e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 744–753

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Substrate effect on bacterial communities from constructed

wetlands planted with Typha latifolia treating
industrial wastewater

Cristina S.C. Calheiros a , Anouk F. Duque a , Alexandra Moura b , Isabel S. Henriques b ,

António Correia b , António O.S.S. Rangel a , Paula M.L. Castro a,∗
a Universidade Católica Portuguesa, Escola Superior de Biotecnologia, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal
b CESAM & Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: Constructed wetlands (CWs) have been recognized as being able to effectively treat
Received 2 September 2008 wastewater from municipal and industrial sources. This study focused on the effect of
Received in revised form different substrates and long-term operation of horizontal subsurface flow CWs treating tan-
31 October 2008 nery wastewater on the bacterial communities. The CWs were planted with Typha latifolia in
Accepted 23 November 2008 three types of substrate: two units with different types of expanded clay aggregates and one
unit with fine gravel. Another unit with expanded clay was left unvegetated. Changes in the
bacterial community related to the type of substrate, different hydraulic loading rates and
Keywords: along CW operation were examined using denaturating gradient gel electrophoresis (DGGE).
Bacterial communities Bacterial enumeration was also performed and several bacterial isolates were retrieved from
Constructed wetland the CWs. Phylogenetic affiliations of those isolates were obtained on the basis of 16S rRNA
DGGE gene sequences and revealed that they were closely related to the genera Bacillus (TM1S1,
Typha latifolia TM1R3, TNR1 and TAR1), Paracoccus (TM1R2), Pseudomonas (TM1R1) and Halomonas (TM1S2).
Industrial wastewater The type of substrate and the presence of T. latifolia had a major effect on the species
richness and the structure of bacterial communities as inferred by numerical analysis of
DGGE profiles.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction often encountered imposing treatment needs in order to avoid

The tannery industry converts rawhide or skin, a putrescible
material, into leather, a stable material, so that it can be used
in the manufacture of a wide range of consumer products.
Most of the steps of the tannery operations are carried out
in water. Consequently, the wastewater treatment is of major
concern. Tannery wastewater composition varies considerably
with the production process that is engaged by the specifi-
cation of the final product. Problems related to high organic
content, and the presence of sulphides and chromium are
negative impacts on the environment (COTANCE, 2002).
Constructed wetlands (CWs) are an effective method for
wastewater treatment (Vymazal, 2005), inclusively for several
types of industrial wastewater (Kadlec et al., 2000). In the case
of the tannery industry these systems have already shown
favorable performance (Calheiros et al., 2007, 2008a). The treat-
ment mechanisms in CWs encompass a mix of physical,
chemical, and biological processes. The vegetation is essential
in wetland treatment systems (Kadlec et al., 2000); however,
the main role in the transformation and mineralization of

