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Appl Microbiol Biotechnol

DOI 10.1007/s00253-011-3759-4

ENVIRONMENTAL BIOTECHNOLOGY

Assessing the impact of alumina nanoparticles

in an anaerobic consortium: methanogenic and humus
reducing activity
Luis H. Alvarez & Francisco J. Cervantes

Received: 6 October 2011 / Revised: 9 November 2011 / Accepted: 10 November 2011

# Springer-Verlag 2011

Abstract The impact of γ-Al2O3 nanoparticles (NP) on regardless if HA was HAImm or HASus. The present study
specific methanogenic activity (SMA) and humus reducing provides a clear demonstration that HA immobilized in γ-
activity (HRA) in an anaerobic consortium was evaluated. Al2O3 NP are effective terminal electron acceptor for mi-
SMA in sludge incubations without γ-Al2O3 was always crobial respiration and suggests that HA could mitigate the
higher compared with those performed in the presence of toxicological effects of metal oxide NP on anaerobic
100 g/L of γ-Al2O3. Nevertheless, the SMA in incubations microorganisms.
with γ-Al2O3 was not completely inhibited, indicating that
some methanogenic microorganisms were physiologically Keywords Anaerobic consortium . Nanoparticles . Humic
active even in the presence of γ-Al2O3 NP during the substances . Humus reduction . Methanogenesis
incubation period (~400 h). SMA and HRA of the anaerobic
consortium were also conducted in the presence of γ-Al2O3
NP coated with humic acids (HA). Microbial HA reduction Introduction
occurred 3.7-fold faster using HA immobilized on γ-Al2O3
NP (HAImm), compared with the control with suspended HA In the last years, the use of materials at nanometric scale (1–
(HASus). Furthermore, immobilized HA decreased the toxi- 100 nm) has allowed the development of nanotechnology.
cological effects of γ-Al2O3 NP on methanogenesis. Scan- These nanomaterials are also usually called nanoparticles
ning electron microscopy (SEM) images revealed cell (NP) and are extensively used for different purposes and
membrane damage in those sludge incubations exposed to applications. For instance, areas such as electronics, materi-
uncoated γ-Al2O3 NP. In contrast, cell damage was not als engineering, food, transportation, cosmetic, energy,
observed in incubations with HA-coated γ-Al2O3 NP. Meth- pharmaceutical, biomedical, automotive, agriculture, fish-
anogenesis out-competed microbial humus reduction ing, manufacturing, security, consumers goods, and envi-
ronmental have introduced the use of NP in their processes
(Biswas and Wu 2005; Sinha et al. 2006). NP offer a great
Electronic supplementary material The online version of this article number of advantages compared with their bulk forms
(doi:10.1007/s00253-011-3759-4) contains supplementary material, (Theron et al. 2008; Tenne 2006; Zhang 2003), and their
which is available to authorized users. use is constantly increasing. Nevertheless, little is known
L. H. Alvarez (*) : F. J. Cervantes about the role of NP on the fate, transport, transformation,
División de Ciencias Ambientales, Instituto Potosino and bioavailability of environmentally relevant substances,
de Investigación Científica y Tecnológica (IPICyT),
and about their toxicity on living organisms in nature and in
Camino a la Presa San José 2055, Col. Lomas 4ª. Sección,
San Luis Potosí, SLP 78216, México environmental bioprocesses.
e-mail: [email protected] The toxicity of several metal oxide NP has been evaluated
on different pure cultures, including Bacillus subtilis, Escher-
F. J. Cervantes
ichia coli, Shewanella oneidensis, Cupriavididus metallidur-
Department of Biotechnology,
Norwegian University of Science and Technology (NTNU), ans, Streptococcus aureus, and Pseudomonas fluorescens
7491 Trondheim, Norway (Jiang et al. 2009; Sadiq et al. 2009; Pelletier et al. 2010;
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Appl Microbiol Biotechnol

