Ecological Engineering 82 (2015) 4956

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Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Bacteria viability and decay in water and soil of vertical subsurface ow

constructed wetlands
P. Foladori a, * , L. Bruni b , S. Tamburini c
a
b
c

Department of Civil, Environmental and Mechanical Engineering, University of Trento, via Mesiano 77, 38123 Trento, Italy
Agenzia per la Depurazione, Autonomous Province of Trento, via Gilli 3, 38122 Trento, Italy
Centre for Integrative Biology (CIBIO), University of Trento, Via delle Regole 101, Mattarello, 38123 Trento, Italy

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 29 April 2014
Received in revised form 4 March 2015
Accepted 6 April 2015
Available online xxx

In this study the functional status of bacterial biomass within a vertical subsurface ow (VSSF)
constructed wetland was examined with the aim to understand the relationship between viable and dead
bacteria in soil and inuent/efuent wastewater and elucidate the large amount of dead cells in the soil
which may affect the long-term behavior of the system. The quantication of viable and dead bacteria in
inuent and efuent wastewater and in the soil of a VSSF was performed at single-cell level by ow
cytometry (FCM). An optimised pre-treatment was applied to soil samples using sodium pyrophosphate
and ultrasonication at a specic energy of 80 kJ/L. Viable and dead cells were detected on the basis of
cellular membrane integrity coupling SYBR-Green I and Propidium Iodide. The bacteria prole in the VSSF
soil depends on the depth and the material grain size. In the upper 010 cm sand layer the number of total
bacteria per gram of dry weight (DW) was higher (1.82
109 cells/gDW) than in the deeper 4050 cm
(4.8
108 cells/gDW) probably due to the vertical feeding and a sieving effect of inuent in the top layers.
Bacterial biomass in the entire VSSF depth was 0.082 mgVSS/gDW or 144 gVSS/m3 (per cubic meter of
VSSF bed). Size of viable bacteria in the VSSF was smaller (0.16 mm3/cell) than typical size of activated
sludge (0.23 mm3/cell), due to lower nutrient conditions and a longer retention time of viable bacteria in
the bed, estimated at around 130 days by mass balance. Dead bacteria were prevalent in the VSSF soil
with a viable/dead bacteria ratio (V/D) of 0.52. The content of dead bacteria might be higher in the soil
due to the presence of unsaturated zones not reached by fresh inuent wastewater (dead-zones),
where moisture and substrate are not so available and bacteria may die. Conversely, the higher V/D ratio
(3.3) in the efuent reects the enrichment of wastewater with viable bacteria during the passage
through the VSSF bed and along preferential water ow, with higher water content and substrate
availability, where the bacterial growth is favored.
2015 Elsevier B.V. All rights reserved.

Keywords:
Wastewater
Vertical subsurface ow constructed
wetland
Flow cytometry
Bacteria
Viability
Decay

1. Introduction
The removal of pollutants in Constructed Wetlands (CW) is
mostly driven by microorganisms which are closely tied to the
cycling of carbon, nitrogen and sulfur (Faulwetter et al., 2009).
Organic matter removal is carried out by heterotrophic bacteria
living under both aerobic and anaerobic conditions, while nitrogen
removal in CWs is the result of the combination of nitrication and
denitrication, even though the signicant role of anaerobic
oxidation of ammonium was demonstrated in the last years (Truu
et al., 2009). Although the high relevance of bacteria communities
that live in CW and in vertical subsurface ow CWs (VSSF) is widely
recognised and well documented (Faulwetter et al., 2009; Truu

* Corresponding author. Tel.: +39 0461 282683.

E-mail address: paola.foladori@ing.unitn.it (P. Foladori).
http://dx.doi.org/10.1016/j.ecoleng.2015.04.058
0925-8574/ 2015 Elsevier B.V. All rights reserved.

et al., 2009; Kadlec and Wallace, 2009), the quantication and the
distribution of bacteria in such systems (both in water and in soil)
have not been investigated sufciently and some microbiological
aspects remain unknown (Tietz et al., 2008; Langergraber, 2011).
Conventional cultivation-based methods produce heavily
biased results and strong underestimations when applied to
quantifying bacteria in CWs due to the uncultivability of a large
fraction of bacteria (Decamp and Warren, 2001; Alexandrino et al.,
2007).
Molecular and biochemical approaches can lead to more indepth microbiological investigations, but they are not yet
completely exploited in the CWs eld (Faulwetter et al., 2009).
Among them, uorescent staining of microbial nucleic acids,
uorescent in-situ hybridisation of 16S rRNA gene sequences
(FISH), polymerase chain reaction (PCR or PCR-DGGE) or new
emerging metagenomic approach have been proposed in the
literature to investigate microbial diversity and abundance in

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P. Foladori et al. / Ecological Engineering 82 (2015) 4956

wastewater treatment and CWs (Decamp and Warren, 2001;

