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Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
MICROBIAL COMMUNITY AND POLLUTANTS SURVEY IN SEDIMENTS OF BIOLOGICALLY
IMPORTANT WETLANDS IN LERMA, MEXICO
Arturo ABURTO-MEDINA
1
*, Derik CASTILLO
1
, Irmene ORTÍZ
2
, Ernesto HERNÁNDEZ
3
,
Rurik LIST
1
and Eric ADETUTU
4
1
Departamento de Ciencias Ambientales, Universidad Autónoma Metropolitana Lerma, Av. Hidalgo Pte. 46,
Col. La Estación, Lerma de Villada, Estado de México, C.P. 52006
2
Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana Unidad Cuajimalpa, Av.Vasco
de Quiroga 4871, Col. Santa Fe Cuajimalpa, Cuajimalpa de Morelos, México, D.F., C.P. 05300
3
Centro Nacional de Investigación y Capacitación Ambiental (CENICA), Periférico 5000, Col. Insurgentes
Cuicuilco, Coyoacán, México, D.F., C.P. 04530
4
Royal Melbourne Institute of Technology, RMIT University, GPO Box 2476, Melbourne VIC 3001, Australia
* Autor de correspondencia: aarturo1309@gmail.com
(Received March 2014; accepted October 2014)
Key words:
microbial diversity, metals, semimetals, wetlands, conservation, marsh
ABSTRACT
Wetlands pollution is of great concern given their importance to conservation and as
water and food sources for the local population. Therefore, microbial and chemical
investigations were carried out on the Lerma (Chimaliapan) and Almoloya del Río
(Chiconahuapan; both Ramsar sites) wetland samples in Mexico for risk assessment
purposes. 16S rRNA-based clone library analyses showed the prevalence of Entero-
bacteriaceae, specifcally the genus
Shigella
and
E. coli
species in both wetlands.
While faecal coliform levels in both wetlands were below the accepted limit, higher
total coliform counts (> 2400 MPN) were observed in ~ 40 % of the sampled sites
at Lerma. Other microorganisms detected included organisms similar to those ob-
tained from hydrocarbon-contaminated environments with alkanes and poly-aromatic
hydrocarbons being detected in the sediments. Detected metals were below United
States Environmental Protection Agency (USEPA) limits, decreasing in the sequence:
Al > Fe > Mn >V> Zn > Pb > Ni > Cr > Cu > Co > Tl > As > Be (except Cr at Al-
moloya). However, Al concentrations were signifcantly above the permissible limits
(2700 mg/kg) of the National Oceanic and Atmospheric Administration of the United
States in both wetlands. This study therefore demonstrates that increased health risks
and potential For metal biomagnifcation by edible species could be associated with
the use of wetland water resources.
Palabras clave:
diversidad microbiana, metales, semimetales, humedales, conservación, ciénega
RESUMEN
La contaminación de las ciénegas es de gran relevancia debido a su importancia en la
conservación del ambiente y a que son fuente de recursos para las poblaciones locales.
Por lo tanto, realizamos análisis químicos y microbiológicos en muestras de las ciénegas
Rev. Int. Contam. Ambie. 31 (1) 7-22, 2015
A. Aburto-Medina
et al.
8
de Lerma (Chimaliapan) y Almoloya del Río (Chiconahuapan; ambos sitios Ramsar)
ubicadas en México para estimar el riesgo que pueden representar estos cuerpos de agua
debido a la contaminación. Librerías de clones basadas en el gen 16S rRNA demostraron
la dominancia de miembros de Enterobacteriaceae, específcamente especies parecidas
a
E. coli
y del género
Shigella
en ambas ciénegas. Aunque en general los niveles de
coliformes fecales estuvieron dentro de los límites permisibles en ambas ciénegas, los
números de coliformes totales excedieron la norma (>2400 NMP) en ~40 % de los sitios
muestreados en la ciénega de Lerma. Otros microorganismos detectados fueron simi-
lares a aquellos observados en sitios contaminados con hidrocarburos, específcamente
con alcanos e hidrocarburos poli-aromáticos detectados en los sedimentos. Los valores
de metales estuvieron debajo de los límites permitidos por la Agencia de Protección
Ambiental de los Estados Unidos (USEPA) y disminuyeron de la siguiente forma:
Al > Fe > Mn >V> Zn > Pb > Ni > Cr > Cu > Co > Tl > As > Be, (excepto Cr en Almo-
loya). Sin embargo, las concentraciones de Al estuvieron signifcativamente por arriba
de los límites permitidos (2700 mg/kg) por la Administración Oceánica y Atmosférica
de los Estados Unidos en ambas ciénegas. Este estudio por lo tanto, demuestra que
un incremento en los riesgos a la salud y el potencial de biomagnifcación de metales
podrían estar asociados al uso de los recursos de las ciénegas.
INTRODUCTION
At the time of the Spanish conquest, the high-
lands of central Mexico had large wetland systems
that covered signifcant portions oF the valleys and
closed basins. Gradually, these were drained
and converted into agricultural lands and urbanized.
According to The Ramsar Convention on Wetlands,
wetlands are areas of marsh, fern, peat land or
water, whether natural or artifcial, permanent or
temporary, with water that is static or ±owing, Fresh,
brackish or salt, including areas of marine water
of which the depth at low tide does not exceed six
metres (Halls 1997). The Ramsar convention lists
25 important wetlands in Mexico and one of them
is the system of marshes at the headwaters of the
Lerma river, that are located in the valley of Toluca,
central Mexico. These marshes cover 3000 ha and
are the last remnants of a former 27 000 ha lake,
which drained over several decades, caused the loss
of biodiversity and the extinction of at least one
species, the Lerma Grackle (
Quiscalus palustris
).