Simon-Deckers et al. 2009; Baek and An 2011). However, Al2O3 NP coated with humic acids (γ-Al2O3-HA). SMA
there are few reports showing the toxicological effects and HRA experiments were conducted using a methano-
of NP on anaerobic sludge, as those obtained in upflow genic consortium previously reported to have the ability to
anaerobic sludge bed (UASB) reactors. For example, use HS as TEA.
Luna-del Risco et al. (2011) and Nyberg et al. (2008) evalu-
ated the effect of metal oxides (CuO and ZnO) and fullerenes,
respectively, on the production of biogas in anaerobic sludge.
Materials and methods
The toxicity of NP on bacteria can be attributed to their
chemical composition, by the fact that they can release toxic
Humic acids and nanoparticles
ions (Brunner et al. 2006), and particle surface catalyzed
reactions (Limbach et al. 2007), producing reactive oxygen
HA were extracted from soil of a coal mine, according to the
species, or due to stress caused by their physical character-
protocol described by the International Humic Substances
istics as surface, size, and shape.
Society (IHSS) (http://ihss.gatech.edu/ihss2/index.html).
Introduction of NP to ecosystems may also have a sig-
The yield of extraction was approximately 35% (unpuri-
nificant impact in relevant environmental processes, includ-
fied), and its content (%) of carbon (59.36), hydrogen
ing biotic and abiotic systems. In aquatic environments, the
(2.8), nitrogen (1.22), sulfur (0.41), and oxygen (29.13)
surface of released metal oxide NP can become negatively
was similar to Standard Leonardite (1S104H) reported by
charged when natural organic matter (e.g., humic substances
IHSS. The electron carrying capacity (ECC) of extracted
(HS)) is adsorbed, altering the fate and transport of both NP
HA was evaluated by chemical (Ratasuk and Nanny 2007)
and HS (Yang et al. 2009). On the other hand, it is well
and biological (with Geobacter sulfurreducens) methods,
known that microorganisms can use dissolved HS as termi-
using the ferrozine technique described by Lovley et al.
nal electron acceptor (TEA) for the anaerobic oxidation of
(1996). The obtained ECC values were 392.4±14 micro-
organic substrates (Lovley et al. 1996), and reduced HS can
electron equivalents (μEq)/L and 160.2±17 μEq/L,
rapidly transfer electrons to reduce Fe(III) and Mn(IV)
respectively.
oxides (Lovley et al. 1996; Nevin and Lovley 2000), acting
γ-Al2O3 NP was purchased from Inframat Advanced
as redox mediators (RM). In addition, the ability of micro-
Materials (Manchester, CT, USA). According to the suppli-
organisms to reduce solid-phase HS has recently been
er, this material has a purity of 99.99%, average particle size
reported (Roden et al. 2010). These authors showed that
of 20–50 nm, and surface area (BET) >150 m2/g. γ-Al2O3
solid-phase HS significantly accelerated Fe(III) reduction
was used as received without previous modification. Surface
by shuttling electrons from bacteria to metal oxide surfaces,
charge distribution of γ-Al2O3 NP was determined using a
representing a new mechanism for extracellular electron
mass balance based on pH change, as described by Alvarez
transfer in sediments. Nevertheless, to our knowledge, the
et al. (2010).
capacity of HS adsorbed on metal oxide NP to act as TEA
and RM has never been evaluated. However, previous stud-
ies have reported the immobilization of quinoid model com- Anaerobic consortium
pounds (main redox functional groups in HS) on NP of Al
(OH)3, which were demonstrated as effective solid-phase The anaerobic consortium studied was granular sludge col-
RM accelerating the reductive decolorization of an azo dye lected from a full-scale UASB reactor treating effluents
by anaerobic consortia (Alvarez et al. 2010). This report was from a factory of candies (San Luis Potosí, Mexico). The
the first demonstration that humic analogous immobilized sludge, which contains 6% of volatile suspended solids
on metal oxide NP can act as RM. As mentioned by Van der (VSS), was acclimated in a lab-scale UASB reactor (1.5 l)
Zee and Cervantes (2009), immobilization of RM is a pre- operated at a hydraulic residence time of 12 h and with an
requisite for their use in anaerobic reactors in order to treat organic loading rate of 1 kg chemical oxygen demand
wastewaters containing electron-accepting contaminants. (COD)/m3 d. Glucose was used as a sole energy source for
The results obtained from this study provide important the UASB reactor, which showed stable efficiencies in terms
evidence about the potential to use immobilized HS (on of COD removal (>90%) during steady-state conditions.
NP) as a solid-phase RM. Nevertheless, the impacts of NP The basal medium used during acclimatization was accord-