∗
Corresponding author. Tel.: +351 22 5580059; fax: +351 22 5090351.
E-mail address: plcastro@esb.ucp.pt (P.M.L. Castro).
0925-8574/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ecoleng.2008.11.010
 e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 744–753
nutrients and organic pollutants is played by microorganisms
(Baptista, 2003; Stottmeister et al., 2003). The functional com-
position of the different bacterial groups in subsurface flow
CWs has not been fully explored (Ibekwe et al., 2003). Accord-
ing to Baptista (2003) these functional bacterial groups may be
largely determined by the environmental conditions and the
nature of the system feed.
Culture-dependent methods are known to be insufficient
for studying natural microbial community composition since
numerous microorganisms are uncultured under laboratory
conditions (Ward et al., 1990). The denaturating gradient gel
electrophoresis (DGGE) analysis of 16S rRNA genes amplified
by polymerase chain reaction (PCR) is useful for profiling com-
plex microbial populations (Muyzer et al., 1993). Examples
of studies of microbial communities in CWs using DGGE are
those of Ibekwe et al. (2003), who characterized the micro-
bial communities composition in CWs for dairy wastewater
treatment, of Truu et al. (2005), who investigated the varia-
tion of microbiological parameters within a CW for domestic
wastewater treatment, of Baptista et al. (2003), who analyzed
the microbial mechanisms of carbon removal in subsurface
flow wetlands, and of Nicomrat et al. (2006), who assessed
the microbial community in a CW receiving acid coal mine
drainage.
The aim of this study was to analyze the effect of
different substrates and long-term operation of horizon-
tal subsurface flow CWs treating tannery wastewater on
the dynamics of the bacterial communities. The bacte-
rial abundance was evaluated by plate counting and the
dynamics of the bacterial communities was evaluated by
DGGE electrophoresis of 16S rRNA gene amplification prod-
ucts. Numerical analysis of DGGE profiles allowed the
estimation of species richness along the operation of each
CW.
2. Methods
2.1. CWs set-up and physico-chemical analysis
The setup conditions of the CWs, located at a tannery wastew-
ater treatment plant in Portugal, are described in Calheiros et
al. (2008a). Briefly, the CWs consisted of three units planted
with Typha latifolia in different substrates: unit one (U1) and
unit three (U3) had expanded clay aggregates being respec-
tively Filtralite® MR 3–8 (FMR) and Filtralite® NR 3–8 (FNR) (from
maxit, Argilas Expandidas, SA, Portugal), and unit two (U2) had
fine gravel: AGH 4–8 (FG) (from Areipor-Areias Portuguesas,
Lda, Portugal). An unvegetated unit (Uc), with the expanded
clay aggregate FMR as substrate, was also included (Fig. 1). The
CWs operated with horizontal subsurface flow and had a sur-
face area of 1.2 m2 . They received tannery wastewater after
primary treatment under different hydraulic loading rates.
The two units with FMR, U1 and Uc, had been in operation
for 17 months during which two hydraulic regimes, 3 and
6 cm d−1 , were tested (Calheiros et al., 2007). The units U2
and U3 had an acclimation period of 3 weeks before the tan-
nery wastewater was applied. After that, all the systems were
operated for 31 months under different hydraulic loadings
and interruptions in feed (18, 6 and 8 cm d−1 ). Maintenance
745
Fig. 1 – Schematic representation of the constructed
wetlands (CWs).
of the systems was carried out according to Calheiros et al.
(2008a).
Wastewater samples were collected periodically from the
inlet and outlet of the CW units and physico-chemical param-
eters were determined based on Standard Methods (APHA,
1998): chemical oxygen demand (COD; Closed Reflux, Titri-
metric Method), biochemical oxygen demand (BOD5 ; 5-day
BOD Test), total suspended solids (total suspended solids, TSS;
dried at 103–105 ◦ C method), Kjeldahl nitrogen (TKN; Kjeldahl
method), nitrate nitrogen (NO3 − –N; nitrate electrode method),
ammonia nitrogen (NH3 –N; phenate method), total phospho-
