Bacteria viability and decay in water and soil of vertical subsurface flow constructed wetlands

Ecological Engineering 82 (2015) 49–56

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Bacteria viability and decay in water and soil of vertical subsurface flow 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 flow (VSSF) constructed wetland was examined with the aim to understand the relationship between viable and dead bacteria in soil and influent/effluent wastewater and elucidate the large amount of dead cells in the soil which may affect the long-term behavior of the system. The quantification of viable and dead bacteria in influent and effluent wastewater and in the soil of a VSSF was performed at single-cell level by flow cytometry (FCM). An optimised pre-treatment was applied to soil samples using sodium pyrophosphate and ultrasonication at a specific 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 profile in the VSSF soil depends on the depth and the material grain size. In the upper 0–10 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 40–50 cm (4.8
108 cells/gDW) probably due to the vertical feeding and a sieving effect of influent 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 influent 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 effluent reflects the enrichment of wastewater with viable bacteria during the passage through the VSSF bed and along preferential water flow, with higher water content and substrate availability, where the bacterial growth is favored. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Wastewater Vertical subsurface flow 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 nitrification and denitrification, even though the significant 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 flow CWs (VSSF) is widely recognised and well documented (Faulwetter et al., 2009; Truu

* Corresponding author. Tel.: +39 0461 282683. E-mail address: [email protected] (P. Foladori). http://dx.doi.org/10.1016/j.ecoleng.2015.04.058 0925-8574/ ã 2015 Elsevier B.V. All rights reserved.

et al., 2009; Kadlec and Wallace, 2009), the quantification and the distribution of bacteria in such systems (both in water and in soil) have not been investigated sufficiently 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 field (Faulwetter et al., 2009). Among them, fluorescent staining of microbial nucleic acids, fluorescent 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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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 quantified (Decamp and Warren, 2001). When the absolute quantification of bacteria is done under epifluorescence 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 quantification 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, fluorescent signals (related to cell properties) and light scattering signals (related to cell size and biovolume) for each bacteria cell. In the field of CWs, FCM has rarely been exploited to investigate bacteria abundance or to distinguish specific properties of bacteria such as viability, activity or decay and only rare publications are available in the field (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 profiles of bacterial biomass within a VSSF CW, and to compare them with the biomass in the influent and effluent 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-profiles of viable and dead bacteria in influent and effluent wastewater during typical cycles; (2) the depth-profiles 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 influent wastewater, effluent 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 influent and effluent 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 flow rate effluent 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 significant difference in bacterial biomass in all the layers of planted and unplanted VSSF systems. 2.2. Sampling and chemical analyses in influent and effluent wastewater Samples of influent and effluent wastewater were collected weekly. Effluent concentrations were measured in 6.6-h-composite-samples obtained by sampling aliquots proportional to effluent flow rate. Intensive monitoring campaigns were conducted monthly during the typical VSSF cycle to obtain time-profiles of effluent concentrations in the intervals 0–5 min, 5–10 min, 10–20 min, 20–30 min, 30 min1 h, 1–2 h, 2–4 h, 4–6 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 filtration on a 0.45-mm-membrane. The mean concentrations in influent and effluent wastewater are indicated in Table 1 and they are in accordance with typical performances expected from VSSF systems. The VSSF wetland demonstrated high efficiency in TSS removal (>74%), total COD and filtered COD removal (>81%). Nitrification occurred in the VSSF wetland due to the aerobic conditions originated by the intermittent loading mode. Influent 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 influent and effluent wastewater by Membrane Filter Techniques. The original samples were diluted to 102 to 106 and volumes of 10–100 mL (in triplicate) were filtered on 0.45-mm membrane filters to obtain plates with 20–100CFUs. 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 Ø15–30 mm, (2) 0.05 m medium gravel Ø7–15 mm, (3) 0.5 m sand Ø1–3 mm, (4) 0.05 m medium gravel Ø7–15 mm (layers are graphically indicated in Supplementary material SPM1). Porosity of both sand and gravel was 0.30. The VSSF was a conventional down-flow 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 influent and effluent wastewater in the VSSF CW.