Sawaittayothin and Polprasert, 2007; Alexandrino et al., 2007;
Tietz et al., 2008; Krasnits et al., 2009; Faulwetter et al., 2009; Zhao
et al., 2010; Zhang et al., 2012; Adrados et al., 2014; Ligi et al., 2014;
Lv et al., 2014). In these microbiological investigations, bacteria
viability and death have rarely been quantied (Decamp and
Warren, 2001). When the absolute quantication of bacteria is
done under epiuorescence or confocal microscope, the analyses
result as time-consuming and labor-intensive, requiring several
hours to analyse few samples.
Flow cytometry (FCM) has been proposed for the absolute
quantication of bacteria in environmental samples at a rate of
thousands of cells in few minutes, with high accuracy and
precision (Porter et al., 1997; Steen, 2000; Vives-Rego et al.,
2000; Bergquist et al., 2009; Foladori et al., 2010). FCM is a multiparametric, single-cell and rapid analysis which acquires, simultaneously, uorescent signals (related to cell properties) and light
scattering signals (related to cell size and biovolume) for each
bacteria cell. In the eld of CWs, FCM has rarely been exploited to
investigate bacteria abundance or to distinguish specic properties
of bacteria such as viability, activity or decay and only rare
publications are available in the eld (Scholz et al., 2001; Gagnon
et al., 2007; Chazarenc et al., 2009).
The objective of this study was to explore the functional status
and the depth proles of bacterial biomass within a VSSF CW, and
to compare them with the biomass in the inuent and efuent
wastewater. The ultimate objective is to provide a better
understanding of which portion of the bacterial biomass is
responsible for the turnover of elements (living or viable biomass)
and which portion is inactive in the soil (dead biomass, which
might have a role in the long-term clogging mechanisms).
In this study, viability and death of bacteria were investigated
applying FCM with the aim of evaluating: (1) the time-proles of
viable and dead bacteria in inuent and efuent wastewater during
typical cycles; (2) the depth-proles of viable and dead bacteria in
gravel and sand layers; (3) some aspects affecting the physiological
status of bacteria cells when removed from wastewater and
retained in the soil.
To our knowledge, the FCM analysis of viability and decay of
bacteria in VSSF CW systems, comparing simultaneously inuent
wastewater, efuent wastewater and bacteria attached to soil
inside the bed, has not been reported yet in the literature, while
Decamp and Warren (2001) analysed viable and dead bacteria only
in inuent and efuent wastewater. This paper aims to contribute
to advancing knowledge in microbiological aspects of VSSF CWs by
considering viable and dead bacteria both in water and soil and by
providing a bacteria biomass balance and some aspects about the
retention of bacteria in the VSSF system.
2. Materials and methods

cycle, resulting in a time-variable ow rate efuent during the

cycle. The hydraulic loading rate applied to the VSSF was 63 L m2
d1 on average during the 1-year monitoring period, while the
applied organic load was correspondent to 3.2 m2/PE. The plant has
been in operation since 2009 and this research was carried out
after three years of operation. The VSSF CW was unplanted, but
Tietz et al. (2007) showed no statistically signicant difference in
bacterial biomass in all the layers of planted and unplanted VSSF
systems.
2.2. Sampling and chemical analyses in inuent and efuent
wastewater
Samples of inuent and efuent wastewater were collected
weekly. Efuent concentrations were measured in 6.6-h-composite-samples obtained by sampling aliquots proportional to efuent
ow rate. Intensive monitoring campaigns were conducted
monthly during the typical VSSF cycle to obtain time-proles of
efuent concentrations in the intervals 05 min, 510 min,
1020 min, 2030 min, 30 min1 h, 12 h, 24 h, 46 h and 6 h.
Routine analyses were: total Chemical Oxygen Demand (COD),
Total Suspended Solids (TSS), Total Kjeldhal Nitrogen (TKN), NH4N, NO3-N and total P (APHA, 2005). Filtered COD was measured
after ltration on a 0.45-mm-membrane. The mean concentrations
in inuent and efuent wastewater are indicated in Table 1 and
they are in accordance with typical performances expected from
VSSF systems. The VSSF wetland demonstrated high efciency in
TSS removal (>74%), total COD and ltered COD removal (>81%).
Nitrication occurred in the VSSF wetland due to the aerobic
conditions originated by the intermittent loading mode. Inuent
wastewater temperature varied in range from 7 to 20  C during one
year of monitoring.
2.3. Plate counts
Total Coliforms, Escherichia coli and Faecal Streptococci, which
are common microbiological indicators in wastewater, were
measured for a comparison in inuent and efuent wastewater
by Membrane Filter Techniques. The original samples were diluted
to 102 to 106 and volumes of 10100 mL (in triplicate) were
ltered on 0.45-mm membrane lters to obtain plates with
20100CFUs.
Total Coliforms were measured after growth on Coliforms-E.
coli agar (C-EC agar, Biolife, Italy) for 24  2 h at 36  1  C (Italian
Standards APAT CNR IRSA Manual 29/2003 Methods 7010C). E. coli
were measured after growth on Tryptone Bile X-glucuronide
medium (TBX, Oxoid, UK) after 21  3 h at 44  1  C according to
Italian Standards (APAT CNR-IRSA, 2003a). Faecal Streptococci
were measured by growth on Slanetz and Bartley medium (Oxoid,
UK) for 44  4 h at 36  1  C according to Italian Standards (APAT
CNR-IRSA, 2003b).

2.1. The VSSF CW plant

The outdoor above-ground VSSF pilot plant was located at
739 m a.s.l. (Province of Trento, Italy). The screened municipal
wastewater underwent settlement in an Imhoff tank before being
pumped up into the VSSF wetland. The VSSF wetland had a surface
area of 2.25 m2 and was characterised by the following layers,
starting from the bottom: (1) 0.2 m coarse gravel 1530 mm, (2)
0.05 m medium gravel 715 mm, (3) 0.5 m sand 13 mm, (4)
0.05 m medium gravel 715 mm (layers are graphically indicated
in Supplementary material SPM1). Porosity of both sand and gravel
was 0.30.
The VSSF was a conventional down-ow system with a single
feeding per cycle applied on top of the bed (6.6 h/cycle, 3.6 cycles/
day) and wastewater was discharged by free drainage for the entire

Table 1
Concentrations of the main parameters in inuent and efuent wastewater in the
VSSF CW.