rus (total P; manual digestion and flow injection analysis
for total phosphorus) and pH. The sulphate determination
(SO4 2− ; turbidimetric method) was done based on the method
of the Association of Official Analytical Chemists (AOAC,
1995). The analyses were done immediately after sample col-
lection otherwise samples were properly stored. Dissolved
oxygen (DO) and conductivity were registered with a WTW
handheld multi-parameter instrument 340i at the inlet and
outlet of the units. The substrates used in the units were
analyzed for organic matter content, based on Houba et al.
(1995).
2.2. Bacterial enumeration
Microbiological analyses were performed simultaneously with
physico-chemical analysis. Colony forming units (CFUs) were
determined based on the surface-plate counting procedure.
Briefly, three subsamples were pooled to form two compos-
ite samples (10 g) of plant roots and substrate (from a depth
between 10 and 15 cm) of each CW, being placed separately
in sterile tubes with 10 mL of saline solution (0.15 mol L−1
NaCl) and shaken on a vortex mixer for 1 min at room tem-
perature. Serial dilutions were made in duplicate and 0.1 mL
of each dilution was spread onto nutrient agar (LABM, UK).
Plates were incubated at 25 ◦ C for 4 days after which CFU
were counted. The same procedure was used for bacterial
enumeration of the wastewater at the inlet and outlet of the
CWs.
746 e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 744–753
2.3. Bacteria isolation, DNA extraction and RAPD
typing
Different bacterial colonies were isolated based on size, mor-
phology and pigmentation, from nutrient agar plates using
a streak-plate procedure. DNA of each isolate was obtained
by picking a colony with a sterile toothpick, suspending the
cells in 20 ␮L sterile water and incubating for 10 min at 100 ◦ C
(Henriques et al., 2006b). Molecular typing of bacterial isolates
was performed by random amplified polymorphic DNA (RAPD)
analysis. Amplification was performed in 25 ␮L reaction mix-
tures containing: 0.75 U Taq polymerase, 1.5 mM MgCl2 , 0.2 mM
of each dNTP, 1.0 ␮M primer M13 (MWG-Biotech AG) and
0.5 ␮L of crude cell lysates. The thermal cycling profile was
as follows: initial denaturation (94 ◦ C for 5 min); 45 cycles of
denaturation (94 ◦ C for 1 min), annealing (34 ◦ C for 2 min), and
extension (72 ◦ C for 2 min); and a final extension (72 ◦ C for
10 min) (Silva et al., 2006). The reactions were carried out
in a Bio-Rad iCycler Thermal Cycler (Bio-Rad Laboratories,
Richmond, CA, USA) using Taq polymerase and nucleotides
purchased from MBI Fermentas (Vilnius, Lithuania). Polymor-
phic DNA fragments were analyzed by electrophoresis in a
1% agarose gel in Tris–acetate–EDTA (TAE) buffer, after stain-
ing with ethidium bromide. Gel image was acquired using
a Molecular Image FX apparatus (Bio-Rad Laboratories, Her-
cules, CA, USA).
2.4. DNA sequence and phylogenetic analysis
Isolates displaying unique RAPD profiles were subsequently
identified by 16S rRNA gene sequencing analysis. Amplifica-
tion was performed with universal bacterial primers 27F and
1492R, as described by Lane (1991). PCR products were puri-
fied with Jetquick PCR Product Purification Spin Kit (Genomed,
Löhne, Germany). DNA sequencing was conducted under
BigDyeTM terminator cycling conditions, and analyzed using
an automatic sequencer 3730xl (Macrogen Inc., Seoul, Korea).
To determine the phylogenetic affiliation, similarity searches
were performed using the BLAST program (Altschul et al.,
1997).
2.5. Analysis of bacterial communities of substrate
and roots from CWs
Genomic DNA from substrate and root samples (six subsam-
ples were pooled to form one composite sample of plant roots
and substrate for each CW) was extracted using the Ultra
CleanTM Soil DNA Isolation Kit (MO BIO Laboratories, Inc.,
USA), according to the manufacturer’s protocol. PCR amplifi-
cation of bacterial 16S rRNA gene fragments was performed
using primers 338F GC and 518R, as described previously
(Henriques et al., 2006a). Nested PCR amplifications were per-
formed using as template 1 ␮L of the DNA amplicon obtained
