Science of the Total Environment 580 (2017) 17–25

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Science of the Total Environment

journal homepage: www.elsevier.com/locate/scitotenv

Removal of pollutants and pathogens by a simplified treatment scheme

for municipal wastewater reuse in agriculture
Marco De Sanctis a,⁎, Guido Del Moro a, Silvia Chimienti a, Pierangela Ritelli a,
Caterina Levantesi b, Claudio Di Iaconi a
a
Water Research Institute, CNR, Via F. De Blasio 5, 70123 Bari, Italy
b
Water Research Institute, CNR, Via Salaria Km 29.300, 00015 Monterotondo, RM, Italy

H I G H L I G H T S G R A P H I C A L A B S T R A C T

• Wastewater reuse in agriculture re-

quires a compact and reliable treatment
scheme.
• E. coli content after biological treatment
was lower than 103 MPN/100 mL.
• Protozoa concentration in plant effluent
was lower than 1 cysts/L.
• Plant effluent wouldn't need chemical
disinfection for its reuse in many
countries.

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

Article history: The availability of high quality water has become a constraint in several countries. Agriculture represents the
Received 14 October 2016 main water user, therefore, wastewater reuse in this area could increase water availability for other needs.
Received in revised form 30 November 2016 This research was aimed to provide a simplified scheme for treatment and reuse of municipal and domestic
Accepted 1 December 2016
wastewater based on Sequencing Batch Biofilter Granular Reactors (SBBGRs). The activity was conducted at
Available online 9 December 2016
pilot-scale and particular attention was dedicated to the microbiological quality of treated wastewater to evalu-
Editor: D. Barcelo ate the risk associated to its reuse. The following microorganisms were monitored: Escherichia coli, Salmonella,
Clostridium perfringens, somatic coliphages, adenovirus, enterovirus, Giardia lamblia and Cryptosporidium parvum.
Keywords: The possibility of SBBGR enhancement with sand filtration was also evaluated. The SBBGR removed N 90% of
Disinfection suspended solids and chemical oxygen demand, and 80% and 60% of total nitrogen and phosphorous, respective-
Escherichia coli ly. SBBGR was also effective in removing microbial indicators, from 1 (for C. perfringens) up to 4 (for E. coli) log
Protozoa units of these microorganisms. In particular, the quality of SBBGR effluent was already compatible with the
Virus WHO criteria for reuse (E. coli ≤103 CFU/100 mL). Sand filtration had positive effects on plant effluent quality
Pathogen removal
and the latter could even comply with more restrictive reuse criteria.
Wastewater reuse
© 2016 Elsevier B.V. All rights reserved.

1. Introduction

⁎ Corresponding author. Climate change, population growth and water scarcity represent
E-mail address: [email protected] (M. De Sanctis). major challenges for economies and societies around the world. A

http://dx.doi.org/10.1016/j.scitotenv.2016.12.002
0048-9697/© 2016 Elsevier B.V. All rights reserved.
18 M. De Sanctis et al. / Science of the Total Environment 580 (2017) 17–25