However, despite their reduced size, these marshes
provide raw materials for centuries’ old handcraft
tradition of the manufacture of reed products, and
local people obtain food and forage from the marsh-
es. They also play important roles in ±ood control,
are host to nearly 100 resident and migratory bird
species (Vázquez 2004) and are refuge to endemic
and endangered fsh, birds, and a micro-endemic
and critically endangered amphibian
Ambystoma
lermaense
(Lyons
et al
. 1995, Aguilar and Casas
2009, Colón
et al
. 2009). A wide variety of aquatic
plants, including endemic species (Ramos 1999) are
also found in these wetlands. Recent studies have
identifed the aquatic vascular ±ora and changes in
plant diversity (Zepeda-Gómez
et al
. 2012) as well
as the ecology of epilithic diatoms (Segura-García
et al
. 2012). As a result of their importance, these
marshes were designated a federally protected area
(Refugio de Flora y Fauna Silvestre Ciénegas de
Lerma; SEMARNAT 2002).
Given that the marshes are sources of water and
direct and indirect food resources to local people
(Pérez-Ortiz and Valdez 2006), pollution of water
and sediments may pose health hazards. Marshes can
experience an advanced process of pollution due to
industrial, agricultural and municipal run offs and a
decline of water volumes due to aquifer exploitation
(Esteller and Díaz-Delgado 2002, Gómez-Salazar
2012). The pollution in the Lerma River and the
Alzate dam is mainly due to excessive amounts of or-
ganic matter and heavy metals. Previous studies have
measured the metal concentrations in sediments of
the river (Tejeda
et al
. 2006, Zarazúa
et al
. 2011) and
the Alzate dam (Ávila-Pérez
et al
. 1999) and most of
them are above the USEPA contamination threshold.
Another study has established the ecological risk of
nine metals (López-Galván
et al
. 2010) in different
zones of the basin and Cu and Pb showed the highest
risks in the dissolved phase (Gómez-Salazar 2012).
However, there is no information of the contami-
nants’ identity or the microbial communities in these
wetlands. Therefore this study aims to characterize
the microbial community in the wetlands (Almoloya
and Lerma) for health risk assessment purposes and
CHARACTERIZATION OF THE MICROBIAL COMMUNITY IN THE LERMA WETLANDS
9
to identify the organic and inorganic contaminants
in sediments obtained from the marshes of these
wetlands in order to evaluate potential impacts of
anthropic activities.
MATERIALS AND METHODS
Site description and sampling
Samples were collected from the Almoloya del
Río (Chiconahuapan) and Lerma (Chimaliapan)
marshes in central Mexico. Detailed descriptions of
the marshes have been previously reported (Zepeda-
Gómez
et al
. 2012). The geographic coordinates for
each of the sampled sites and the relative position of
the samples in the wetlands are indicated in
table I
and
fgure 1
, respectively. Sampling points were
selected in order to cover most areas of each wetland
with sampling being carried out during the rainy sea-
son that spans from late May to early October (an-
nual precipitations range between 800 to 1200 mm).
Water samples were collected in sterile 1-litre glass
bottles. Sediments from the bottom of the wetlands
were collected in 50 mL sterile centrifuge tubes in
triplicates after which, they were frozen with dry
ice and protected from light. Domestic and indus-
trial wastewater is discharged into both wetlands
although the vast majority is discharged mainly
into the river.
Sample preparation and total metal analyses
Metal analyses were performed at the Mexican
Centre for Training and Research on Environmental
Issues, Centro Nacional de Investigación y Ca-
pacitación Ambiental (CENICA, in spanish). Both,
Lerma and Almoloya sediments were air-dried prior
to analysis and sieve was not required. For all analy-
ses, ultrapure deionized (nanopure) water was used
throughout. All reagents were of analytical grade or
higher purity. A rigorous quality control system was
used including reagent blanks, duplicate samples and
certifed international reFerence materials (Channel
Sediment BCR 320R from BCR). Precision and
accuracy were better than 10 % for all analyzed
components and the blanks were below the method
detection limit (MDL).
Trace elements in sediments
Total trace elements were determined in 0.5 g of
dried sediment using inductively coupled plasma
atomic emission spectroscopy (ICP - AES) iCAP
6500, Thermo Scientifc according to the USEPA
6010 method (USEPA 2007a). This was carried out
after microwave assisted acid digestion in an oven
Anton Paar, MULTIWAVE 3000 model, following
the USEPA 3051 procedure (USEPA 2007b).
Identifcation oF semivolatile organic compounds
The organic compounds were extracted from dried
sediments by sonication (method 3550C, USEPA
2007c) using 3 g of soil and 95 mL of a mixture
1:1
n
-hexane-ketone (Mallinckrodt, HPLC grade).
The extracts were concentrated by roto-evaporation
and changed to
n
-hexane as a solvent. The recovery
percentage of this extraction method was validated
using a soil sample with known concentrations of
17 reference compounds. CENICA provided this
spiked sample and the percentage of recovery of all
compounds was higher than 90 %. Regular controls
with this soil were performed to ensure the quality
of the data. The extracts were analyzed to identify
organic compounds by gas chromatography (method
8270D, USEPA 2007d) coupled with a mass spectrum
detector (Agilent 6890N, MSD 5975B, USA) using
a 5 MS column (Agilent, USA). The temperatures
of the detector and injector were 250 ºC and 220 ºC,
respectively. The initial and fnal temperatures oF the
oven were 70 ºC and 250 ºC at a rate of 7 ºC/min.
Helium was used as the carrier gas and the mass scan
range was From 50 to 450 z/m at 70 eV. Identifcation
was made using the NIST05 Mass Spectral Library.