after the first amplification round and using the same primers
and conditions applied in the first PCR amplification.
DGGE analysis was performed on a DCodeTM Universal
Mutation Detection System (Bio-Rad Laboratories, Hercules,
CA, USA). Samples containing approximately equal amounts
of nested-PCR amplicons were loaded onto 8% (w/v) poly-
acrylamide gels (37.5:1, acrylamide/bis-acrylamide) in 1× TAE
buffer using a denaturing gradient ranging from 35% to 60%
(100% denaturant solution is defined as 7 M urea and 40% (v/v)
formamide (Muyzer et al., 1993)). A standard marker was also
included in all gels, to serve as an indicator of the analysis
quality. The standard marker was constructed using bacte-
rial isolates (obtained as described above) selected to cover
an adequate range of bands. Electrophoresis conditions and
image acquisition were as described previously (Henriques et
al., 2006b).
DGGE profiles, concerning the presence and intensity of the
bands, were analyzed using GelCompar® II software (Version
4.6; Applied Maths, Sint-Martens-Latem, Belgium). Detected
band patterns were transferred to an absence/presence
matrix. The binary matrix was transformed into a similar-
ity matrix using the Bray-Curtis measure. Dendrograms were
generated by unweight pair group mean average (UPGMA)
cluster analysis. Cluster analysis and multidimensional scal-
ing diagram (MDS) were performed using PRIMER 5 for
Windows (Version 5.2, 2001, PRIMER-E Ltd.) (Clarke and Gorley,
2001).
DGGE banding data were used to estimate diversity, H
(Shannon and Weaver, 1963) and equitability, E (Pielou, 1975)
indexes to describe possible changes in the dominance among
phylotypes (Fromin et al., 2002).
2.6. Nucleotide sequence accession numbers
The 16S rRNA gene sequences of bacterial isolates obtained
in this study were deposited in GenBank under the
accession numbers TM1R1: EU430693, TM1R2: EU430694,
TM1R3: EU430695, TM1S1: EU430696, TM1S2: EU430697, TNR1:
EU430700, TAR1: EU430701.
2.7. Data analysis
Statistical analysis was performed using the software SPSS
(SPSS Inc., Chicago, IL, USA; Version 12.0). When applicable,
the data were analyzed through one-way analysis of variance
(ANOVA) and Student’s t-test. To detect the statistical signifi-
cance of differences (p < 0.05) between means of observation,
the Duncan test was performed. When applicable, values were
presented as the mean ± standard error.
3. Results
3.1. Physico-chemical characterization
In Fig. 2 the removal of several wastewater components by
the CWs is illustrated. COD, BOD5 and TSS inlet concentration
varied between 835–2261, 430–850 and 43–110 mg L−1 , respec-
tively. In general, the FMR unit U1 presented higher removal
levels of organic matter, followed by the FNR unit U3, the fine
gravel unit U2 and the FMR unvegetated unit Uc.
The inlet concentrations of TKN, NH3 and SO4 2− ranged
between 102–160, 60–98 and 78–1070 mg L−1 , respectively.
The outlet concentrations of TKN, NH3 and SO4 2− ranged
between 57–115, 35–65 and 32–1005 mg L−1 , respectively. For
NO3 − the inlet varied between 17–59 mg L−1 and the out-
let between 9–47 mg L−1 . Total phosphorus ranged between
 e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 744–753
Fig. 2 – Removal efficiency of (A) COD, BOD5 and TSS and (B)
TKN, NH3 and SO4 2 , by the constructed wetlands (CWs).
Dispersion bars represent standard error of the mean. U1:
CW with Typha latifolia planted in Filtralite® MR 3–8; U2: CW
with T. latifolia planted in fine gravel, AGH 4–8; U3: CW with
T. latifolia planted in Filtralite® NR 3–8; Uc: unvegetated unit
with Filtralite® MR 3–8. One-way ANOVA was performed in
order to compare the performance in terms of removal
efficiency between units for each parameter. Columns
marked with different letters differed significantly
according to Duncan’s multiple range tests at of p < 0.05.
0.13–0.83 mg L−1 at the inlet and between 0.06–0.77 mg L−1 at
the outlet.
DO at the inlet and outlet was low, varying between
0.17–0.99 mg L−1 and 0.09–0.88 mg L−1 , respectively. The pH
