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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.
∗
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 ) 744–753 745
2.3. Bacteria isolation, DNA extraction and RAPD buffer using a denaturing gradient ranging from 35% to 60%
typing (100% denaturant solution is defined as 7 M urea and 40% (v/v)
formamide (Muyzer et al., 1993)). A standard marker was also
Different bacterial colonies were isolated based on size, mor- included in all gels, to serve as an indicator of the analysis
phology and pigmentation, from nutrient agar plates using quality. The standard marker was constructed using bacte-
a streak-plate procedure. DNA of each isolate was obtained rial isolates (obtained as described above) selected to cover
by picking a colony with a sterile toothpick, suspending the an adequate range of bands. Electrophoresis conditions and
cells in 20 L sterile water and incubating for 10 min at 100 ◦ C image acquisition were as described previously (Henriques et
(Henriques et al., 2006b). Molecular typing of bacterial isolates al., 2006b).
was performed by random amplified polymorphic DNA (RAPD) DGGE profiles, concerning the presence and intensity of the
analysis. Amplification was performed in 25 L reaction mix- bands, were analyzed using GelCompar® II software (Version
tures containing: 0.75 U Taq polymerase, 1.5 mM MgCl2 , 0.2 mM 4.6; Applied Maths, Sint-Martens-Latem, Belgium). Detected
of each dNTP, 1.0 M primer M13 (MWG-Biotech AG) and band patterns were transferred to an absence/presence
0.5 L of crude cell lysates. The thermal cycling profile was matrix. The binary matrix was transformed into a similar-
as follows: initial denaturation (94 ◦ C for 5 min); 45 cycles of ity matrix using the Bray-Curtis measure. Dendrograms were
denaturation (94 ◦ C for 1 min), annealing (34 ◦ C for 2 min), and generated by unweight pair group mean average (UPGMA)
extension (72 ◦ C for 2 min); and a final extension (72 ◦ C for cluster analysis. Cluster analysis and multidimensional scal-
10 min) (Silva et al., 2006). The reactions were carried out ing diagram (MDS) were performed using PRIMER 5 for
in a Bio-Rad iCycler Thermal Cycler (Bio-Rad Laboratories, Windows (Version 5.2, 2001, PRIMER-E Ltd.) (Clarke and Gorley,
Richmond, CA, USA) using Taq polymerase and nucleotides 2001).
purchased from MBI Fermentas (Vilnius, Lithuania). Polymor- DGGE banding data were used to estimate diversity, H
phic DNA fragments were analyzed by electrophoresis in a (Shannon and Weaver, 1963) and equitability, E (Pielou, 1975)
1% agarose gel in Tris–acetate–EDTA (TAE) buffer, after stain- indexes to describe possible changes in the dominance among
ing with ethidium bromide. Gel image was acquired using phylotypes (Fromin et al., 2002).
a Molecular Image FX apparatus (Bio-Rad Laboratories, Her-
cules, CA, USA). 2.6. Nucleotide sequence accession numbers
2.4. DNA sequence and phylogenetic analysis The 16S rRNA gene sequences of bacterial isolates obtained
in this study were deposited in GenBank under the
Isolates displaying unique RAPD profiles were subsequently accession numbers TM1R1: EU430693, TM1R2: EU430694,
identified by 16S rRNA gene sequencing analysis. Amplifica- TM1R3: EU430695, TM1S1: EU430696, TM1S2: EU430697, TNR1:
tion was performed with universal bacterial primers 27F and EU430700, TAR1: EU430701.
1492R, as described by Lane (1991). PCR products were puri-
fied with Jetquick PCR Product Purification Spin Kit (Genomed, 2.7. Data analysis
Löhne, Germany). DNA sequencing was conducted under
BigDyeTM terminator cycling conditions, and analyzed using Statistical analysis was performed using the software SPSS
an automatic sequencer 3730xl (Macrogen Inc., Seoul, Korea). (SPSS Inc., Chicago, IL, USA; Version 12.0). When applicable,
To determine the phylogenetic affiliation, similarity searches the data were analyzed through one-way analysis of variance
were performed using the BLAST program (Altschul et al., (ANOVA) and Student’s t-test. To detect the statistical signifi-
1997). cance of differences (p < 0.05) between means of observation,
the Duncan test was performed. When applicable, values were
2.5. Analysis of bacterial communities of substrate presented as the mean ± standard error.
and roots from CWs
Genomic DNA from substrate and root samples (six subsam- 3. Results
ples were pooled to form one composite sample of plant roots
and substrate for each CW) was extracted using the Ultra 3.1. Physico-chemical characterization
CleanTM Soil DNA Isolation Kit (MO BIO Laboratories, Inc.,
USA), according to the manufacturer’s protocol. PCR amplifi- In Fig. 2 the removal of several wastewater components by
cation of bacterial 16S rRNA gene fragments was performed the CWs is illustrated. COD, BOD5 and TSS inlet concentration
using primers 338F GC and 518R, as described previously varied between 835–2261, 430–850 and 43–110 mg L−1 , respec-
(Henriques et al., 2006a). Nested PCR amplifications were per- tively. In general, the FMR unit U1 presented higher removal
formed using as template 1 L of the DNA amplicon obtained levels of organic matter, followed by the FNR unit U3, the fine
after the first amplification round and using the same primers gravel unit U2 and the FMR unvegetated unit Uc.
and conditions applied in the first PCR amplification. The inlet concentrations of TKN, NH3 and SO4 2− ranged
DGGE analysis was performed on a DCodeTM Universal between 102–160, 60–98 and 78–1070 mg L−1 , respectively.