on anaerobic consortium need to be considered prior to ing to the following composition (g/L): NaHCO3 (1.68),
using them in wastewater treatment systems. NH4Cl (0.3), KH2PO4 (0.2), MgCl2•6H2O (0.03), CaCl2
The two main goals of this study are (1) to evaluate the (0.1), and 1 mL/L of trace element solution. The trace
inhibitory effects of NP of alumina (γ-Al2O3) on specific element solution contained (mg/L) FeCl2•4H2O, (2,000);
methanogenic activity (SMA) and (2) to evaluate the humus H3BO3, (50); ZnCl2, (50); CuCl2•2H2O,(38); MnCl2•4H2O
reducing activity (HRA) and SMA in the presence of γ- (500); (NH4)6Mo7O24•4H2O, (50); AlCl3•6H2O, (90);
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CoCl2•6H2O, (2,000); NiCl2•6H2O, (92); Na2SeO•5H2O, inoculated with 2 g VSS/L for SMA and 1 g VSS/L for
(162); EDTA, (1,000); and 1 mL/L of HCl (36%). HRA with anaerobic sludge previously disintegrated with
sterile needles (Microlance 3, 25 G5/8, 0.5×16 mm). The
Adsorption of HA on γ-Al2O3 NP controls for SMA were a control in the absence of γ-Al2O3
and an endogenous control incubated without external elec-
The capacity of NP of γ-Al2O3 to adsorb HA was deter- tron donor. The HRA was conducted with sacrificial bottles,
mined by adsorption isotherms using the batch equilibrium including controls with suspended HA (not attached to NP
technique. Different concentrations of HA (100 to 5,800-mg of γ-Al2O3). All experiments were carried out by triplicate
total organic carbon (TOC)/L) were prepared using deion- and were incubated at 27 °C and 160 rpm. Incubation
ized water and then adjusting the pH to 4 with 0.1-M HCl. conditions were selected in order to resemble realistic sce-
Then, 0.2 g of γ-Al2O3 and 10 mL of HA solution (previ- narios commonly found in anaerobic digesters. For instance,
ously filtered with 0.22 μm) were mixed in polypropylene pH and temperature values correspond to those commonly
vials. The vials were placed on a shaker (160 rpm at 27 °C) prevailing in anaerobic wastewater treatment facilities
for three days until the equilibrium was accomplished. After (Monroy et al. 2000). No stabilizers were used to prevent
centrifugation (3,200 rpm, 10 min), the supernatant was NP agglomeration in sludge incubations supplied with NP
analyzed in order to determine the equilibrium concentration of γ-Al2O3 so that exposure of the microbial consortium to
of HA in terms of TOC, and the adsorption capacity was NP could be performed at conditions expected in anaerobic
determined by a mass balance. digesters. Agglomeration of NP can prevent their washout
According to the data obtained from adsorption iso- from bioreactors and could be caused by the presence of
therms, 40 g of γ-Al2O3 was prepared in order to evaluate organic compounds and pH variations (Limbach et al.
SMA and HRA in the presence of immobilized HA. After 2008), which is desirable in the context of the present study.
this procedure, the material was washed with deionized SMA and HRA were determined on the maximum slope
water at pH 4 and dried in an oven at 40–45 °C. Then, in observed on linear regressions considering at least three
order to evaluate the adsorption strength of HA on NP, the sampling points. The coefficient of determination (R2) was
saturated material was exposed several times to the basal higher than 0.95 for most of the activities calculated.
medium described previously. Finally, the material was
characterized by Fourier transform infrared (FTIR) spec- Analytical methods
trometry and energy dispersive X ray (EDX) analysis,
according to previously described methods (Cervantes et Concentration of produced methane was measured by
al. 2010). injecting 100 μL with a gas-tight syringe (Hamilton Co.,
Reno, NV, USA) in a gas chromatograph (GC, 6890 N
SMA and HRA of anaerobic sludge Network GC System, Agilent Technologies, Waldbronn,
Germany) equipped with a thermal conductivity detector
Activity assays of sludge incubations in the presence of γ- and a column Hayesep D (Alltech, Deerfield, Illinois,
Al2O3 were conducted in serum glass bottles of 120 and USA) with the following dimensions: 10'×1/8"×0.085".
60 mL, with a working volume of 50 and 10 mL for SMA Temperatures of the injection port, oven, and the detector
and HRA, respectively. The concentration of NaHCO3 in were 250, 60, and 250 °C, respectively. Nitrogen was used
the basal medium was changed to 5,000 mg/L to create the as carrier gas, with a flow rate of 12 mL/min. HA reduction
proper buffer capacity (pH 7.2). Glucose, acetate, and hy- of immobilized and suspended samples was measured in an
drogen at 1 g COD/L were used as model substrates during anaerobic chamber with an atmosphere composed of N2–H2
SMA assays, and different concentrations (25, 50, 75, and (95%/5%) and using the ferrozine method described by