values were quite stable at the outlet (7.23–8.68) although the
inlet values varied between 4.76 and 8.26. Conductivity varied
between 4.74–9.64 mS cm−1 at the inlet and 5.00–9.61 mS cm−1
at the outlet.
The organic matter content was determined for the three
substrates as being 0.30% for FMR, 0.30% for FNR and 0.04% for
FG. At the inlet and outlet zone of each unit the organic matter
content was also determined as being, respectively: 0.86 and
0.63% for U1, 0.37 and 0.17% for U2, 1.00 and 0.71% for U3 and
0.89 and 0.71% for Uc.
3.2. Plate-counting and bacterial isolation
The enumeration of bacteria in the units, including roots and
substrate, is shown in Fig. 3. One-way ANOVA was applied
to compare the CFUs between the planted units and no sig-
nificant differences were found. When Student’s t-test was
applied to compare bacterial counts from roots and substrate
of each unit significant differences were seen for units U2 and
U3, although for U1 no significant differences were found.
747
The number of aerobic bacteria present in the wastewa-
ter at the inlet and outlet of the CWs was also determined.
Samples collected periodically (February, July, September,
December 2005 and February, June, October 2006) showed a
variation between 3.9 × 101 and 4.2 × 105 for the inlet, between
3.2 × 103 and 2.2 × 106 for the outlet of the vegetated units (U1,
U2 and U3) and between 2.6 × 104 and 1.2 × 106 for the outlet
of the unvegetated unit (Uc).
Dominant bacterial isolates obtained from the plates were
further molecular typed by RAPD-PCR. Seven different RAPD
types were then characterized through sequencing of the 16S
rRNA encoding gene. According to BLAST results, 2 strains
were affiliated with ␥-Proteobacteria, 4 with Firmicutes and 1
with ␣-Proteobacteria. These strains were isolated from plant
roots (TM1R1, TM1R2, and TM1R3) and substrate (TM1S1 and
TM1S2) of unit U1 and from plant roots of unit U2 (TAR1) and
unit U3 (TNR1) (Table 1).
3.3. DGGE analysis of bacterial community
Analysis of bacterial communities from substrate and root
samples, collected along the CWs operation, was performed
by PCR–DGGE. Two examples of banding patterns for the 16S
rRNA gene PCR–DGGE amplicons are presented in Fig. 4, corre-
sponding to units U1 and U2. Each gel presents the variability
within each CW at different sampling periods correspond-
ing to different hydraulic conditions. Reproducibility of PCR
amplification and DGGE was confirmed in replicates.
Table 2 shows, for each CW, the number of bands in each
lane and the Shannon and equitability indexes (calculated by
using the DGGE profiles) correspondent to the different sam-
pling times within each gel. The total number of different band
positions in the four gels was 68 (58 in gel U1, 50 in gel U2, 51 in
gel U3 and 43 in gel Uc). The Shannon’s diversity index (H) was
in average of 1.10 ± 0.07 for U1, 1.08 ± 0.03 for U2, 1.14 ± 0.05
for U3 and 1.01 ± 0.07 for Uc. The equitability index (E) was in
average of 0.77 ± 0.05 for U1, 0.82 ± 0.02 for U2, 0.80 ± 0.02 for
U3 and 0.73 ± 0.05 for Uc.
Fig. 5 shows clustering analysis of DGGE banding patterns.
In general, samples clustered mostly according to the place of
collection (substrate or root) and the type of substrate, being
the temporal factor less relevant in bacterial assemblage com-
position.
Multidimensional scaling diagrams of similarity matrices
(Fig. 6), calculated from the DGGE patterns of samples, showed
that the bacterial community changed according to the differ-
ent substrates. Concerning the analysis between the FMR units
U1 and the unvegetated control (Uc), differences between
community profiles within each unit are clear, with a higher
dispersion for the vegetated unit U1.
4. Discussion
This study intended to investigate the bacterial communi-
ties in CWs established with different substrates, namely
expanded clay aggregates and fine gravel. Close attention was
given to bacteria, although it is known that fungi may also play
an important role in the function of wetlands (Baptista, 2003).
748 e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 744–753