Mutation Detection System (Bio-Rad Laboratories, Hercules, The outlet concentrations of TKN, NH3 and SO4 2− ranged
CA, USA). Samples containing approximately equal amounts between 57–115, 35–65 and 32–1005 mg L−1 , respectively. For
of nested-PCR amplicons were loaded onto 8% (w/v) poly- NO3 − the inlet varied between 17–59 mg L−1 and the out-
acrylamide gels (37.5:1, acrylamide/bis-acrylamide) in 1× TAE 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 ) 744–753 747
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 propagation for the expanded clay aggregates (Calheiros et al.,
wastewater passing through the wetland is of major impor- 2008a). The macrophytes are important in the wetlands since
tance since it can have a relevant effect on the vegetation they provide structure and a source of reduced carbon for the
(Calheiros et al., 2007, 2008a,b) and in the bacterial commu- microbes that mediate most of the pollutant transformations
nities that inhabit these ecosystems. The wastewater content occurring in the wetlands (Kadlec et al., 2000). Collins et al.
varies in terms of nutrients and toxic substances and can (2004) concluded that plants do have an effect on water qual-
limit or promote bacterial activity and growth. The main role ity, in part because they affect bacterial assemblages. Higher
concerning the direct degradation of organic chemicals in pollutant removals, in terms of COD and BOD5 , were achieved
wastewater treatment is played by microorganisms despite in expanded clay planted units after long-term operation. The
the capacity of plants to detoxify xenobiotics (Stottmeister similar behavior of the expanded clay systems (U1 and U3)
et al., 2003). The organic compounds degradation in the hori- concerning the pollutant removal may be attributed to the fact
zontal subsurface flow wetlands is carried out aerobically and that they may have similar functional group of microorgan-
anaerobically at different extents by bacteria attached to plant isms.
roots and substrate surfaces (Kadlec et al., 2000). The substrate is an important wetland component since
The three tested substrates have proven to be adequate it supports plant growth, establishment of microbial biofilms
for T. latifolia development although there was higher plant 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 (%)
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 at this point the wastewater organic loading was higher than
greater surface area for treatment contact and biofilm devel- at the outlet.
opment. Each substrate used here has different characteristics The number of CFU found in the vegetated units is within
in terms of pH, electrical conductivity, porosity, and organic the range of what has been published by Truu et al. (2005)
matter content. The higher organic matter content verified at for aerobic heterotrophic bacteria in horizontal subsurface
the inlet substrate of all units was attributed to the fact that 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
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
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 in the future, to undertake anaerobic enumeration consid-
Epilobium hirsutum. The fact that there were no significant dif- ering the fact that the organic degradation can occur both
ferences in bacterial numbers between the substrate and the aerobically and anaerobically in the CW systems. Attempts
roots in unit U1 was attributed to the diffuse propagation of T. to evaluate the relative importance of different microbial
latifolia, since this unit was established for units longer than reactions on organic matter removal (in terms of COD), in hor-
U2 and U3. Significantly higher numbers of CFUs were fre- izontal subsurface flow CWs treating urban wastewater, have
quently found in the wastewater samples collected from the been carried out using a 2D simulation model (Ojeda et al.,
CWs outlet when compared to the inlet, which can be due to 2008). They indicated that the microbial anaerobic reactions
the fact that the water passing through the wetland washes involved in organic matter removal (methanogenesis and sul-
out bacteria from the substrate. Although in this study an aer- phate reduction) occurred over larger areas of the wetlands
obic plate count method was followed it would be interesting, 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 ) 744–753 751
ing its design and operation. A more extensive investigation Calheiros, C.S.C., Rangel, A.O.S.S., Castro, P.M.L., 2008a. Evaluation
may also be undertaken at other levels, mainly those related of different substrates to support the growth of Typha latifolia
to the detection of microbial genes encoding enzymes linked in constructed wetlands treating tannery wastewater over
long-term operation. Bioresour. Tecnhol. 99, 6866–6877.
to degradation pathways, and analysis of their quantitative
Calheiros, C.S.C., Rangel, A.O.S.S., Castro, P.M.L., 2008b. The
expression within each CW. effects of tannery wastewater on the development of different
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