Statistical analyses
The statistical comparison of all heavy metal
concentrations in sediments between both wetlands
was performed using Hotelling’s T
2
(Rencher 2002),
that corresponds to the multivariate version of the
Student’s t test.
TABLE I.
COORDINATES OF THE SAMPLED SITES CO-
LLECTED BY GPS
Code
GPS positioning
Elevation
(m)
L1
N19º14.069´, W99º29.539´
2580
L2
N19º14.415´, W99º29.924´
2580
L3
N19º14.453´, W99º29.963´
2584
L4
N19º14.570´, W99º30.059´
2578
L5
N19º14.845´, W99º29.456´
2585
L6
N19º16.007´, W99º30.093´
2589
L7
N19º15.164´, W99º31.106´
2593
A1
N19º09.212´, W99º29.764´
2575
A2
N19º09.245´, W99º29.555´
2574
A3
N19º09.175´, W99º29.635´
2575
A4
N19º09.322´, W99º29.682´
2575
A5
N19º09.458´, W99º30.020´
2575
A. Aburto-Medina
et al.
10
Determination of total and faecal coliforms
The most probable number (MPN) of total coli-
forms, faecal coliforms and
Escherichia coli
followed
the Mexican Norm NMX-AA-42-1987, which agrees
with the ISO norm: ISO/DP 9308/2.
DNA extraction and PCR
Microbial community DNA was extracted directly
from the slurries collected on September 21 using
the UltraClean Soil DNA kit (MoBio Laboratories,
Solana Beach CA) and PCR amplifcation oF 16S
rDNA genes was performed with the following
primer pairs: 27F (TCT GGT TGA TCC CGC CAG)
and 1392R (ACG GGC GGT GTG TAC; Lane 1991)
for members of the Archaea, and 63F (CAG GCC
TAA CAC ATG CAA GTC) and 1389R (ACG GGC
GGT GTG TAC AAG; Marchesi
et al
. 1998) for the
Bacteria as described previously (Aburto
et al
. 2009).
The cycling conditions for both primer pairs were
as follows: 1 cycle at 94 ºC for 2 min, 30 cycles of
1 min at 94 ºC, 1 min at 55 ºC and 2 min at 72 ºC and
a fnal elongation at 72 ºC For 10 min.
Cloning and sequencing
PCR products were cloned with a CloneJet clon-
ing kit (Thermo) as described in the manufacturer´s
instructions using One Shot TOP10 chemically
competent
E. coli
cells. Recombinant colonies were
identifed as white colonies and recovered From
LB agar plates containing ampicillin (50 mg/mL).
The screening of inserts from transformants was
performed by direct PCR amplification from
colonies using the 16S primers 63F (CAG GCC
TAA CAC ATG CAA GTC) and 1389R (ACG
GGC GGT GTG TAC AAG). Heating at 94 ºC
for 10 min preceded standard cycling conditions
(as above). Amplifed inserts were grouped on the
basis of restriction fragment length polymorphism
(RFLP) patterns using a combination of the two
restriction endonucleases
Cfo
I and
Alu
I (Sigma) at
37 ºC for 3 h. Representative clones of each RFLP
group were sequenced, giving between 45 and 50
clones per sample. The insertions were sequenced
by the Sanger method using primer 63 F and were
performed at the Biotechnology Institute (Instituto
–116
32
28
24
20
16
12
1000
0
1000
Kilometers
32
28
24
20
16
12
–112
–108
–104
–100
–96
–92
–88
100º30'
D.F.
20º00'
19º00'
19º30'
18º30'
20º00'
19º00'
19º30'
18º30'
99º30'
98º30'
99º00'
100º00'
100º30'
99º32'00''
99º31'20''
99º29'20''
N
E
W
S
99º30'40''
99º28'40''
99º28'00''
99º28'30''
99º29'30''
99º30'30''
99º30'31''
99º29'00''
99º30'00''
99º31'00''
99º28'30''
99º29'30''
99º30'30''
99º30'31''
99º29'00''
99º30'00''
99º31'00''
19º16'00''
19º10'00''
19º9'30''
19º8'30''
19º9'00''
19º8'00''
19º10'00''
19º9'30''
19º8'30''
19º9'00''
19º8'00''
19º15'30''
19º15'00''
19º14'30''
19º14'00''
19º13'30''
19º16'00''
T
enango-Lerma
Acueduct
o
P
ro
f
.
Carlo
Hank
González
Te
nn
g
o-Le
r
ma
19º15'30''
19º15'00''
19º14'30''
19º14'00''
19º13'30''
99º30'00''
99º32'00''
99º31'20''
99º29'20''
99º30'40''
99º28'40''
99º28'00''
99º30'00''
99º30'
98º30'
99º00'
100º00'
–116
–112
–108
–104
–100
–96
–92
–88
Toluca
Lerma wetland
Almoloya wetland
Lerma
Metepec
Lerma
Capulhuac
Tianguistenco
Mexicaltzingo
San Mateo Atenco
0.7
0
0.7
1.4 Kilometers
Kilometers
1012
L6
State of Mexico
Lerma wetland
Almoloya wetland
L7
L3
L5
L4
L2
L1
San Mateo Atenco
A5
A4
A1
A3
A2
70
70
140
0K
ilometers
Fig. 1.
Location of sampled sites. L: Lerma, A: Almoloya. No samples were collected from the south region of the Almoloya wetland
due to heavy vegetation. GPS coordinates of each sampled site is shown in
table I
. L1 to L7 and A1 to A5 represent different
sampling points in Lerma and Almolya wetlands respectively.