Fig. 3 – Bacterial enumeration analyzed by plate counts and expressed in CFUs g−1 for substrate and plant roots of
constructed wetlands (CWs). Dispersion bars represent standard error of the mean. U1: CW with T. latifolia planted in
Filtralite® MR 3–8; U2: CW with T. latifolia planted in fine gravel, AGH 4–8; U3: CW with T. latifolia planted in Filtralite® NR 3–8;
Uc: unvegetated unit with Filtralite® MR 3–8.

The complex physico-chemical composition of the tannery
wastewater passing through the wetland is of major impor-
tance since it can have a relevant effect on the vegetation
(Calheiros et al., 2007, 2008a,b) and in the bacterial commu-
nities that inhabit these ecosystems. The wastewater content
varies in terms of nutrients and toxic substances and can
limit or promote bacterial activity and growth. The main role
concerning the direct degradation of organic chemicals in
wastewater treatment is played by microorganisms despite
the capacity of plants to detoxify xenobiotics (Stottmeister
et al., 2003). The organic compounds degradation in the hori-
zontal subsurface flow wetlands is carried out aerobically and
anaerobically at different extents by bacteria attached to plant
roots and substrate surfaces (Kadlec et al., 2000).
The three tested substrates have proven to be adequate
for T. latifolia development although there was higher plant
propagation for the expanded clay aggregates (Calheiros et al.,
2008a). The macrophytes are important in the wetlands since
they provide structure and a source of reduced carbon for the
microbes that mediate most of the pollutant transformations
occurring in the wetlands (Kadlec et al., 2000). Collins et al.
(2004) concluded that plants do have an effect on water qual-
ity, in part because they affect bacterial assemblages. Higher
pollutant removals, in terms of COD and BOD5 , were achieved
in expanded clay planted units after long-term operation. The
similar behavior of the expanded clay systems (U1 and U3)
concerning the pollutant removal may be attributed to the fact
that they may have similar functional group of microorgan-
isms.
The substrate is an important wetland component since
it supports plant growth, establishment of microbial biofilms
and influences the hydraulic processes (Stottmeister et al.,