100 g/L) of γ-Al2O3 were also tested. On the other hand, Lovley et al. (1996). Briefly, Fe(III) citrate was mixed with
experiments with 100 g/L of γ-Al2O3 uncoated (only for the sacrificial samples to obtain a final concentration of
SMA) and coated with HA (for HRA and SMA) were also 10 mM and allowed to react for 15 min. Then, equal volume
conducted using 7-g COD/L of acetate in order to evaluate of 0.5-M HCl was added to the sample, and an aliquot was
microbial activity under conditions not limited by substrate. taken for spectrophotometric (562 nm) determination of Fe
The HA concentration used in HRA and SMA either in (II). The Fe(II) concentration was used to calculate the
soluble or immobilized form was ~3,750 mg TOC/L, which number of equivalents of electrons transferred from HA
was established according to the adsorption capacity samples to Fe(III). Results were corrected for a control
achieved on γ-Al2O3 NP. After sealing the bottles with untreated with Fe(III) citrate.
rubber stoppers and aluminum caps, anaerobic conditions TOC concentrations of HA were measured in a TOC-
were established by saturating the headspace with a gas meter (Shimadzu Co. Model TOC-VCSN), using previously
mixture of N2/CO2 (80%/20%). Then, the bottles were filtered samples (0.22 μm). VSS and COD concentrations
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were determined according to Standard Methods (APHA On the other hand, after the material was exposed to
1985). Glucose and acetate concentrations were measured seven desorption cycles with basal medium, the adsorp-
using a capillary electrophoresis ion analyzer (Agilent tion capacity value decreased 53.5% with respect to the
G1600A, Waldbronn, Germany) equipped with a capillary initial value. This decrement can be explained because
column (50 μm ID×72 cm) under the following conditions: the pH value (7.0) of the basal medium changes the
temperature, 15 °C; current, 45 μA; and negative polarity. A surface charge of γ-Al2O3 particles (SD, Fig. S1), which
buffer composed of 25 mM of 2,6-pyridinecarboxylic acid promotes a decrease of attraction forces, and, consequently,
and 5 mM of hexadecy-trimethyl ammonium bromide (pH the desorption of HA. However, after the fourth desorption
12) was used as eluant. Samples were also filtered (0.22 μm) cycle, the material becomes stable (SD, Fig. S3). The FTIR-
before processing the electrophoresis analyses. Finally, dis- ATR spectra confirmed that quinone moieties remained avail-
solved Al was measured by ICP-OES (Varian 730 series, able once HA were adsorbed on γ-Al2O3 particles (Fig. 1).
Palo Alto, CA, USA). Certainly, the spectral signal of the carbonyl (C0O) groups at
1,700–1,630 cm-1 associated to quinone groups (Salavagione
Scanning electron microscopy et al. 2004) appeared on the surface of γ-Al2O3 particles in
which HA were previously immobilized. These function-
In order to observe the interactions between NP and the al groups were also detected in the spectra of HA, but
anaerobic consortium studied, incubations with 2 g/L of γ- not in γ-Al2O3 lacking HA. In addition, EDX analysis
Al2O3 were prepared. One drop of suspension was placed on confirmed adsorption of HA on γ-Al2O3 because an
the top of a pin, and then, the sample was dried at room increment in the content of C and N was detected in
temperature in 12 h. Finally, the sample was covered with a the surface of HA-amended γ-Al2O3, compared with their
thin layer of gold to make their surface conductive. counterpart lacking HA; also, a decrease in the content of Al
and O occurred once HA were adsorbed on this material (data
not shown).
Results
SMA of anaerobic sludge in the presence of γ-Al2O3 NP
Immobilization of HA on γ-Al2O3 NP
Methane production was observed under all concentrations
Surface charge distribution of NP of γ-Al2O3 at different pH of γ-Al2O3 NP and with the three substrates tested (SD,
values is given in the supplementary data (SD, Fig. S1). The Table S1 and Fig. S4). In all cases, SMA was higher in the
pH point of zero charge (pHPZC) of γ-Al2O3 was 8.1; this absence of NP and was consistently affected with the incre-
indicates that the surface of γ-Al2O3 is positively charged ment of γ-Al2O3 concentration, decreasing 62.2% and
below this pH value and negatively charged at pH values 84.6% at 100 g/L of γ-Al2O3 when glucose and acetate
higher than 8.1. According to surface charge distribution, an
appropriate value for the adsorption of HA on γ-Al2O3 is 4
(Yang et al. 2009).
γ-Al2O3
Adsorption isotherm indicated that the maximum adsorp-
tion capacity (Qmax) of HA on γ-Al2O3 was ~93 mg TOC/g
(SD, Fig. S2). Qmax value was achieved using a concentra-
Transmittance (%)