CHARACTERIZATION OF THE MICROBIAL COMMUNITY IN THE LERMA WETLANDS
11
de Biotecnología), National Autonomous Univer-
sity of Mexico (Universidad Nacional Autónoma
de México, UNAM). The length of the obtained
sequences was 600 to 700 nucleotides in average.
Phylogenetic analyses
The sequences obtained were compared with the
European Bioinformatics Institute and Genebank
databases by online FastA searches (Pearson 1988)
and the Check Chimera programme (Cole 2003)
for detecting chimeras. Sequence alignments were
carried out using MUSCLE (Edgar 2004) and align-
ment curation carried out using G-Block (Castresana
2000). Phylogenetic trees were constructed by Mr
Bayes (Bayesian inference) using Markov Chain
Monte Carlo parameters with 100 000 tree genera-
tions carried out. Tree sampling was performed after
every 10 generations (Huelsenbeck 2001) with the
fnal tree being viewed in TreeDyn (Chevenet
et al
.
2006, Dereeper
et al
. 2008, Dereeper
et al
. 2010)
RESULTS
Total and faecal coliforms
Total and faecal coliforms determined with the
MPN are shown in
table II
. Most of the sampling
points (A1-A5 and L1-L4) total coliform values were
below the limit (1000 MPN/100 ml) established by
the Mexican Norm for Residual Waters to be reused
in public services (NOM-003-SEMARNAT-1997)
(SEMARNAT 1997). However, the total coliform
values of sampling points L5, L6 and L7 in the Lerma
wetland were signifcantly above (> 2400 MPN) the
Norm limit.
Metals, semimetals and other elements
Concentrations for Ag, Al, As, Be, Cd, Co, Cr,
Cu, Fe, Mn, Ni, Pb, Sb, Se, Tl, V and Zn were ob-
tained for each of the sampling points within the
Almoloya (A1-A5) and Lerma (L1-L7) wetlands
(
Table III
). The concentrations of Ag, Cd, Sb, Se,
As, Be and Tl were below the detection level for
all sampling points (data not shown) except for A4
where 4.42, 1.88 and 14.6 mg/kg were detected for
As, Be and Tl, respectively. Moreover, Tl was also
detected in L7. The rest of the metal concentrations
were above detection level in all sampling points
except for Zn at a few locations (L3, L4, A1, A4;
data not shown).
A statistical comparison of all element values
between both wetlands revealed an overall similarity
between them (p = 0.08) and a description of these
values using means and standard error of means is
displayed in
Fgure 2
. The overall similarity of the
studied wetlands suggests that their inFuence area
is substantially larger than both studied sites. The
inFuence area includes wastewater discharges ±rom
industrial, agricultural and household sources.
Organic compounds
A total of 24 organic compounds were detected
in the sediments of both wetlands and they include
polyaromatic hydrocarbons, phthalates, fatty ac-
ids and higher alkanes (C15-C24) among others
(
Table IV
). The higher alkanes were detected in all the
sampling points (A1-A5, L1-L7), while sediments of
sampling point L3 recorded the highest number
of compounds with 12, followed by A1, L5 and
L1 each with 11 and A3, A5, L2 and L4 each with
nine compounds. Seven organic compounds were
detected in A4 and six in L6 and L7. The detection
quality of most of the compounds was above 95 %
(
Table IV
).
Microbial diversity
in situ
In order to identify the microbial diversity and
their potential in the wetlands that give origin to
the Lerma river, a clone library was created for
each wetland: Almoloya (A) and Lerma (L). 16S
rRNA gene PCR amplicons were obtained from the
environmental samples using prokaryotic
primers,
and the main bacterial phylogenetic groups are sum-
marized in
Fgure 3
. No archaeal amplicons were
obtained. The Gammaproteobacteria followed by
the Firmicutes dominates the Almoloya sample. Op-
erational taxonomic units were formed on the basis
of RFLP results and a total of 13 clones were 98 %
similar to the uncultured
Shigella
sp. clone C199 (A4;
TABLE II.
MPN OF TOTAL AND FAECAL COLIFORMS IN
EACH OF THE SAMPLES SITES. A: ALMOLO-
YA, L: LERMA
Code
Faecal coliforms
(MPN/100 ml)
Total coliforms
(MPN/100 ml)
A1
< 3
< 3
A2
< 3
9
A3
< 3
120
A4
< 3
9
A5
< 3
< 3
L1
< 3
150
L2
< 3
240
L3
< 3
240
L4
< 3
< 3
L5
< 3
> 2400
L6
93
> 2400
L7
7
>2400
A. Aburto-Medina
et al.
12
Fig. 4
). On the other hand 11 clones (A7;
Fig. 5
) were
95 % similar to those (afFliated to the ±irmicutes)
found in a study that analyzed the microbial compo-
sition of patients with in²ammatory bowel diseases
(IBD; Li
et al
. 2012). Other nine clones (A5;
Fig. 4
)
were related to a Gammaproteobacteria isolated
from soil contaminated with polyaromatic hydro-
carbons (unpublished data). The rest of the clones
(5 and 7 respectively) were similar to an uncultured
Betaproteobacterium clone from epiphytic bacterial
communities on native plant species in a midwestern
Michigan creek (Olapade
et al
. 2011) and to other
uncultured microorganisms from the bacterial com-
munity detected in different developmental stages of
the house²y (Wei
et al
. 2013). The most abundant
phylogenetic group of Bacteria in the Lerma wetland
was also the Gammaproteobacteria (
Fig. 3
). Some
of the organisms detected in the Lerma wetland
TABLE III.