COD
Filtered COD
TSS
TKN
NH4-N
NO3-N
Total P
No. samples

Inuent concentration
(mg/L)

Efuent concentration
(mg/L)

Removal rate
(g m2 d1)

544
282
161
72.7
59.7
2.4
8.6
5584

89
51
41
17.1
12.9
32.6
5.4
5585

28.9
14.7
7.6
3.5
3.0

0.2

P. Foladori et al. / Ecological Engineering 82 (2015) 4956

2.4. Outline of bacterial soil detachment

Samples of soil (granular medium within the bed) were taken
randomly in the VSSF wetland by a corer. Six sampling campaigns
were performed. After sampling, the soil cores were transported to
the lab to perform FCM analysis on the same day. Aliquotes of soil
underwent dry-weight analysis (DW) carried out at 105  C until a
constant weight was achieved.

2.4.1. Washing cycles

For the detachment of bacteria from the soil, a 0.1 M sodium
pyrophosphate solution (Na4P2O7 solution) was used to loosen the
strong hydrogen bindings and van der Waals, electrostatic and
chemical forces that tie cells and particles together (Amaltano
and Fazi, 2008; Tietz et al., 2007). Briey, a 100-mL-ask was lled
with 520 g of wet soil (Fig. 1, step A) and then with 50 mL of
Na4P2O7 solution for the rst washing step (Fig. 1, step B). The ask
was placed in a shaker at 150 rpm for 15 min. The entire liquid part
was then separated from the soil, and the soil underwent a second
washing step with a further amount of 50 mL Na4P2O7 solution for
15 min. Finally, the liquid part was completely separated from the
soil and added to the liquid part separated in the rst washing step,
thus obtaining a nal suspension with a volume of 100 mL (Fig. 1,
step C).
To conrm the maximum recovery of bacterial cells from soil
samples, uorescent microscopic analyses were performed on soil
samples after washing steps.

Influent and
effluent wastewater

51

2.4.2. Sample sonication

The bacterial suspension underwent a dispersion step with the
aim of separating bacteria aggregates and obtaining a single-cell
suspension suitable for FCM analysis. The ask containing the 100mL-suspension, surrounded by cooling water to avoid an increase
of temperature, underwent sonication (Branson 250 Digital Ultrasonier, frequency of 20 kHz) at specic energy (Es = time
power/
volume of liquid) of up to 100 kJ/L (Fig. 1, step D). The power used
for Es calculation was the actual power transferred to the bacterial
suspension measured by calorimetry (Mason et al., 1992). As
shown in Fig. 2, the optimal Es value of 80 kJ/L (Es = 210s
38
W/100 mL) permitted the recovery of the maximum number of
free viable bacteria minimising the disruption of bacteria. This
value is in agreement with the optimal Es value found for activated
sludge by Foladori et al. (2007).
The inuence of the wet soil weight placed in the 100-mL-ask
on the accuracy of the FCM analysis was evaluated calculating the
coefcient of variation (CV) on three replicates for various amounts
of soil (sand in particular) used in the analysis (details are indicated
in Supplementary material SPM3). A weight equal or greater than
10 g had to be used to obtain a CV lower than 10% in the
measurement of viable and dead cells.
2.5. Fluorescent staining and FCM analysis
FCM analysis was applied to: (1) inuent and efuent
wastewater collected as described in Section 2.2; (2) bacteria
suspensions recovered from VSSF soil as described in Section 2.4.

Cores of the
VSSF-CW soil

Dry-weight analysis

Step A.
100-mL-flask filled with 5-20 g of wet soil

Step C.
Final suspension (100 mL volume)
Step D.
Sonication at Es of 80 kJ/L

Pre-treatment
of the soil samples

Step B.
Addition of 50 mL Na4P2O7 solution
and washing (2x)

Dilution and filtration on 20-m-filter

FCM analysis

Step E.
Fluorescent staining of cells with
SYBR-Green I + Propidium Iodide
Step F.
FCM analysis ( > 20,000 cells)

Number of viable
and dead bacteria

Biomass of viable and dead

bacteria (as COD)

Calculation of
bacteria biomass

Biovolume
of bacteria

Fig. 1. Flow chart of the pre-treatment, FCM analysis and biomass calculation in VSSF soil samples and wastewater.

52

P. Foladori et al. / Ecological Engineering 82 (2015) 4956

100

Free viable cells (% of N max)

90
80
70
60
50
bacteria
activateddisagg
sludgeregated
from activated sludge
(data from Foladori et
al., 2007)

40
30
20

bacteria detached and

disaggregated from the
VSSF soil (this paper)

10

Regions were identied in the dot plots to distinguish viable and

dead cells (Fig. 3).
Green and red uorescences were collected with logarithmic
gain, while the forward angle light scattering (FALS) was collected
with linear gain, in order to permit the conversion of FALS into
bacteria biovolume (data not shown) according to Foladori et al.
(2008).
A Students t test, producing a p value, was applied to evaluate
the signicant difference in the number of bacteria and the
bacteria biovolume, assuming signicance at p < 0.05.
2.6. Calculation of bacteria biomass

0
0

20

40

60

80

100

120

140

Specific energy, Es (kJ L-1)

Fig. 2. Free viable bacteria detached and disaggregated from the VSSF soil as a
function of Es and comparison with disaggregation of activated sludge.
Free viable cells are expressed as a percentage of the maximum number of cells
recovered (Nmax).