tion of HA of ~2,300 mg TOC/L, with a mass/volume ratio Humic acids

of 20. The isotherm showed an initial slope for the lower
concentrations of HA tested, and then, a plateau and the
Qmax value were observed. The adsorption followed a typ-
ical Langmuir isotherm, also observed in other studies γ-Al2O3 + humic acids
(Yang et al. 2009; Kang and Xing 2008; Wang and Xing
2005). Electrostatic attraction could explain the adsorption
of HA by γ-Al2O3 NP. According to the surface charge
distribution (SD, Fig. S1), the positive charges on the sur- 1700-1630 cm-1
face of γ-Al2O3 (at pH 4) and the negative charges of HA, quinone moieties

due to unprotonated groups (e.g., carboxyl, phenolic), pro- 2400 2100 1800 1500 1200

moted an electrostatic attraction (Tombacz et al. 2004). Wavenumber (cm -1)

Once the HA are adsorbed on γ-Al2O3, the surface of this Fig. 1 FTIR spectra of HA, γ-Al2O3, and γ-Al2O3 coated with HA.
NP becomes less positively charged as reported by Yang et Arrows indicate the spectral signals for quinone (1,700–1,630 cm-1)
al. (2009). group
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were supplied as electron donors, respectively, compared Table 1 Impact of γ-Al2O3 NP on SMA and HRA by anaerobic
sludge (values are given in micro-electron equivalents (μEq)/g VSS h)a
with their corresponding controls lacking NP. Nevertheless,
the SMA observed in incubations with hydrogen was less Condition SMA
affected, decreasing only 14.2% compared to those incuba-
tions lacking γ-Al2O3 NP. The decrease in SMA could be 0–167 h 168–287 h 288–385 h
associated to inhibitory effects caused by exposure of this
Without γ-Al2O3 967±9.5 1,754±112 1,965±285
consortium to γ-Al2O3 NP, but also to substrate limitation
With γ-Al2O3 518±66 541±83 559±49
due to substrate adsorption on NP, especially in incubations
SMA HRA
with glucose and acetate. Electrophoretic analysis revealed
0–96 h 97–166 h 0–166 h
that there were no glucose and acetate remaining in those
HASus without γ-Al2O3 1,759±150 2,678±73 6.0±0.28
incubations with γ-Al2O3 at the end of the incubation period,
HAImm on γ-Al2O3 768±26 1,084±34 22.3±4.1
but the amount of produced methane (recovery ranging
between 24% and 93%) did not agree with the total consump- a
Experimental conditions: acetate added, 7.03±0.07 g COD/L; acetate
tion of substrate, suggesting that these substrates were partly concentration after pre-incubation period (after adsorption on γ-Al2O3
adsorbed on γ-Al2O3, thus limiting the availability of electron NP), 6.51±0.17 g COD/L; γ-Al2O3 concentration, 100 g/L. The results
are the mean of triplicate ± standard deviation
donor during SMA tests. Further experiments revealed that the
adsorption capacity of acetate on γ-Al2O3 is 1.4 g COD/g; this
phenomenon could play an important role in affecting SMA,
which was not evident when a gaseous substrate like hydrogen them (mean of 539±14 μEq/g VSS h); this indicates that some
was used as electron donor. methanogenic microorganisms were physiologically active
SMA was also conducted using 100 g/L of γ-Al2O3 and even in the presence of γ-Al2O3 NP. These results clearly
high concentration of acetate in order to evaluate the confirm that γ-Al2O3 NP had a toxicological effect on some
activity under conditions not limited by substrate. Before methanogenic microorganisms.
inoculating with the anaerobic consortium, bottles were
preincubated for 24 h to achieve equilibrium of acetate by
adsorption on γ-Al2O3 NP. During this period, the concentra- SMA and HRA of anaerobic sludge in the presence of HA
tion of acetate decreased from 7.03±0.08 g COD/L to 6.5±
0.17 g COD/L. SMA was calculated for each phase during the SMA and HRA of the anaerobic consortium were conducted
incubation period; each phase represents the replacement of in the presence of 100 g/L of γ-Al2O3 NP coated with HA,