CONTENT OF METALS, SEMIMETALS AND OTHER ELEMENTS IN THE SEDIMENTS OF THE ALMOLOYA
AND LERMA WETLANDS AND COMPARISON WITH THE US EPA LIMITS
Code
Elements (mg/kg)
Al
As
Be
Co
Cr
Cu
Fe
Mn
Ni
Pb
Tl
V
Zn
Max L
54 341
< DL
< DL
15
38
26
35 853
1127
41
71
12
197
81
Max A
26 433
4
1
9.4
48
12
13 821
215
24
22
14
271
65
Min L
6 600
< DL
< DL
2.7
6.2
4.2
9 156
66
9.6
2.3
< DL
18
23
Min A
6 466
< DL
<DL
2
6
4
9 156
66
9
2
< DL
18
23
Av L
22 372
< DL
< DL
6
15
14
15 973
319
18
19
1
75
41
Av A
11 128
0.5
0.1
3
28
5
7 142
185
13
13
2
161
18
EPA
----
8
---
---
75
50
25 000
500
---
60
---
---
200
Max: maximum, Min: minimum value, Av: Average value, L: Lerma, A: Almoloya, EPA: USEPA Guideline (USEPA 1977),
DL: Detection limit.
Detection limits for Ag, Be, Cd, Co, Cr, Ni, Y: 1 mg/kg
Detection limits for As, Cu, Mn, Pb, Sb: 2 mg/kg
Ag, Cd, Sb and Se were below detection limit at all sampling points
3E4
(A)
(B)
2E4
mg/kg
1E4
0
Al
Fe
Metal
400
200
mg/kg
0
Mn
V
Metal
60
(C)
(D)
40
mg/kg
20
0
Cr
Zn
Ni
Pb
Metal
15
10
5
mg/kg
0
Co
Cu
Metal
Fig. 2.
Comparison of average heavy metal concentrations in the Almoloya
(slash-Flled boxes) and Lerma (backslashed-Flled boxes) wetlands
for each metal. Metals whose averages were similar were grouped
together in the same panel. The bar height indicates average, while
the whiskers indicate one standard error of the mean (SEM) above
and below the average. Due to the accumulation of type I error, it
is not advisable to do pairwise comparisons, and a multivariate test
was employed (see text).
CHARACTERIZATION OF THE MICROBIAL COMMUNITY IN THE LERMA WETLANDS
13
TABLE IV.
ORGANIC COMPOUNDS IDENTIFIED BY GC-MS FROM SEDIMENTS OF THE
LERMA AND ALMOLOYA WETLANDS
Sample
Quality (%) Compound
CAS number
A1-A5
L1-L3
L5
91
87-91
91
Benzenecarboxylic acid
000065-85-0
L1
L3
L4
92
81
91
Naphthalene, 2,6-dimethyl-
000581-42-0
A1-A5
L1-L7
94-98
96-98
2,5-Cyclohexadiene-1,4-dione, 2,6-bis(1,1-dimethylethyl)-
000719-22-2
A1
A5
L1
L3
93
91
93
94
Phenol, 2,6-bis(1,1-dimethylethyl)-4-ethyl-
004130-42-1
A1
A3
A4
L2-L7
95
94
93
90-96
Diethyl phthalate
000084-66-2
A1-A5
L1-L5
94
94-98
Dibutyl phthalate
000084-74-2
A1-A5
L2
L5
90-98
91
92
Benzyl butyl phthalate
00085-68-7
A1
L1-L4
94
80-93
Benzophenone
000119-61-9
A5
L2
L3
L5
97
92
97
94
Phenanthrene, 3,4,5,6-tetramethyl-
007343-06-8
A2-A4
L1-L5
L7
98-99
97-99
93
7,9-Di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione
082304-66-3
L1
L7
95
83
1-Pentadecanol
000629-76-5
A1-A5
L1-L7
91-99
90-97
Higher alkane hydrocarbons a*
A3
L1
L3-L6
91
96
91-96
Cyclotetradecane
000295-17-0
A1
A5
L1
L5
L6
92
93
90
91
96
Cyclopentadecane
000295-48-7
A1
A3
A5
L2-L7
95
91
90
92-99
1,19-Eicosadiene
014811-95-1
A1
A5
L2
L4
98
93
90
96
1,21-Docosadiene
053057-53-7
* Pentadecane (000629-62-9), eicosane (000544-76-3), heptadecane (000629-78-7), nonadecane (000629-
92-5), hexadecane (000112-95-8), heneicosane (000629-94-7), tetracosane (000646-31-1), pentacosane
(000629-99-2)
A. Aburto-Medina
et al.
14
Almoloya (45)
Lerma (50)
5%
12%
27%
55%
70%
3%
4%
11%
11%
Fig. 3.
Main bacterial phylogenetic groups represented in two wetland samples.
(
) Gammaproteobacteria (
) Firmicutes (
) Chlorofexi (
) Beta-
proteobacteria (
) Uncultured.
The number of clones screened for each library is indicated in brackets
after the wetland name.
Proteus vulgaris
(DQ885257.1)
Proteus mirabilis
(JF430796.1)
Klebsiella oxytoca
(Y17655.1)
Enterobacter aerogenes
(KC429778.1)
Enterobacter cloacae
(EF219421.2)
Salmonella bongori
(KC329819.9)
Salmonella enterica
(JQ694165.1)
Shigella dysenteriae
(JF346892.1)
Shigella flexneri
2a (444439567)
Serratia marcescens
(FJ971877.1)
Serratia liquefaciens
(FJ971876.1)
Yersinia pseudotuberculosis
(AM182402.1)
Yersinia pestis
(EF165975.1)
(AF233876.1)
0.2
Comamonas
denitrificans
Shigella boydii
(AY696659.1)
Escherichia coli
DSM (HE97978270.1)
Escherichia coli
ATCC 25922 (X80724.1)
A4
(13) (HG423446)
0.96
0.99
0.89
0.86
0.95
0.96
0.96
0.63
0.62
0.94
0.51
1
1
1
0.8
L18
(7) (HG423452)
L3
(3) (HG423450)
L2
(3) (HG423451)
L4
(20) (HG423449)
A5
(9) (HG423447)
Klebsiella terrigena
(Y17658.1)
Klebsiella pneumoniae
(Y17656.1)
Fig. 4.