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

Influent concentration (mg/L)

Effluent concentration (mg/L)

Removal rate (g m2 d1)

544 282 161 72.7 59.7 2.4 8.6 55–84

89 51 41 17.1 12.9 32.6 5.4 55–85

28.9 14.7 7.6 3.5 3.0 – 0.2 –

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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 (Amalfitano and Fazi, 2008; Tietz et al., 2007). Briefly, a 100-mL-flask was filled with 5–20 g of wet soil (Fig. 1, step A) and then with 50 mL of Na4P2O7 solution for the first washing step (Fig. 1, step B). The flask 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 first washing step, thus obtaining a final suspension with a volume of 100 mL (Fig. 1, step C). To confirm the maximum recovery of bacterial cells from soil samples, fluorescent 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 flask containing the 100mL-suspension, surrounded by cooling water to avoid an increase of temperature, underwent sonication (Branson 250 Digital Ultrasonifier, frequency of 20 kHz) at specific 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 influence of the wet soil weight placed in the 100-mL-flask on the accuracy of the FCM analysis was evaluated calculating the coefficient 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) influent and effluent 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-µm-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.

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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 identified in the dot plots to distinguish viable and dead cells (Fig. 3). Green and red fluorescences 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 Student’s t test, producing a p value, was applied to evaluate the significant difference in the number of bacteria and the bacteria biovolume, assuming significance 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 filtered through 20-mm filters with the aim of excluding coarse solids (for example silt) which risk clogging the cytometer nozzle. Viable and dead bacteria cells were identified on the basis of their cellular membrane integrity, applying the double fluorescent staining with Propidium Iodide and SYBR-Green I (Ziglio et al., 2002): viable cells are defined as those with an intact membrane, while cells with a permeabilised membrane are classified 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 fluorescence whilst dead bacteria emit red fluorescence (Fig. 1, Step E). Stained bacteria were analysed with an Apogee A40 flow 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 fluorescence and red fluorescence in order to exclude non-fluorescent 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 flow constructed wetlands (Langergraber et al., 2009). M was calculated taking into account the biovolume (V) of each bacteria, according to the following expression: M½gCOD ¼ 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 coefficient 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-profiles of viable and dead bacteria in the influent and effluent wastewater The profiles of viable and dead bacteria in the effluent from the VSSF wetland after the feeding of influent wastewater, measured in different runs are shown in Fig. 4. The highest presence of dead bacteria was observed in the influent wastewater taken from the Imhoff tank used as a pre-treatment. After only 5 min of feeding, viable and dead bacteria in the influent wastewater (9.9
1010 and 7.7
1010 cells/L, respectively, on average in all the monitored runs) were reduced immediately in the effluents 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 fluorescence to distinguish viable and dead bacteria in water and soil: (A) influent wastewater; (B) 0–10 cm sandy layer in the VSSF; (C) effluent wastewater.

P. Foladori et al. / Ecological Engineering 82 (2015) 49–56

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

10–20 min after feeding the concentration of viable and dead bacteria in the effluents formed a peak. The highest flow 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 effluent. Successively, the bacteria concentration in the VSSF effluents decreased progressively, both for viable and dead cells (Fig. 4), due to the lower flow 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 effluent and an increase in the ratio of viable/dead bacteria (V/D), which was about 1.0 in the influent wastewater, but increased remarkably up to 3.0–3.9 in the effluent in the first 30 min, indicating a higher amount of viable bacteria in the effluent. The concentration of viable bacteria measured by FCM in the influent and effluent 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 flow rate, a removal efficiency of 84–95% for total coliforms, E. coli and faecal streptococci was observed in the VSSF CW, in agreement with other findings in the literature reporting efficient removal of about

0.8–1.7 log-units (Arias et al., 2003). The concentrations of viable bacteria and the culturable groups differed demonstrably by 2–3 orders of magnitude in the influent and of 3–4 orders in the effluent. The percentage of culturable faecal indicators with respect to viable bacteria was higher in influent wastewater, while it was lower in the effluent due to two main phenomena: (i) retention of a part of influent bacteria, including culturable faecal indicators, in the bed and enrichment of effluent with viable bacteria grown directly in the bed itself; (ii) loss of cultivability of a part of influent bacteria, especially those of faecal origin, which may enter a “viable-but-not-culturable” state (VBNC state) before being released into the effluent wastewater. The significant precence of VBNC bacteria in CW effluent was highlighted several times (inter alia Decamp and Warren, 2001; Alexandrino et al., 2007). The release in the effluent of viable bacteria of non-faecal origin is the reason for their apparently lower removal efficiency than the cultivable ones; similar observations were made by Decamp and Warren (2001). Bacteria size and biovolume of bacteria in influent and effluent wastewater was estimated from FALS signals acquired by FCM according to Foladori et al. (2008). The mean biovolume of viable bacteria in the influent wastewater was 0.19 mm3/cell (diameter of the equivalent sphere was 0.71 mm), significantly higher than the biovolume of viable bacteria in the VSSF effluent (0.17 mm3/cell) with similar values during the entire 6.6-h-cycle.