produced methane by saturating the headspace with a gas representing ~3.75 g TOC/L of immobilized HA (HAImm).
mixture of N2/CO2 (80%/20%) and then continuing with This concentration of HA was also provided in incubations
incubation. For almost 400 h, sludge incubations with γ- without γ-Al2O3, but in suspended form (HASus). The estab-
Al2O3 showed a stable methane production, but always lower lished concentrations of HA and γ-Al2O3 NP for these
than that obtained with the control lacking NP (Fig. 2 and incubations are very distant from what could be expected
Table 1). The stable methane production in the presence of γ- in natural ecosystems. However, they were intentionally
Al2O3 was evidenced by the values obtained of SMA during selected for two purposes: (1) to have quantifiable levels
the three phases of experiment, which are very close among to accurately measure the microbial reduction of HAImm and
HASus and (2) to establish HA concentrations demanded for
450
phase 1 phase 2 phase 3 redox-mediating reactions in anaerobic bioreactors since
HA immobilized in metal oxide NP have been proposed as
solid-phase redox mediator for the anaerobic biotransforma-
CH4 (m-Eq/L)

300 tion of priority pollutants (Alvarez et al. 2010).

After 166 h of incubation, HA reduction was 2.4-fold
higher using HAImm compared with the control with HASus
150 at the end of the incubation period (Fig. 4a). Conversely,
methane production was higher by 3.6-fold and 1.6-fold
during the first and second phases, respectively, when
0 HASus was provided as compared with incubations with
0 100 200 300 400
Time (h) HAImm (Fig. 4b). SMA and HRA obtained in sludge incu-
bations are given in Table 1. When HAImm was provided,
Fig. 2 Concentration of methane produced by anaerobic sludge using
acetate. (Empty circles) Experiment with 100 g/L of γ-Al2O3; (filled
SMA decreased 56% and 59% during the first and second
circles) control lacking γ-Al2O3. Values are given in milli-electron phases, respectively, compared with the treatment lacking
equivalents (m-Eq)/L NP, but with HASus. Nevertheless, HRA in incubation with
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Appl Microbiol Biotechnol

HAImm was 73% faster than the control with HASus was observed in those sludge incubations exposed to
(Table 1). uncoated γ-Al2O3 NP (Fig. 3a). The fact that cell damage
SMA values obtained in the presence of HA-coated γ- was not observed in incubations with HA-coated γ-Al2O3 NP
Al2O3 NP (e.g., HAImm) were higher than those obtained (Fig. 3b) suggests that HA mitigated the toxicological effects
with incubations using uncoated γ-Al2O3 (Table 1). These of γ-Al2O3 NP. On the other hand, interaction of NP with cell
results can be explained by the fact that adsorption of HA on membrane could be attributable to electrostatic attraction
the surface of γ-Al2O3 NP decreases their toxicological between positive charges of γ-Al2O3 NP and negative charges
effects. NP of γ-Al2O3 coated with HA could enhance Al on the membrane of bacteria. Charges are promoted because
(Al3+) sorption, thus decreasing also the toxicological the pH of the basal medium is 7.2, which is lower than the
effects of this ion. Previous studies showed that association pHPZC of γ-Al2O3 (SD, Fig. S1). Finally, Fig. 3b shows HA
(adsorption) of HA with zero-valent iron NP dramatically particles adsorbed on cell membrane, which was common
mitigated their toxicological effects on bacterial cultures only in those images from incubation with HA-coated γ-
(Chen et al. 2011). On the other hand, Fig. 4 and Table 1 Al2O3 NP. This phenomenon could explain why HRA was
show that methanogenesis was the preferable pathway over higher in the treatment with HAImm compared with sludge
HA reduction to oxidize acetate in both experiments with incubations supplied with HASus (Fig. 4a).
HAImm and HASus.