All samples were diluted in Phosphate Buffer Saline (PBS) to

obtain a concentration of 103 to 104 cells/mL, suitable for FCM
analysis. Aliquots of 1 mL of these suspensions were ltered
through 20-mm lters with the aim of excluding coarse solids (for
example silt) which risk clogging the cytometer nozzle.
Viable and dead bacteria cells were identied on the basis of
their cellular membrane integrity, applying the double uorescent
staining with Propidium Iodide and SYBR-Green I (Ziglio et al.,
2002): viable cells are dened as those with an intact membrane,
while cells with a permeabilised membrane are classied as dead
cells (Nebe-von-Caron et al., 2000). In particular, cells were stained
with 10 mL of Propidium Iodide (PI, stock solution concentration
1 mg/mL; Invitrogen, USA; lex = 536 nm, lem = 617 nm) and 10 mL
of SYBR-Green I (SYBR-I, 1:30 dilution of commercial stock;
Invitrogen, USA; lex = 495 nm, lem = 525 nm) and incubated in the
dark for 15 min at room temperature prior to FCM analysis (Ziglio
et al., 2002). SYBR-I is capable of staining all cells, whereas the
polarity of PI allows it to penetrate only cells with permeabilised
membranes (dead cells). In dead cells, the simultaneous staining
with SYBR-I and PI activates energy transfer between the dyes and,
consequently, viable bacteria emit green uorescence whilst dead
bacteria emit red uorescence (Fig. 1, Step E).
Stained bacteria were analysed with an Apogee A40 ow
cytometer (Apogee Flow Systems, UK) equipped with an Ar laser at
488 nm (Fig. 1, Step F). The counting rate was of about 100 cells/s
and more than 20,000 cells were analysed for each sample to
ensure accurate results. Thresholds were set on green uorescence
and red uorescence in order to exclude non-uorescent debris.

The biomass of each bacterial cell (M) was expressed as

Chemical Oxygen Demand (COD) as indicated in the best-known
mathematical models used to describe biochemical transformation and degradation processes in subsurface ow constructed
wetlands (Langergraber et al., 2009). M was calculated taking into
account the biovolume (V) of each bacteria, according to the
following expression:
MgCOD V
Cs
1015

1:48
0:53

where the carbon content per unit of cell volume (Cs) is 310 fgC

mm3 (Fry, 1990), the carbon content is 53% of dry weight of cells

(derived from the empirical formula of bacteria composition,

C5H7NO2) and the coefcient 1.48 is used to convert the dry weight
of cells into COD. Finally, the entire bacteria biomass in a sample
was calculated summing up M of all bacteria cells (Foladori et al.,
2010) and expressing it per gram of DW (gCOD/gDW) or per unit of
soil volume (gCOD/m3).
3. Results and discussion
3.1. Time-proles of viable and dead bacteria in the inuent and
efuent wastewater
The proles of viable and dead bacteria in the efuent from the
VSSF wetland after the feeding of inuent wastewater, measured in
different runs are shown in Fig. 4. The highest presence of dead
bacteria was observed in the inuent wastewater taken from the
Imhoff tank used as a pre-treatment. After only 5 min of feeding,
viable and dead bacteria in the inuent wastewater (9.9
1010 and
7.7
1010 cells/L, respectively, on average in all the monitored
runs) were reduced immediately in the efuents discharged from
the VSSF wetland by gravity, with a reduction always above 40%
and 80% for viable and dead bacteria respectively (Fig. 4). At

Fig.3. FCM dot plot of red versus green uorescence to distinguish viable and dead bacteria in water and soil: (A) inuent wastewater; (B) 010 cm sandy layer in the VSSF; (C)
efuent wastewater.

P. Foladori et al. / Ecological Engineering 82 (2015) 4956

53

Fig. 4. Proles of viable and dead bacteria in the VSSF in two different runs (1-year monitoring).

1020 min after feeding the concentration of viable and dead

bacteria in the efuents formed a peak. The highest ow rate
discharged from the VSSF bed at this time increased tangential
shears, the detachment of bacteria from the granular medium and
an increase of the bacteria concentration in the efuent.
Successively, the bacteria concentration in the VSSF efuents
decreased progressively, both for viable and dead cells (Fig. 4), due
to the lower ow rate of the wastewater percolating through the
vertical bed and a limited detachment of bacteria retained in the
bed.
During the typical 6.6-h-cycle, the progressive percolation
of wastewater through the VSSF bed caused an increase in
the proportion of viable bacteria in the efuent and an increase in
the ratio of viable/dead bacteria (V/D), which was about 1.0 in the
inuent wastewater, but increased remarkably up to 3.03.9 in the
efuent in the rst 30 min, indicating a higher amount of viable
bacteria in the efuent.
The concentration of viable bacteria measured by FCM in the
inuent and efuent wastewater was compared with some
groups of culturable faecal indicators, often considered in
routinary microbiological analyses (Table 2, data of 1-year
monitoring). In average samples weighted by ow rate, a removal
efciency of 8495% for total coliforms, E. coli and faecal
streptococci was observed in the VSSF CW, in agreement with
other ndings in the literature reporting efcient removal of about

0.81.7 log-units (Arias et al., 2003). The concentrations of viable

bacteria and the culturable groups differed demonstrably by 23
orders of magnitude in the inuent and of 34 orders in the
efuent. The percentage of culturable faecal indicators with
respect to viable bacteria was higher in inuent wastewater, while
it was lower in the efuent due to two main phenomena: (i)
retention of a part of inuent bacteria, including culturable faecal
indicators, in the bed and enrichment of efuent with viable
bacteria grown directly in the bed itself; (ii) loss of cultivability of a
part of inuent bacteria, especially those of faecal origin, which
may enter a viable-but-not-culturable state (VBNC state) before
being released into the efuent wastewater. The signicant
precence of VBNC bacteria in CW efuent was highlighted several
times (inter alia Decamp and Warren, 2001; Alexandrino et al.,
2007). The release in the efuent of viable bacteria of non-faecal
origin is the reason for their apparently lower removal efciency
than the cultivable ones; similar observations were made by
Decamp and Warren (2001).
Bacteria size and biovolume of bacteria in inuent and efuent
wastewater was estimated from FALS signals acquired by FCM
according to Foladori et al. (2008). The mean biovolume of viable
bacteria in the inuent wastewater was 0.19 mm3/cell (diameter of
the equivalent sphere was 0.71 mm), signicantly higher than the
biovolume of viable bacteria in the VSSF efuent (0.17 mm3/cell)
with similar values during the entire 6.6-h-cycle.

