
gests that HS supported on c-Al2O3 NP remain in the
amended UASB reactor effectively increasing the
reduction of RR2.
Conclusions
Co-immobilization of HRM and HS supported on c-
Al2O3 NP was achieved in the present study by a
granulation process in a UASB reactor. Larger granules
were produced using NP coated with HS as compared
with uncoated NP. HS-enriched granules achieved
generally higher decolorization and COD removal
297
efficiencies, as compared to the control reactor operated
in the absence of HS, in long term operation and
applying high concentrations of the recalcitrant azo dye
RR2. This co-immobilizing technique could be attrac-
tive for its application in anaerobic wastewater treat-
ment systems for the reductive biotransformation of
several contaminants, such as nitroaromatics, poly-
halogenated compounds, metalloids, among others.
Acknowledgments This study was financially supported by
the Council of Science and Technology of Mexico (Grant SEP-
CONACYT 155656) and by the Marcos Moshinsky Foundation.
We thank the technical assistance of Dulce Partida, Guillermo
Vidriales, Carmen Rocha and Héctor Amézquita.
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