Interaction of γ-Al2O3 with anaerobic bacteria Discussion

Figure 3 shows the interaction between γ-Al2O3 NP and The aim of this study was to evaluate the impact of γ-Al2O3
bacteria. SEM images revealed aggregates of NP ranging NP in an anaerobic consortium through methanogenic and
100–1,000 nm in both coated (with HA) and uncoated γ- humus reducing activities. The adverse impact of γ-Al2O3
Al2O3, which could decrease their adverse impact on the NP on anaerobic consortium was evidenced by a lower
anaerobic consortium evaluated. Cell membrane damage methane production in comparison to the control lacking
γ-Al2O3 NP. Previously, Luna-del Risco et al. (2011) and
Nyberg et al. (2008) evaluated the effect of metal oxides
(CuO and ZnO) and fullerene, respectively, on methane
production by anaerobic consortia. In the case of metal
oxides, inhibition of methane production can partially be
attributed to the release of toxic ions (Cu and Zn) from NP,
as well as its size (Luna-del Risco et al. 2011). Nevertheless,
fullerene did not show adverse effects on an anaerobic
community because no changes in methane production were
observed (Nyberg et al. 2008). In the present study, inhibi-
tion of methane production in the anaerobic consortium
studied could be associated to the size of γ-Al2O3 NP. Jiang
et al. (2009) compared the toxicity of Al2O3 on bacteria at
micro- and nanoscale. These authors found that Al2O3 at
nanoscale showed higher toxicity, with a mortality rate of
57% to Bacillus subtilis, 36% to Escherichia coli, and 70%
to Pseudomonas fluorescens, while their bulk counterpart
showed no toxicity. Thus, because agglomeration of γ-
Al2O3 (Fig. 3a) was not controlled in the present study, their
toxicity could be lower compared with individual particles,
as mentioned by Simon-Deckers et al. (2009). Conversely,
no toxicological effects by Al2O3 NP were observed on
Cupriavididus metallidurans, which indicates that this mi-
croorganism has suitable defense mechanisms to this NP
(Simon-Deckers et al. 2009). The great diversity of micro-
Fig. 3 SEM images of interaction of bacteria with γ-Al2O3 (a) and γ- organisms that are present in anaerobic consortia (Díaz et al.
Al2O3 coated with HA (b). Image A: yellow arrow indicates cell
membrane damage, only appreciated in those incubations with uncoat-
2003), and especially with distinctive characteristics among
ed γ-Al2O3. Image B: surface of bacteria is covered with HA-coated γ- them, could be responsible why SMA was not completely
Al2O3 NP, only observed in these incubations inhibited by γ-Al2O3 NP. For this reason, SMA becomes
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Fig. 4 Humic acid reduction (a)

phase 1 phase 2
and concentration of methane
2.0
A 300 B
produced (b) by anaerobic

Reduced HA (m-Eq/L)
sludge. (Empty circles) Experi-
ment with 100 g/L of γ-Al2O3

CH4 (m-Eq/L)
1.5
coated with HA; (filled circles) 200
control lacking γ-Al2O3 but
suspended HA. In all cases, 1.0
HA concentration
was ~3,750 mg TOC/L. Values 100
are given in milli-electron 0.5
equivalents (m-Eq)/L