Table 2
Viable and total bacteria measured by FCM in inuent and efuent wastewater (average samples weighted by ow rate), compared with groups of culturable faecal indicators
and relative ratios.

FCM
Plate counts

Ratios

Viable bacteria (cells/L)

Total (viable + dead) bacteria (cells/L)
Total coliforms (CFU/L)
Escherichia coli (CFU/L)
Faecal streptococci (CFU/L)
Total coliforms/Viable bacteria x 100
Escherichia coli/Viable bacteria x 100
Faecal streptococci/Viable bacteria x 100

Inuent concentration

Efuent concentration

Removal efciency

9.3
1010
1.7
1011
5.2
108
1.1
108
4.2
107
0.56%
0.12%
0.05%

3.6
1010
4.5
1010
2.6
107
9.2
106
6.8
106
0.07%
0.02%
0.02%

62%
73%
95%
92%
84%

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P. Foladori et al. / Ecological Engineering 82 (2015) 4956

3.2. Depth-proles of viable and dead bacteria in the VSSF granular

medium
The proles of viable and dead bacteria at various depths of the
VSSF CW are shown in Fig. 5. The depth has a great inuence on the
distribution of bacteria content and this is consistent with other
observations in the literature (inter alia Tietz et al., 2008).
The bacteria content in the upper 5 cm gravel layer (715 mm)
was signicantly lower compared to the 010 cm sand layer (1
3 mm), which offers a larger surface per unit of dry weight for
bacterial attachment. The highest content of viable bacteria was
found in the upper 010 cm sand layer, as a consequence of two
main phenomena: (i) the vertical feeding which causes a higher
availability of organic matter and nutrients and a consequent
stimulation of microbial growth (Tietz et al., 2008; Faulwetter
et al., 2009); (ii) the ltration of inuent wastewater and the
entrapment of bacteria embedded in solids.
The V/D ratio was calculated as viable cells over dead cells. In
the upper 5 cm gravel layer the ratio V/D was low (0.38), due to the
fact that a larger fraction of bacteria entering the wetland with the
wastewater are already dead. Truu et al. (2009) indicated that dead
or damaged bacteria are removed from the wastewater more
efciently than undamaged cells.
The V/D ratio increased in the upper 010 cm sand layer, where
V/D ratio was 0.73, which was the maximum value along the depth
of the bed. In this layer, the growth of viable bacteria is favored due
to the higher availability of organic matter and nutrients and the
larger surface for bacterial attachment. In the layers from 10 to
50 cm the V/D ratio was always lower than 0.5. The mean V/D ratio
in the entire bed was 0.52. The considerable accumulation of dead
bacteria in the VSSF soil, as shown in Fig. 5, suggests that bacteria
undergo slow cellular lysis or are hard to lyse and take longer to
decompose. By comparison, bacteria in activated sludge present a
V/D ratio of around 4.1 (Foladori et al., 2010), which is 8 times
higher the V/D ratio in the VSSF wetland. A slower autolysis rate is
probably the reason of the accumulation of dead cells inside the
bed. This may contribute to the mechanisms involved in clogging,
in addition to biomass production, deposition/ltration of incoming particulates and chemical precipitation (Kadlec and Wallace,
2009).
The prevalence of dead bacteria inside the VSSF bed compared
to viable ones was, to our knowledge, not observed before.
Microbiological methods applied to CWs are in fact not designed
specically to detect dead cells, but are used mainly to quantify
vital cellular properties (such as the presence of RNA or ATP

content) or to count total cells independently on their viability or

death (such as DNA staining or some PCR-based methods).
However, Sawaittayothin and Polprasert (2007), using FISH
analysis of bacteria populations in CWs, indicated that the EUB
338 mix probes (specic for 16S rRNA) detected about 50% of total
bacteria identied with DAPI staining, probably suggesting that the
remaining 50% could be made up of inactivated or dead bacterial
cells. In a horizontal subsurface CW with relatively high organic
loads, Krasnits et al. (2009) reported that viable cells (FISH-stained
cells) represent 6080% of the total cells enumerated by DAPI
staining.
In the sand layer the mean biovolume of the dead bacteria was
0.14 mm3/cell, whilst that of the viable bacteria was 0.16 mm3/cell.
These values are similar to the biovolume of 0.13 mm3/cell
indicated by Tietz et al. (2007) in wetlands for bacteria observed
under epiuorescence microscope. By comparison, bacteria in
activated sludge have a biovolume of 0.23 mm3/cell (Foladori et al.,
2010) or 0.25 mm3/cell (Frlund et al., 1996), which is consistently
larger than the biovolume of bacteria in the VSSF wetlands. The
smaller size may be due to the development of bacterial
communities with different cell size and to an environment in
CWs with relatively lower nutrient content than activated sludge.
As an example, the volumetric organic load applied in this VSSF
wetland was approximately 0.1 kgCOD m3 d1, while activated
sludge for carbon oxidation and nitrication is exposed to typical
loads of around 1.0 kgCOD m3 d1. In general, it is well known that
bacteria reduce their size as a consequence of nutrient limited
environments (Robertson et al., 1998; Blagodatskaya and Kuzyakov, 2013).
The amount of total bacteria in the VSSF soil was calculated as
the sum of both viable and dead cells. The density of total bacteria
at various depths of the VSSF system was compared to the results
obtained by Tietz et al. (2008) in unplanted VSSF wetlands (the
comparison is graphically shown in Supplementary material
SPM4). Both total bacterial proles appear similar except for the
rst sand layer, which could be attributed to the different grain size
of sand used in these two researches: sand 13 mm and d10 of
1.0 mm in this work and sand 0.064 mm and d10 of 0.2 mm used
by Tietz et al. (2008). The use of sand with a smaller d10 may
enhance the ltration and the accumulation of inuent bacteria
and organic solids in the upper centimeters of the bed. Conversely,
in a coarse sand, the transport and penetration of bacteria through
the bed is increased. A potential advantage of the use of coarse
material is the reduction of clogging risk in the upper layers.
However, a disadvantage may be a higher efuent concentration of
bacteria due to the reduced retainment capacity of the bed. The
mean density of total bacteria in the entire depth (weighted by the
layer depths) of our VSSF system was 1.0
109 cells/gDW, very
close to the value obtained by Tietz et al. (2008) (1.4
109 cells/
gDW).
3.3. Comparison between viable and dead bacteria in wastewater and
in soil