Table 2 Viable and total bacteria measured by FCM in influent and effluent wastewater (average samples weighted by flow 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

Influent concentration

Effluent concentration

Removal efficiency

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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3.2. Depth-profiles of viable and dead bacteria in the VSSF granular medium The profiles of viable and dead bacteria at various depths of the VSSF CW are shown in Fig. 5. The depth has a great influence 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 (Ø7–15 mm) was significantly lower compared to the 0–10 cm sand layer (Ø1– 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 0–10 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 filtration of influent 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 efficiently than undamaged cells. The V/D ratio increased in the upper 0–10 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/filtration 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 specifically 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 (specific for 16S rRNA) detected about 50% of total bacteria identified 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 60–80% 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 epifluorescence microscope. By comparison, bacteria in activated sludge have a biovolume of 0.23 mm3/cell (Foladori et al., 2010) or 0.25 mm3/cell (Frølund 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 nitrification 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 profiles appear similar except for the first sand layer, which could be attributed to the different grain size of sand used in these two researches: sand Ø1–3 mm and d10 of 1.0 mm in this work and sand Ø0.06–4 mm and d10 of 0.2 mm used by Tietz et al. (2008). The use of sand with a smaller d10 may enhance the filtration and the accumulation of influent 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 effluent 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. Profiles 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, quantified 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 field (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 0.022–1.69 mgC/ gDW, corresponding to about 0.06–4.6 mgCOD/gDW. For instance,

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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 influent wastewater converted into COD, was 3.1 gCOD m3 d1 whilst, in the effluent 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 effluent 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 effluent 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 5–25 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

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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 flow in CWs, for instance using multiphysics modelling, permits to avoid negative effects on the treatment efficiency of the system as a whole (Rajabzadeh et al., 2015). Conversely, bacteria growth seems rather favored in water films along the preferential water flow directions. Here the fluid 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 effluent microbial population which will be enriched in viable cells, whilst dead bacteria remain confined in the dead zones excluded from the main water flow. The V/D ratio in the effluent will subsequently appear higher than the V/D ratio in the soil and also than the V/D in the influent wastewater. The hypothesis that bacteria in the effluent wastewater may largely originate from the detachment of biofilm grown on soil rather than as a fraction of the influent bacterial community was confirmed 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 influent and the effluent bacterial communities, suggesting that the community structure in the system and in the effluent wastewater is related to other conditions developed inside the bed rather than the influent wastewater microbiology. 4. Conclusions In this study, viable and dead bacteria were quantified by CM in influent, effluent wastewater and in the VSSF soil. The main conclusions are: 1) The study has confirmed that bacteria profile 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 influent wastewater (0.93 on average) and in the effluent wastewater (3.3 on average). This large discrepancy indicates that the dead cells accumulated within the VSSF soil, while effluent wastewater was remarkably enriched in viable cells. These observations could be explained assuming the hypothesis of preferential flow paths formed during percolation through the unsaturated VSSF soil (a theoretical schema of this hypothesis is shown in Supplementary material SPM5). The wastewater flow path may follow preferential directions due to the variable moisture content, the presence of small channels closed to flow and the variable biofilm thickness around the sand grains. The consequence of this is the possible formation of: (1) zones of preferential water flow, with high water content and substrate availability, where the growth of viable bacteria is favored; (2) zones where fluid 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 flow, biofilm 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 flux, 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) identified dead zones in the upper layers where water flows near the distribution holes (individual points), while Rajabzadeh et al. (2015) identified dead

2)

3)

4)

5)

systems depends on the depth: the number of viable cells in the upper 0–10 cm sand layer was 3.7 times higher than in the deeper 40–50 cm, due to the vertical feeding and a sieving effect of influent in the top layers. The study has confirmed the removal in the VSSF system of a significant part of influent 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 influence on the long-term behavior of the VSSF systems. In the effluent the viable bacteria were 3.3 times the dead ones: effluent wastewater transports viable bacteria during the passage through the VSSF bed, probably along preferential water flow where the bacterial growth is favored. The hypothesis that dead bacteria may remain confined in the dead zones excluded from the main water flow 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., Sánchez, O., Arias, C., Becares, E., Garrido, L., Mas, J., Brix, H., Morato, J., 2014. Microbial communities from different types of natural wastewater

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