0.0 0
0 40 80 120 160 0 40 80 120 160
Time (h) Time (h)

stable (Fig. 2) after ~400 h of incubation, indicating that not but not with acetate (Liu et al. 2011), which indicates that
all methanogenic microorganisms in the anaerobic consor- methane production in the presence of alternative electron
tium were affected by γ-Al2O3 NP. Furthermore, a concen- acceptors, such as ferric iron and HA, varied with methano-
tration of 0.28±0.09 mg/L of Al was detected in incubations genic substrate and species involved. On the other hand,
with 100 g/L of γ-Al2O3, and it represents the highest level HAImm was more effective to serve as TEA than HASus by
of Al measured with respect to all experiments. This could the anaerobic consortium studied (Fig. 4). During the last
be another adverse mechanism causing inhibition of SMA years, it was suspected that HA in solid phase were able to
on the anaerobic consortium evaluated. Illmer and Erlebach effectively act as TEA (Scott et al. 1998; Heitmann and
(2003) evaluated the toxicity effect of Al (0.8–2.4 mg/L) on Blodau 2006). Recently, it has been demonstrated that micro-
bacteria, and they observed a partial or complete growth bial and chemical reduction of solid-phase HS is possible. For
inhibition according to the different concentrations tested. instance, values (mmol/L) of 3.5–4.0 and 2.0 for microbial
Al released from γ-Al2O3 can induce both production of (with Geobacter sulfurreducens and Shewanella putrefaciens)
intracellular reactive oxygen species, causing damage in and chemical reduction, respectively, using solid-phase HS
the cell membrane (Simon-Deckers et al. 2009), and/or were obtained (Roden et al. 2010). Reduction of HA immo-
production of free radicals under dark conditions (Green bilized on γ-Al2O3 NP presented in this study could represent
and Howman 2005), which could also affect cellular viability. a similar form to reduce solid-phase HS.
Figure 3a confirms that exposure of anaerobic bacteria present The results of the present study could explain, in part, the
in the studied anaerobic sludge to γ-Al2O3 NP caused cell role of released γ-Al2O3 NP on aquatic ecosystems. Ad-
membrane damage, which was not observed in sludge incu- sorption of HA can modify size distribution, bioavailability,
bations lacking γ-Al2O3 NP. toxicity, fate, transport, and cell penetration and transport
Methanogenesis was the preferable pathway over microbi- within the organisms of NP (Christian et al. 2008). It is well
al HA reduction to oxidize acetate in both experiments with know that HS have a significant relevance in aquatic envi-
HAImm and HASus (Fig. 4, Table 1). These results did not ronments because of their role in several chemical and
agree with previously reported by the fact that it has been biological redox reactions and in adsorption of different
demonstrated that quinones and HA inhibited methanogenesis chemicals (ions and organic and inorganic compounds). In
on several methanogenic sludge samples (Cervantes et al. aquatic environments, HS adsorbed on NP could also par-
2000; 2008; Keller et al. 2009; Minderlein and Blodau ticipate in redox reactions as evidenced here. Indeed, HA
2010). However, when acetate was used as electron donor, adsorbed on γ-Al2O3 NP were deposited on the surface of
several consortia were not capable of reducing the humic bacteria promoting a better HA–bacteria interaction, thus
model compound anthraquinone-2,6-disulfonate (AQDS), achieving a faster reduction of HA as compared to incuba-
but they could use it as TEA using hydrogen as electron donor tions performed in the absence of γ-Al2O3 NP. In addition,
(Cervantes et al. 2000). In addition, methane production was coating γ-Al2O3 NP with HA decreases their toxicological
not inhibited under Fe(III)-reducing conditions when acetate effects; therefore, the interaction between γ-Al2O3 NP and
was used by different methanogenic species in comparison HA, which are expected to occur in aquatic environments
with the use of H2/CO2 (van Bodegom et al. 2004). More considering the increasing discharge of NP to the environ-
recently, it has been reported that Methanosarcina barkeri was ment, significantly affects the mobility and toxicity of these
able to reduce ferric iron minerals with H2/CO2 and methanol, NP (Limbach et al. 2008). Although the concentration of γ-
 Author's personal copy
Appl Microbiol Biotechnol

Al2O3 NP and HA established in the present study is not close Baek YW, An YJ (2011) Microbial toxicity of metal oxide nanopar-
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of Gladis Labrada (for the SEM images), María Del Carmen Rocha (for Díaz E, Amils R, Sanz JL (2003) Molecular ecology of anaerobic
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(CONACYT) for the scholarship provided to support the doctoral Illmer P, Erlebach C (2003) Influence of Al on growth, cell size and
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