Phylogenetic tree of the Gammapro-
teobacteria based on partial
16S rDNA
sequences showing the relationships
among bacterial clone sequences from
this study and members of the Ente-
robacteriaceae family of the Gamma-
proteobacteria found in the database.
Mr Bayes (Bayesian inference) using
Markov Chain Monte Carlo parameters
with 100 000 tree generations carried out.
Outgroup:
Comamonas denitrificans.
Clones from this study are in bold, A:
Almoloya, L: Lerma. A number in pa-
renthesis indicates the number of clones
obtained for each sequence. Accession
numbers of the sequences retrieved from
the databases are also indicated in pa-
renthesis. 16S rRNA sequence similarity
over the region 800 to 1390 based on
E.
coli
numbering.
CHARACTERIZATION OF THE MICROBIAL COMMUNITY IN THE LERMA WETLANDS
15
were similar to uncultured microorganisms from
diverse environments such as a lake in Panama (two
clones), from a brewery wastewater treatment plant
(three clones) and (as in Almoloya wetland) to plant-
associated bacterial populations in freshwater from
a Michigan creek (fve clones; Olapade
et al
. 2011).
Five other clones have close a±fliation with an un
-
cultured microorganism detected in a polychlorinated
biphenyl (PCB) - contaminated core sediment from
the Grasse River in New York undergoing dechlorina-
tion (Xu
et al
. 2012), while one was highly similar
to another found in a plutonium contaminated soil
(Kimber
et al
. 2012). Moreover, although two clones
were similar to an
E. coli
isolate from healthy people
gastric juices (Delgado
et al
. 2013) seven more were
similar to uncultured bacteria detected in mammalian
gut in²ammation, and the majority o± clones (20
clones) were related to a
Shigella fexneri
strain.
Fig. 5.
Phylogenetic tree of the Firmicutes based on partial
16S rDNA sequences showing the rela-
tionships among bacterial clone sequences from this study and members of the Firmicutes
found in the database. Mr Bayes (Bayesian inference) using Markov Chain Monte Carlo
parameters with 100 000 tree generations carried out. Outgroup:
Comamonas denitriFcans
.
Clones from this study are in bold, A: Almoloya, L: Lerma. A number in parenthesis indica-
tes the number of clones obtained for each sequence. Accession numbers of the sequences
retrieved from the databases are also indicated in parenthesis. 16S rRNA sequence similarity
over the region 800 to 1390 based on
E. coli
numbering.
Comamonas denitrificans
(AF233876.1)
Clostridium botulinum
(X73442.1)
Clostridium septicum
(CSU59278)
Clostridium tetani
(X74770.1)
Lactobacillus plantarum
(AM279758.2)
Lactobacillus casei
(D16551.1)
Bacillus subtilis
(AB018486.1)
Bacillus anthracis
(AB190217.1)
Bacillus cereus
(AJ310098.1)
Staphylococcus epidermidis
(AJ717377.1)
Staphylococcus epidermidis
(AJ344227.1)
Staphylococcus epidermidis
(AM697667.2)
Staphylococcus aureus
(DQ2679498.1)
Staphylococcus aureus
(Y15856.1)
Staphylococcus haemolyticus
(L37600.1)
Staphylococcus saprophyticus
(L20250.1)
Enterococcus faecalis
(KC510232.1)
Enterococcus faecalis
(AB036835.1)
Streptococcus pneummoniae
(AB002522.1)
Erysipelothrix
tonsillarum
(AB034201.1)
Selenomonas ruminantium
(AB017195.1)
0.4
Streptococcus pyogenes
(AB002521.1)
Listeria monocytogenes
(JF967617.1)
L5
(1) (HG423453)
1
1
1
1
0.64
0.64
0.99
0.99
0.97
0.74
0.55
0.68
0.9
0.9
0.87
1
1
1
1
1
1
1
A7
(11) (HG423448)
A. Aburto-Medina
et al.
16
DISCUSSION
Metals, semimetals and other elements in sedi-
ments
The mean of the values detected in this study for
As, Cr, Cu, Ni, Pb and Zn were below the ones ob-
tained previously (Pérez-Ortiz 2005) in both wetlands
except for Cr (Almoloya) suggesting a movement
of the sediments due to the water Fow. Also, these
values are below the threshold of contamination as
established by the USEPA (USEPA 1977) and permit
aquatic life although with the lowest toxicity effect
of Cr according to the Ontario Ministry of the Envi-
ronment and Energy (OME 1976). In addition, these
values are also below the ones reported for sediments
in the Lerma River (Tejeda
et al
. 2006, Zarazúa
et al
.
2011) and the Alzate dam (Ávila-Pérez
et al
. 1999).
This suggests that the high contamination reported
in these studies was due to discharges from domes-
tic and industrial activities along the river course
(Ávila-Pérez
et al
. 1999). However, Al concentrations
were the highest among all metals in both wetlands
and they are signi±cantly above the permissible
limits (2700 mg/kg) of the National Oceanic and
Atmospheric Administration of the United States
(NOAA 1999). A recent study analyzed Al toxicity
in zooplankton (rotifers, cladocerans and copepods)
at the Lerma wetland suggesting that these organisms
are good candidates for biological monitoring since
they are more sensitive than other groups and have
a high ecological relevance (García-García
et al
.