Fig. 5. Proles of viable and dead bacteria at various depths of the VSSF wetland
(upper gravel layer and main sand layer are distinguished) expressed per unit of dry
weight (DW) of the soil.

The bacteria biomass in wastewater and soil, quantied

according to the Section 2.6, was expressed per unit of DW or
per unit of volume in the VSSF. The size of bacteria and the biomass
estimations have rarely been reported in the CW eld (Tietz et al.,
2007; Tietz et al., 2008).
The mean biomass of total bacteria (viable + dead) in the VSSF
soil was 213 gCOD/m3 and 0.12 mgCOD/gDW (weighted by the
layer depths). This latter value is very similar to the value of
approximately 0.10 mgCOD/gDW calculated by us from the data of
Tietz et al. (2007). These results are in the range of biomass values
in VSSF systems reviewed by Truu et al. (2009) of 0.0221.69 mgC/
gDW, corresponding to about 0.064.6 mgCOD/gDW. For instance,

P. Foladori et al. / Ecological Engineering 82 (2015) 4956

the total amount of microbial biomass expected in common soils

ranges from 0.1 to 4.0 mg of cell dry weigh per g of soil
(Blagodatskaya and Kuzyakov, 2013).
On average, the total bacterial biomass fed into the VSSF CW
with the inuent wastewater converted into COD, was 3.1 gCOD
m3 d1 whilst, in the efuent wastewater during the entire 6-h
cycle, it was 0.77 gCOD m3 d1. The difference between input and
output (2.3 gCOD m3 d1) is the mean total bacteria biomass
removed every day from wastewater and accumulated in the VSSF
bed. This corresponds to 1.1% of the total biomass present in the
bed which is also produced by the bacteria growth. The total
bacteria biomass daily leaving the VSSF bed with the efuent
wastewater was only 0.36% of the total biomass present in the bed.
Considering viable bacteria, the viable biomass removed every day
from wastewater was 1.5% of the viable biomass in the bed, while
the viable biomass left in the efuent was 0.77%. Although the
concept of solid retention time (sludge retention time or sludge
age, common in activated sludge systems; Henze et al., 2008) is not
used in wetlands, a ballpark balance of viable biomass was here
calculated, obtaining a rough mean retention time of the viable
biomass in the bed of around 130 days. For instance, this value is far
longer than solid retention time in activated sludge systems, where
sludge age is typically in the range of 525 days. It has been
demonstrated that the observed growth yield in biological
processes is inversely dependent on the solid retention time and
endogenous respiration (Lawrence and McCarty, 1970; van
Loosdrecht and Henze, 1999; Liu and Tay, 2001). A decrease in
the observed growth yield of the bacterial biomass is thus expected
in the VSSF bed as a consequence of the longer solid retention time
in these systems.
3.4. Hypothesis of dead and viable bacterial distribution in
unsaturated VSSF soil

55

zones near the bottom in an area opposite the outlet where the
water velocity is low. Understanding and preventing conditions
which lead to dead zones and channeled ow in CWs, for instance
using multiphysics modelling, permits to avoid negative effects on
the treatment efciency of the system as a whole (Rajabzadeh
et al., 2015).
Conversely, bacteria growth seems rather favored in water lms
along the preferential water ow directions. Here the uid velocity
causes tangential shear and detachment of bacteria from the zones
where the viable bacteria are more abundant. The consequence of
this elution of viable bacteria from the soil is the shift in the
efuent microbial population which will be enriched in viable
cells, whilst dead bacteria remain conned in the dead zones
excluded from the main water ow. The V/D ratio in the efuent
will subsequently appear higher than the V/D ratio in the soil and
also than the V/D in the inuent wastewater.
The hypothesis that bacteria in the efuent wastewater may
largely originate from the detachment of biolm grown on soil
rather than as a fraction of the inuent bacterial community was
conrmed recently by Adrados et al. (2014). Adrados et al. (2014),
using the PCR-DGGE molecular technique, observed that in VSSF
wetlands there was almost no relation between the inuent and
the efuent bacterial communities, suggesting that the community
structure in the system and in the efuent wastewater is related to
other conditions developed inside the bed rather than the inuent
wastewater microbiology.
4. Conclusions
In this study, viable and dead bacteria were quantied by CM in
inuent, efuent wastewater and in the VSSF soil. The main
conclusions are:
1) The study has conrmed that bacteria prole in the VSSF