Table 1 – Phylogenetic affiliation of bacterial strains isolated from the constructed wetlands.
Isolate NCBI Phylogenetic Closest relative (accession no.) Similarity Origin
accession no. affiliation (%)

Pseudomonas stutzeri ST27MN3 (U26419) 99 Sea

TM1R1 EU430693 ␥-Proteobacteria
Pseudomonas stutzeri 19smn4 (U22426) 99 Sea
Paracoccus sp. G30 (DQ667122) 98 Antarctic seawater
TM1R2 EU430694 ␣-Proteobacteria
Paracoccus sp. G29 (DQ667121) 98 Antarctic seawater
Bacillus pumilus MLA14 (EF462914) 99 n.d.
TM1R3 EU430695 Firmicutes
Bacillus sp. NS88 (EF633174) 99 Phyllostachys edulis
Bacillus sp. BCw063 (DQ492812) 99 Arctic sea water
TM1S1 EU430696 Firmicutes
Bacillus sp. GB02-29 (DQ079002) 99 Hydrothermal
sediments and plumes
Halomonas sp. 3014 (AM110971) 99 Deep sea sediment
TM1S2 EU430697 ␥-Proteobacteria
Halomonas sp. A3-3 (AB219125) 99 Fermented foods
Bacillus sp. BCw063 (DQ492812) 99 Arctic sea water
TNR1 EU430700 Firmicutes
Bacillus sp. GB02-29 (DQ079002) 99 Hydrothermal
sediments and plumes
Bacterium 8-gw3-5 (DQ990048) 99 River waters and
TAR1 EU430701 Firmicutes
shallow groundwater
Bacillus pumilus NUC-F (DQ833752) 99 Agricultural soil
 e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 744–753 749

Fig. 4 – DGGE analysis of 16 rRNA gene fragments of total bacterial population from samples of the root and substrate of
constructed wetlands planted with T. latifolia in different matrixes. (A) Gel image of root and substrate samples collected in
U1 in September 2004 (lanes 1 and 2), February 2005 (substrate, lane 4), June 2005 (lanes 5 and 6), July 2005 (lanes 7 and 8),
July 2006 (lanes 9 and 10), October 2006 (lanes 11 and 12) and (B) gel image of root and substrate samples collected in U2 in
February 2005 (lanes 19 and 20), June 2005 (lanes 21 and 22), July 2005 (lanes 23 and 24), July 2006 (lanes 25 and 26), October
2006 (lanes 27 and 28). A DNA marker (M) was included in all the gels to serve as control.

2003). A porous matrix, such as expanded clay, provides a
greater surface area for treatment contact and biofilm devel-
opment. Each substrate used here has different characteristics
in terms of pH, electrical conductivity, porosity, and organic
matter content. The higher organic matter content verified at
the inlet substrate of all units was attributed to the fact that
at this point the wastewater organic loading was higher than
at the outlet.
The number of CFU found in the vegetated units is within
the range of what has been published by Truu et al. (2005)
for aerobic heterotrophic bacteria in horizontal subsurface
flow CW for domestic wastewater treatment, filled with coarse

Table 2 – Number of bands and Shannon diversity (H) and equitability (E) indexes, calculated for the constructed
wetlands (CWs). U1: CW with Typha latifolia planted in Filtralite® MR 3–8; U2: CW with T. latifolia planted in fine gravel,
AGH 4–8; U3: CW with T. latifolia planted in Filtralite® NR 3–8; Uc: unvegetated unit with Filtralite® MR 3–8.
Samplesa U1 U2 U3 Uc

Nr bands H E Nr bands H E Nr bands H E Nr bands H E

Set R 04 35 1.40 0.91 n.d. n.d. n.d. n.d. n.d. n.d. n.a. n.a. n.a.
Set S 04 36 1.18 0.76 n.d. n.d. n.d. n.d. n.d. n.d. 17 0.82 0.66
Feb R 05 n.d. n.d. n.d. 21 0.94 0.71 17 0.81 0.66 n.a. n.a. n.a.
Feb S 05 26 0.88 0.62 14 0.92 0.80 21 1.05 0.79 25 1.06 0.76
Jun R 05 29 1.02 0.70 22 1.18 0.88 24 1.09 0.79 n.a. n.a. n.a.
Jun S 05 30 1.01 0.68 27 1.20 0.84 22 1.05 0.78 23 1.00 0.74
Jul R 05 26 1.64 1.16 18 1.05 0.84 29 1.18 0.81 n.a. n.a. n.a.
Jul S 05 24 0.76 0.55 19 1.12 0.87 25 1.21 0.87 23 1.29 0.94
Sep S 05 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 21 0.64 0.49
Jun S 06 n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. n.d. 30 1.04 0.71
Jul R 06 22 1.13 0.84 22 1.16 0.86 35 1.30 0.84 n.a. n.a. n.a.
Jul S 06 21 0.94 0.71 25 1.13 0.80 35 1.23 0.80 30 1.02 0.69
Oct R 06 22 1.09 0.81 25 1.10 0.79 33 1.26 0.83 n.a. n.a. n.a.
Oct S 06 22 1.02 0.76 22 1.03 0.77 26 1.19 0.84 28 1.18 0.82