2012). In that study only one species was frequently
found where high concentrations (above 2350 mg/kg)
of Al were recorded in sediments, what is consis-
tent with previous studies reporting the negative
effects of Al on zooplankton and ±sh populations
(Havens and Heath 1989, Havens 1990, Gensemer
and Playle 1999, Cherry
et al
. 2001). Moreover, we
detected sediment concentrations of Al nearly three
times higher than those detected from March 2008
to February 2009 (García-García
et al
. 2012) and
almost ten times higher than the permissible limits
(2700 mg/kg). This might be related to the increase of
Al production and melting by the nearby industries in
the recent years. Additionaly acid rain (consequence
of industrial activities and burning of fossil fuels)
makes the wetland waters more acidic and this, in
turn, increases the solubility of Al (Moore 2006). Al-
though a couple of values detected for Fe and Mn were
above the USEPA limits (both at sampling point L7),
their means were not only below those limits but also
those reported for sediments along the upper course
of the Lerma River (UCLR; Zarazúa
et al
. 2011).
In addition, their source is presumed mainly natural
as was established for the UCLR sediments (Zarazúa
et al
. 2011). The mean of vanadium values for both
wetlands are above the USEPA phytotoxic limit
of 2 mg/kg for soil but within the range of what is
considered typical in sediments (7-500 mg/kg) of
the US irrespective of the parent material (Irwin
et
al
. 1998). The highest values for all metals except
V (2
nd
highest) were observed at sampling point L7
and this may be explained by the fact that it is next
to the recently inaugurated (August 2011) highway
Lerma-Tenango, suggesting its construction and us-
age has had an immediate impact on the wetlands.
The analysis of sediments is important because
it is a direct indicator of the changes throughout
time since the sediments absorb and accumulate
metals that cannot be biodegraded (Rosales-Hoz
et
al
. 2000). A portion of those metals can be trans-
ported vertically to the water column. Zooplankton
is partially responsible for such transport since those
organisms feed by ±ltration and incorporate metals
from the sediments. This incorporation may lead
to biological accumulation and bio magni±cation,
potentially affecting humans through consumption
of wetland-derived species such as ±sh, cray±sh and
axolotl (
Ambystoma lermaense
). Therefore, although
the amounts of metals are in general below the lim-
its permitted by the USEPA for lake sediments and
signi±cantly below the ones observed in the UCLR
and the Alzate Dam sediments, it remains necessary
to investigate the concentration of heavy metals and
other toxic substances in biological tissues from all
trophic levels in order to rule out biological magni±
-
cation. Moreover, the introduction of selected species
of macrophyte into the wetlands should be considered
since it has been a viable solution to the treatment of
highway runoff (Mungur
et al
. 1995).
Organic compounds detection
Among the identi±ed organic compounds, ben
-
zenecarboxylic acid, naphthalene, 2,6-dimethyl-,
2,5-cyclohexadiene-1,4-dione, 2,6-bis (1,1-dimeth-
ylethyl)-, diethyl phthalate, dibutyl phthalate, benzyl
butyl phthalate, benzophenone, pentadecane, eico-
sane, heptadecane, nonadecane, hexadecane and he-
neicosane have been listed as chemicals for potential
endocrine disruptor screening and testing (USEPA
2012). Also 2,5-cyclohexadiene-1,4-dione, 2,6-bis
(1,1-dimethylethyl)-, dibutyl phthalate, pentadecane,
heptadecane, pentacosane and nonadecane have been
found in samples of water with historical contamina-
tion and a related toxicity effect on ±shes (Mancaş
et al
. 2002). Higher alkane hydrocarbons from C
15
CHARACTERIZATION OF THE MICROBIAL COMMUNITY IN THE LERMA WETLANDS
17
to C
24
, benzenecarboxylic acid and 2,5-cyclohexa-
diene-1,4-dione, 2,6-bis (1,1-dimethylethyl)- were
found in all of the samples and some cycloalkanes
(C
14
and C
15
) were also frequently found. Higher
alkane hydrocarbons are the main components of
fuel and lubricating oils while benzenecarboxylic
acid is used as a food preservative due to its inhibi-
tory effects on the growth of mold, yeast and some
bacteria and it is also an important precursor for the
synthesis of many other organic substances. The 2,5-
cyclohexadiene-1,4-dione, 2,6-bis (1,1-dimethyl-
ethyl)-, also known as 2,6-di-tert-butylbenzoquinone
is considered a semi volatile priority organic pollutant
of the steel Fnishing subcategory in the iron and steel
manufacturing (USEPA 2002).
The group of phthalates compounds has also been
reported as endocrine-disrupting chemicals. Dibutyl
phthalate is the predominant phthalic acid ester
present in agricultural soils and it can signiFcantly
affect paddy soil microbial diversity, regarding its
population size and species representation (Zeng
et
al
. 2008, Xie
et al
. 2013). Although the phthalates
found in the samples are not classiFed as either mu
-
tagenic or carcinogenic, they are included in the list
of priority pollutants by the USEPA (Kamrin 2009,
Wang
et al
. 2013).
On the other hand, benzophenone related com-
pounds are utilized as organic UV Flters not only in
cosmetics and personal care products but also in food
packaging, pharmaceuticals, plastics, textiles and
vehicle-maintenance products to prevent photodeg-
radation of polymers and pigments (Cuquerella
et al
.