The mean V/D ratio (viable/dead cells) in the soil was 0.52,
much lower than the V/D ratio in the inuent wastewater (0.93 on
average) and in the efuent wastewater (3.3 on average). This large
discrepancy indicates that the dead cells accumulated within the
VSSF soil, while efuent wastewater was remarkably enriched in
viable cells.
These observations could be explained assuming the hypothesis
of preferential ow paths formed during percolation through the
unsaturated VSSF soil (a theoretical schema of this hypothesis is
shown in Supplementary material SPM5). The wastewater ow
path may follow preferential directions due to the variable
moisture content, the presence of small channels closed to ow
and the variable biolm thickness around the sand grains. The
consequence of this is the possible formation of: (1) zones of
preferential water ow, with high water content and substrate
availability, where the growth of viable bacteria is favored; (2)
zones where uid and air arrive randomly or not at all and the
substrates are limited or absent, where bacteria can not grow,
decay and die.
The presence of dead zones in VSSF systems was observed by
Giraldi et al. (2009) as a result of hydrodynamic studies where
actual and theoretical residence times were compared. Dead
zones, caused by preferential water ow, biolm accumulation,
porosity decrease or clogged regions, were predicted by Rajabzadeh et al. (2015) in a VSSF system using a calibrated model. It is
believed that organic matter ux, required for the growth of
heterotrophic bacteria, may be limited in dead zones, where also
oxygen could become a limiting component (Rajabzadeh et al.,
2015); the result may be a limitation in bacteria survival causing a
subsequent death. Giraldi et al. (2009) identied dead zones in the
upper layers where water ows near the distribution holes
(individual points), while Rajabzadeh et al. (2015) identied dead

2)

3)

4)

5)

systems depends on the depth: the number of viable cells in the

upper 010 cm sand layer was 3.7 times higher than in the
deeper 4050 cm, due to the vertical feeding and a sieving effect
of inuent in the top layers.
The study has conrmed the removal in the VSSF system of a
signicant part of inuent bacteria (viable bacteria and
culturable faecal indicators);
The long retention time of the viable biomass in the VSSF soil
(130 days) explains the low observed growth yield of bacterial
biomass is such systems.
Dead bacteria accumulate in the VSSF soil: the number of dead
cells in the soil were 2 times the viable ones, due to slow
autolysis rate and with a possible inuence on the long-term
behavior of the VSSF systems.
In the efuent the viable bacteria were 3.3 times the dead ones:
efuent wastewater transports viable bacteria during the
passage through the VSSF bed, probably along preferential
water ow where the bacterial growth is favored. The
hypothesis that dead bacteria may remain conned in the dead
zones excluded from the main water ow was proposed.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/j.
ecoleng.2015.04.058.
References
Adrados, B., Snchez, O., Arias, C., Becares, E., Garrido, L., Mas, J., Brix, H., Morato, J.,
2014. Microbial communities from different types of natural wastewater

56

P. Foladori et al. / Ecological Engineering 82 (2015) 4956

treatment systems: vertical and horizontal ow constructed wetlands and

biolters. Water Res. doi:http://dx.doi.org/10.1016/j.watres.2014.02.011.
APAT CNR-IRSA, 2003a. Methods 7030 F. In Metodi analitici per le acque. Manuale
29/2003, Sezione 7000, Metodi per microrganismi. In Italian.
APAT CNR-IRSA, 2003b. Methods 7040C. In Metodi analitici per le acque. Manuale
29/2003, Sezione 7000, Metodi per microrganismi. In Italian.
APHA, AWWA, and WPCF, 2005. Standard Methods for the Examination of Water
and Wastewater. 21st edition. Washington DC, USA.
Alexandrino, M., Grohmann, E., Szewzyk, R., Szewzyk, U., 2007. Application of
culture-independent methods to assess the bacteria removal efciency of
subsurface ow constructed wetlands. Water Sci. Technol. 56 (3),
217222.
Amaltano, S., Fazi, S., 2008. Recovery and quantication of bacterial cells
associated with streambed sediments. J. Microbiol. Methods 75, 237243.
Arias, C.A., Cabello, A., Brix, H., Johansen, N.-H., 2003. Removal of indicator bacteria
from municipal wastewater in an experimental two-stage vertical ow
constructed wetland system. Water Sci. Technol. 48 (5), 3541.
Bergquist, P.L., Hardiman, E.M., Ferrari, B.C., Winsley, T., 2009. Applications of ow
cytometry in environmental microbiology and biotechnology. Extremophiles 13
(3), 389401.
Blagodatskaya, E., Kuzyakov, Y., 2013. Active microorganisms in soil: critical review
of estimation criteria and approaches. Soil Biol. Biochem. 67, 192211.
Chazarenc, F., Gagnon, V., Comeau, Y., Brisson, J., 2009. Effect of plant and articial
aeration on solids accumulation and biological activities in constructed
wetlands. Ecol. Eng. 35 (6), 10051010.
Decamp, O., Warren, A., 2001. Abundance, biomass and viability of bacteria in
wastewaters: impact of treatment in horizontal subsurface ow constructed
wetlands. Water Res. 35 (14), 34963501.
Faulwetter, J.L., Gagnon, V., Sundberg, C., Chazarenc, F., Burr, M.D., Brisson, J.,
Camper, A.K., Stein, O.R., 2009. Microbial processes inuencing performance of
treatment wetlands: a review. Ecol. Eng. 35, 9871004.
Foladori, P., Bruni, L., Andreottola, G., Ziglio, G., 2007. Effects of sonication on
bacteria viability in wastewater treatment plants evaluated by ow cytometry
fecal indicators, wastewater and activated sludge. Water Res. 41, 235243.
Foladori, P., Quaranta, A., Ziglio, G., 2008. Use of silica microspheres having
refractive index similar to bacteria for conversion of ow cytometric forward
light scatter into biovolume. Water Res. 42 (14), 37573766.
Foladori, P., Bruni, L., Tamburini, S., Ziglio, G., 2010. Direct quantication of bacterial
biomass in inuent: efuent and activated sludge of wastewater treatment
plants by using ow cytometry. Water Res. 44 (13), 38073818.
Frlund, B., Palmgren, R., Keiding, K., Nielsen, P.H., 1996. Extraction of extracellular
polymers from activated sludge using a cation exchange resin. Water Res. 30,
17491758.
Fry, J.C., 1990. Direct methods and biomass estimation. Methods Microbiol. 22,
4185.
Gagnon, V., Chazarenc, F., Comeau, Y., Brisson, J., 2007. Inuence of macrophyte
species on microbial density and activity in constructed wetlands. Water Sci.
Technol. 56 (3), 249254.
Giraldi, D., de'Michieli Vitturi, M., Zaramella, M., Marion, A., Iannelli, R., 2009.
Hydrodynamics of vertical subsurface ow constructed wetlands: tracer tests
with rhodamine WT and numerical modelling. Ecol. Eng. 35, 265273.
Henze M., van Loosdrecht M.C.M., Ekama G.A., Brdjanovic D. 2008. Biological
Wastewater Treatment: Principles, Modelling and Design. ISBN:
9781843391883.
Kadlec, R.H., Wallace, S.D., 2009. Treatment Wetlands, 2nd edition CRC Press ISBN
978-1-56670-526-4..
Krasnits, E., Friedler, E., Sabbah, I., Beliavski, M., Tarre, S., Green, M., 2009. Spatial
distribution of major microbial groups in a well established constructed
wetland treating municipal wastewater. Ecol. Eng. 35, 10851089.