n.a., not applicable; n.d., not determined.

a
In samples collumn is presented the month, location (R: root or S: substrate) and year, that each sample was collected.
750 e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 744–753

Fig. 5 – Cluster analysis of microbial communities generated by the analysis of DGGE 16S rRNA gene patterns representing
the genetic similarity of the microbial profiles acquired. Similarities were calculated using the Bray-Curtis measure. (A)
Cluster analysis of bacterial communities from U1 (Filtralite® MR 3–8 substrate), U2 (fine gravel: AGH 4–8 substrate) and U3
(Filtralite® NR 3–8 substrate). (B) Cluster analysis of bacterial communities from U1 and Uc (unvegetated control with
Filtralite® MR 3–8 substrate).

sand and dominated by Scirpus sylvaticus, Urtica dioica and
Epilobium hirsutum. The fact that there were no significant dif-
ferences in bacterial numbers between the substrate and the
roots in unit U1 was attributed to the diffuse propagation of T.
latifolia, since this unit was established for units longer than
U2 and U3. Significantly higher numbers of CFUs were fre-
quently found in the wastewater samples collected from the
CWs outlet when compared to the inlet, which can be due to
the fact that the water passing through the wetland washes
out bacteria from the substrate. Although in this study an aer-
obic plate count method was followed it would be interesting,
in the future, to undertake anaerobic enumeration consid-
ering the fact that the organic degradation can occur both
aerobically and anaerobically in the CW systems. Attempts
to evaluate the relative importance of different microbial
reactions on organic matter removal (in terms of COD), in hor-
izontal subsurface flow CWs treating urban wastewater, have
been carried out using a 2D simulation model (Ojeda et al.,
2008). They indicated that the microbial anaerobic reactions
involved in organic matter removal (methanogenesis and sul-
phate reduction) occurred over larger areas of the wetlands
than anoxic (denitrification) and aerobic reactions.
 e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 744–753
Fig. 6 – Multidimensional scaling diagram of DGGE patterns
of samples collected in the constructed wetlands (CWs) of
(A) U2 and U3 and (B) U1 and Uc. U1: CW with T. latifolia
planted in Filtralite® MR 3–8; U2: CW with T. latifolia planted
in fine gravel, AGH 4–8; U3: CW with T. latifolia planted in
Filtralite® NR 3–8; Uc: unvegetated unit with Filtralite® MR
3–8.
The microbial diversity in the substrate and rhizosphere
is of great importance since it may be influenced by the type
and amount of plants. Most of the bacterial isolates retrieved
from the roots and substrate of the CWs presented here were
similar to environmental isolates reported from sources such
as river and sea waters, sediments and soil. Two isolates affil-
iated with ␥-Proteobacteria were recovered from substrate and
roots from the CWs. Franco et al. (2005), based on enrichment
cultures with soil collected nearby the same CWs, have iso-
lated bacteria able to degrade polyphenols used in the tannery
process being all of them members of ␥-Proteobacteria.
The dynamics of the bacterial community in the CWs over 3
years of operation were analyzed by comparison of 16S rDNA
PCR–DGGE profiles. Data from this study showed that there
was a diverse community of bacteria in the CWs, and that
might influence to different extents the final effluent quality.
Although the FMR units U1 and Uc had been in operation for a
bit longer than U3 and U2, which could indicate that these sys-
tems would be more stable and resistant to the stress caused
751
by the hydraulic alterations and wastewater contaminations,
no direct relationship could be found.
In this study the community composition was not deep-
ened in relation to the identification of bacterial groups.
Despite that, species richness and the structure of bacterial
communities have been inferred by numerical analysis of
DGGE profiles (Moura et al., 2007). DGGE profiles showed a
high variability among bacterial assemblages. The presence
of vegetation seemed to have a major effect on the commu-
nity diversity, as lower bacterial diversity (lower H values) was
observed in the unvegetated unit (Uc). Similarly, Baptista et
al. (2003) reported that microbial communities from inlet and
outlet of horizontal subsurface flow CW with plants are dif-
ferent when compared to an unplanted wetland, treating a
solution of filtered beer. In this study, species richness was
higher in the unit with the expanded clay FNR (U3), followed
by FMR (U1) and FG (U2). The differences observed might be
explained in part by the substrates that differ in characteris-
tics such as pH, conductivity and porosity (Calheiros et al.,
2008a,b). In general, all units showed higher diversity near
root comparing to substrate, which can be related to rhi-
zosphere effects on bacterial communities. Previous studies
have reported the importance of plant metabolites excreted to
the rhizosphere, such as vitamins, that may stimulate micro-
bial growth (Stottmeister et al., 2003). Additionally, Marschner
et al. (2002) have studied the microbial community structure in
the rhizosphere of cluster roots of Lupinus albus and reported
that changes in the community structure may be related with
organic acid plant exudates.
Differences in microbial community structure were
detected by using the equitability index (Pielou, 1975). The
equitability was higher in the unit with the sand FG (U2) fol-
lowed by the units with expanded clay FNR (U3) and then FMR
(U1), and finally the unvegetated unit (Uc). Vacca et al. (2005)
have reported that when using CWs for domestic wastewater
treatment, the rhizosphere of P. australis is colonized by dis-
tinct communities depending on the filter material (sand and
expanded clay) and have concluded that the technology, filter
material and plant have a selective influence on the microbial
community within the CW. The functional diversity may be
linked to certain extents to the ecophysiological roles played
by functional groups although the present study did not allow
such conclusions. In general, equitability was higher in root
samples comparing to substrate samples. Again, this may be
related to the rhizosphere effect on microbial communities
that may promote the growth of certain bacterial groups.
The assessment of microbial communities in CWs has been
addressed by several authors (Baptista et al., 2003; Ibekwe
et al., 2003; Truu et al., 2005; Nicomrat et al., 2006). Ibekwe
et al. (2003) have characterized the microbial communities
and composition in CW for dairy wastewater and reported
that these systems are dependent on microbial communi-
ties for optimal wastewater treatment. Since little information
is available regarding the bacterial communities that inhabit
these ecosystems, the PCR–DGGE technique employed here
was of great importance in obtaining new data concerning
the microbial structure and dynamics of CWs for tannery
wastewater treatment. The study of dynamics of microbial
communities from CWs is thus essential in understanding
these treatment systems, being a valuable tool for improv-
752 e c o l o g i c a l e n g i n e e r i n g 3 5 ( 2 0 0 9 ) 744–753
ing its design and operation. A more extensive investigation
may also be undertaken at other levels, mainly those related
to the detection of microbial genes encoding enzymes linked
to degradation pathways, and analysis of their quantitative
expression within each CW.
5. Conclusions
(1) The type of substrate and the presence of T. latifolia seemed
to have a major effect on the dynamics and diversity of the
bacterial community.
(2) The variations introduced in the systems in terms of
hydraulic regimes and wastewater contamination did not
result in substantial changes in the diversity of the micro-
bial communities along the systems operation.
(3) A high diversity of bacterial populations was found in the
CW units (according to DGGE profiles) and that could con-
tribute to the resilience and resistance of the CWs to stress
created by the wastewater loadings applied.
(4) The type of substrate was more relevant in determining
the bacterial composition than the temporal variability,
with the planted CWs units presenting a high similarity
within the same year for root and substrate.
Acknowledgements
The authors thank Dias Ruivo, Curtumes e Produtos Indus-
triais, Lda for making possible the establishment of this
project and to maxit, Argilas Expandidas, SA that kindly
offered Filtralite® . Cristina S.C. Calheiros, Isabel Hen-
riques and Alexandra Moura wish to thank the research
grants from Fundação para a Ciência e Tecnologia (FCT),
Portugal (SFRH/BDE/15507/2004, SFRH/BPD/21384/2005 and
SFRH/BD/19433/2004). The work was supported by the project
POCI/AMB/60126/2004.
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