2012, Gago-Ferrero
et al
. 2012). These compounds
are considered as emerging environmental pollutants
because of their increasing use over the last decade
related to the concern of skin damage due to UV-solar
radiation. Despite being scattered and limited, current
ecotoxicological data indicate that the potential risk
posed by these widely used chemicals requires further
investigation, particularly terrestrial environments
should be more widely studied to identify their fate
and effects (Gago-Ferrero
et al
. 2012). The presence
of hydrocarbons in the sediments may be due to
natural biological sources such as plant waxes or dia-
genetic transformation of functionalized lipids into
the sediments (Volkman
et al
. 1992). However, we
suggest that they are from anthropic activities since
most of the detected hydrocarbons in the wetlands
sediments are the main components of lubricating
oils. This is supported by the fact that these type of
oils are the major source of hydrocarbon pollution
in many estuaries and coastal zones around Australia
(Volkman
et al
. 1992) but more importantly, runoffs
containing residues of these oils from the Lerma-
Tenango highway may be increasing the hydrocarbon
levels in the sediments.
Microbial diversity
in situ
In general, the microbial diversity observed in
both wetlands is low. This may be due to the ongo-
ing environmental degradation in these water bodies.
The Almoloya wetland harbours microorganisms
similar to an uncultured
Shigella
clone C199 and
to an uncultured Firmicutes previously detected
in patients with IBD. While uncultured bacteria
found in mammalian gut in±ammation (Stecher
et
al
. 2012) and a strain of
Shigella fexneri
were the
closest organisms to the clones retrieved from the
Lerma wetland. Infection by
Shigella
is an impor-
tant cause of morbidity and mortality around the
world. An estimate from the World Health Organi-
zation indicates there are 164 million episodes of
shigellosis per year, which resulted in 1.1 million
deaths, mostly children under 5 years old (Kotloff
et al
. 1999). More importantly, the majority of the
episodes and deaths occur in the developing world
(163 million) and the main isolates correspond to
S. fexneri
, being the serotype 2a the most common
(Kotloff
et al
. 1999). However in order to assess the
pathogenicity of the
Shigella
-related clones detected
in the wetlands, it is necessary to look for the genes
that code for those toxins or virulence and invasion
plasmids (Sansonetti
et al.
1982, Menard
et al.
1993,
Tang
et al.
2005) in future studies.
Dominance of clones related to
Shigella
and to
bacteria found in gut studies (Stecher
et al
. 2012) in
both wetlands is not uncommon since they receive
domestic ef±uents from the surrounding towns and
this is conFrmed by the large number of coliforms
> 2400 MPN 100 mL (
Table II
) recovered from a
sampling point very near a sewage pipe from the San
Pedro Tultepec town that discharges directly into the
Lerma wetland (sampling point L6;
Table I
). The
latter was also found in previous studies (Pérez-Ortiz
2005) and MPN values above the limit from nearby
sampling points L5 and L7.
Although both wetlands were dominated by mi-
croorganisms partially related to Gammaproteobac-
teria, there were other clones related to those found
in hydrocarbon, PCB and plutonium-contaminated
environments. Several clones retrieved from the
Almoloya sediments were related to a Gammapro-
teobacterium previously detected in a polyaromatic
hydrocarbon-contaminated soil and several species of
Gammaproteobacteria have been reported to degrade
polyaromatic hydrocarbons (Boonchan
et al
. 1998,
A. Aburto-Medina
et al.
18
Ma
et al
. 2006, Arun
et al
. 2008). Also, clones
retrieved from the Lerma sediments were closely
related to those afFliated to the Chloro±exi and pre
-
viously
found in a PCB-contaminated core sediment
from a New York river undergoing dechlorination
(Xu
et al
. 2012). Members of the Chloro±exi have
also been found in several hydrocarbon-degrading
consortia in different environments such as soil
(Aburto-Medina
et al
. 2012), a petroleum contami-
nated aquitard (Van Stempvoort
et al
. 2009), a BTEX
contaminated aquifer (Berlendis
et al
. 2010) but more
importantly in dimethyl phthalate and naphthalene-
contaminated sludge (Liang
et al
. 2009, Cao
et al
.
2012); these hydrocarbons were also detected in the
wetland sediments studied (
Table IV
). This may sug-
gest the potential use of these available hydrocarbons
by the microorganisms, however further laboratory
experiments are needed in order to conFrm such
processes.
Another clone was similar to an uncultured
bacterium found in a plutonium-contaminated
soil in the United Kingdom (Kimber
et al
. 2012).
Microbial processes performed by
Clostridium
have the potential to mobilize the plutonium by the
reduction of Pu (IV) to Pu (III)(Francis
et al
. 2008,
Kimber
et al
. 2012). This organism is also capable
of performing hydrocarbon degradation (Sethuna-
than and Yoshida 1973) and PCB dechlorination
(Hou and Dutta 2000), suggesting a potential use
of the available organic compounds detected in the
wetland sediments by this species. However this
bacterium is often found in soils and wastes and it
is involved in the sulphur cycle, therefore there is
also a need for further degradation experiments to
validate those processes.
Despite the pressure on the marshes, contaminant
levels are not extremely high in general suggest-
ing that their self-cleansing system may still be
functional. However, it is necessary to establish the
origin of hydrocarbons and metals detected in the
sediments in order to prevent an increase of their
concentrations. Further experiments are required to
rule out biomagniFcation of metals to protect both
the local human population, who depends on the
wetland and the endangered and endemic species,
which live there.
ACKNOWLEDGMENTS
This research was funded by Universidad Autono-
ma Metropolitana-Lerma and the Consejo Nacional
de Ciencia y Tecnología (CONACyT). The authors
would also like to acknowledge the support of Clau-
dia Granada M.Sc. for her help with the maps and
Mr. Raúl Gutiérrez and Mr. RuFno Rodríguez Salas
for giving access to the wetlands.
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