Langergraber, G., Rousseau, D.P.L., Garca, J., Mena, J., 2009. CWM1: a general model
to describe biokinetic processes in subsurface ow constructed wetlands. Water
Sci. Technol. 59 (9), 16871697.
Langergraber, G., 2011. Numerical modelling: a tool for better constructed wetland
design? Water Sci. Technol. 64 (1), 1421.
Lawrence, A.W., McCarty, P.L., 1970. Unied basis for biological treatment design
and operation. J. Sanitary Eng. Div. 96 (3), 757778.
Ligi, T., Oopkaup, K., Truu, M., Preem, J.-K., Nlvak, H., Mitsch, W.J., Mander, U., Truu,
J., 2014. Characterization of bacterial communities in soil and sediment of a
created riverine wetland complex using high-throughput 16S rRNA amplicon
sequencing. Ecol. Eng. 72, 5666.
Liu, Y., Tay, J.-H., 2001. Strategy for minimization of excess sludge production from
the activated sludge process. Biotechnol. Adv. 19, 97107.
Lv, X., Yu, J., Fu, Y., Ma, B., Qu, F., Ning, K., Wu, H., 2014. A meta-analysis of the
bacterial and archaeal diversity observed in wetland soils. Sci. World J. 112.
doi:http://dx.doi.org/10.1155/2014/437684.
Mason, T.J., Lorimer, J.P., Bates, D.M., 1992. Quantifying sonochemistry: casting some
light on a black art. Ultrasonics 30 (1), 4042.
Nebe-von-Caron, G., Stephens, P.J., Hewitt, C.J., Powell, J.R., Badley, R.A., 2000.
Analysis of bacterial function by multi-colour uorescence ow cytometry and
single cell sorting. J. Microbiol. Meth. 42, 97114.
Porter, J., Deere, D., Hardman, M., Edwards, C., Pickup, R., 1997. Go with the owuse
of ow cytometry in environmental microbiology. FEMS Microbiol. Ecol. 24, 93
101.
Rajabzadeh, A.R., Legge, R.L., Webe, K.P., 2015. Multiphysics modelling of ow
dynamics, biolm development and wastewater treatment in a subsurface
vertical ow constructed wetland mesocosm. Ecol. Eng. 74, 107116.
Robertson, B.R., Button, D.K., Koch, A.L., 1998. Determination of the biomasses of
small bacteria at low concentrations in a mixture of species with forward light
scatter measurements by ow cytometry. Appl. Environ. Microbiol. 64, 3900
3909.
Sawaittayothin, V., Polprasert, C., 2007. Nitrogen mass balance and microbial
analysis of constructed wetlands treating municipal landll leachate. Bioresour.
Technol. 98, 565570.
Scholz, M., Xu, J., Dodson, H.I., 2001. Comparison of lter media, plant communities
andd microbiology within constructed wetlands treating wastewater
containing heavy metals. J. Chem. Technol. Biotechnol. 76, 827835.
Steen, H.B., 2000. Flow cytometry of bacteria: glimpses from the past with a view to
the future. J. Microbiol. Methods 42, 6574.
Tietz, A., Kirschner, A., Langergraber, G., Sleytr, K., Haberl, R., 2007. Characterisation
of microbial biocoenosis in vertical subsurface ow constructed wetlands. Sci.
Total Environ. 380, 163172.
Tietz, A., Langergraber, G., Watzinger, A., Haberl, R., Kirschner, A.K.T., 2008. Bacterial
carbon utilization in vertical subsurface ow constructed wetlands. Water Res.
42, 16221634.
Truu, M., Juhanson, J., Truu, J., 2009. Microbial biomass, activity and community
composition in constructed wetlands. Sci. Total Environ. 407, 39583971.
van Loosdrecht, M.C.M., Henze, M., 1999. Maintenance, endogenous respiration,
lysis, decay and predation. Water Sci. Technol. 39 (1), 107117.
Vives-Rego, J., Lebaron, P., Nebe-von Caron, G., 2000. Current and future applications
of ow cytometry in aquatic microbiology. FEMS Microbiol. Rev. 24, 429448.
Zhang, T., Shao, M.-F., Ye, L., 2012. 454 Pyrosequencing reveals bacterial diversity of
activated sludge from 14 sewage treatment plants. ISME J. 6 (6), 11371147.
Zhao, Y., Liu, B., Zhang, W., Hu, C., An, S., 2010. Effects of plant and inuent C:N:P
ratio on microbial diversity in pilot-scale constructed wetlands. Ecol. Eng. 36,
441449.
Ziglio, G., Andreottola, G., Barbesti, S., Boschetti, G., Bruni, L., Foladori, P., Villa, R.,
2002. Assessment of activated sludge viability with ow cytometry. Water Res.
36 (2), 460468.