recent study aimed to assess the impact of climate change on water of microbiological and physical-chemical parameters. An overview of
scarcity (Gosling and Arnell, 2016) highlighted that about 2 billion peo- the current water quality parameter applied in EU countries is provided
ple are currently living under water scarcity condition in several areas of in Sanz and Gawlik (2014) and Paranychianakis et al. (2015). E. coli is
the world. Considering that N 70% of the water withdrawn globally is the most common microbiological parameter and its maximum concen-
used for agricultural irrigation (UNESCO – United Nations Educational tration range between 10 and 250 CFU/100 mL for Italy and France re-
Scientific and Cultural Organization, 2003), the reuse of reclaimed spectively, in case of unrestricted agricultural reuse (i.e. irrigation of
urban wastewater has received more and more attention in recent crops which are eaten uncooked). Referring to the physical-chemical
years (Levine and Asano, 2004; Pereira et al., 2011). parameters they usually include: total suspended solids (TSS), biochem-
The use of compact and simple wastewater treatment schemes ical oxygen demand (BOD5), chemical oxygen demand (COD), pH,
would facilitate its actual reuse in agriculture. In fact, the treatment Heavy metals, electrical conductivity (EC), sodium adsorption ration
plants could be realized nearby of cultivated fields. Thus the treated (SAR), chloride, nitrogen, phosphorus and several priority substances.
wastewater would not need to be transported for long distance from As an illustration the quality limits for wastewater reuse in Italy are de-
big centralized treatment plants. However, reuse needs to be safely im- tailed as Supplementary material (Table S.1).
plemented to prevent transmission of pathogens and to avoid endan- The effectiveness of the proposed treatment has been evaluated in
gering public health and the environment. terms of some main gross parameters such as TSS, COD, BOD5, nitrogen,
From a technological point of view, a multi barrier treatment phosphorus, pH, EC, and SAR. Furthermore, from the microbiological
scheme, including several steps, has been proposed for protecting public point of view, a wide group of pathogens and indicators of faecal con-
health when reclaimed wastewater effluents are used for irrigation. tamination were considered including Escherichia coli and Salmonella
Such a complex treatment scheme, however, is hardly attainable in (representative of bacteria), Clostridium perfringens spores (representa-
small rural communities where compact and simple (short treatment tive of spore-forming bacteria), somatic coliphages, human enterovi-
line) plants are required. Furthermore, taking into account the signifi- ruses and adenoviruses (representative of viruses), Giardia lamblia and
cant fluctuations of wastewater composition and flow rate occurring Cryptosporidium parvum (representative of protozoa). In addition to
in small communities, a treatment line ensuring stable performances these parameters the other compounds required by Italian regulation
is also required. A recent system known as Sequencing Batch Biofilter (e.g. heavy metals) were analysed during the experimental trial also.
Granular Reactor (SBBGR), developed by the Water Research Institute
(IRSA) of the Italian National Research Council (CNR), seems to fulfil 2. Materials and methods
these needs. This system is able to carry out the entire wastewater treat-
ment train (i.e., primary, secondary and tertiary treatment) in a single 2.1. Pilot plant description
stage with excellent treatment performances in removing organic pol-
lutants, suspended solids and nitrogen and with a very low solid resi- The pilot plant used in the present study consisted of a SBBGR unit
dues production (Di Iaconi et al., 2014). In a recent study carried at followed by a sand filter as shown in Fig. 1. The main feature of SBBGR
laboratory scale, the authors have shown that SBBGR is also able to per- lies in the complete separation of biomass and wastewater, where the bio-
form an efficient disinfection action producing an effluent with a quality mass is confined in a dedicated compartment (also known as biofilter)
higher than that of conventional treatment systems based on primary with a volume of about 120 L (diameter: 22 cm; height: 320 cm) and filled
and secondary treatments (De Sanctis et al., 2016). However, SBBGR with plastic filling material (features: 7 mm high, 11 mm diameter,
treatment needed to be chemically or physically enhanced for improv- 650 m2/m3 specific area, 0.95 g/cm3 density, 0.7 bed porosity and
ing effluent quality and complying with the standards of agriculture 50–80 mm3 voids dimension) entrapped between two perforated plates.
reuse in European countries (De Sanctis et al., 2016; Paranychianakis
et al., 2015; Sanz and Gawlik, 2014).
The present experimentation was planned on the base of the previ-
ous one with the multiple purposes of investigating the effect of the pro-
cess scale-up and different plant configuration, and overcoming the
need for chemical or physical enhancement.
In particular, differently from De Sanctis et al., 2016, a pilot plant was
used for this experimentation. Moreover, a new SBBGR design (two-
compartment configuration vs single compartment plant used in De
Sanctis et al., 2016) is proposed in the present paper treating a raw sew-
age. This plant configuration was chosen as it reflects that one of a hypo-
thetical full scale plant. Furthermore, in order to avoid the enhancement
of SBBGR treatment with chemical (i.e. addition of chlorine, peracetic
acid, ozone) or physical (i.e. UV radiation) process, evaluated in De
Sanctis et al. (2016), a combination of SBBGR treatment and sand filtra-
tion (SF) was assessed. Considering that SF is an easy to handle and cost
effective disinfection technique able to reduce up to 1–2 log units of co-
liforms and viruses (Elliott et al., 2008; Harrison et al., 2000; Schuler et
al., 1991), its combination with SBBGR treatment could allow a safe
wastewater reuse in absence of conventional tertiary disinfection. This
would avoid the release in the environment of hazardous by-products
generated during tertiary disinfection (i.e. chlorine addition; Watson
et al., 2012). Indeed, a recent study by da Costa et al. (2014) evaluated
the effect of tertiary wastewater disinfection on algae, insects, shellfish
and fish. The research showed that all the disinfection strategies evalu-
ated (i.e. chlorine, peracetic acid, ozone and UV) increased the toxicity
of a WWTP secondary effluent. Fig. 1. A photograph of the treatment plant used in the present study. a: aerator; b:
Currently a common EU directive on wastewater reuse is still lacking biofilter; c: recirculation pump; d: filling pump; e: blower; f: drawing valve; g: sand
and each country set its own quality parameters considering a number filter; h: PLC.
 M. De Sanctis et al. / Science of the Total Environment 580 (2017) 17–25 19

The wastewater is stored in a different compartment (also known as aer- Table 2

ator) with a volume of about 180 L (diameter: 27 cm; height: 320 cm) Composition of the domestic wastewater fed to the SBBGR plant (in terms of average
values and standard deviations of the main gross parameters; n. samples: 190).
and where aeration is also provided. Wastewater is continuously
recirculated through the biofilter, allowing biological degradation process- Parameters Concentration [mg/L] Parameters Concentration [mg/L]
es. This way, the treated wastewater, being free of biomass, can easily be COD 535 ± 201 TN 73 ± 26
disposed of without the traditional sedimentation step. The two compart- BOD5 321 ± 115 NH3 52 ± 19
ments (i.e., biofilter and aerator) are connected at the top by means of TSS 237 ± 128 P-tot 11 ± 5
a pipe. The SBBGR unit was operated in sequential mode on the base of
6 h–treatment cycles, each including three sequential phases: filling, recir-
samples was also monitored (twice in each season; 10 samples). The anal-
culation and drawing. During the 0.5 h filling phase, 60 L of raw wastewa-
ysis of sodium, calcium and magnesium (needed for calculating SAR) was
ter were pumped into the aerator. In the recirculation phase, the
carried out by means of atomic absorption spectrophotometer analysis.
wastewater was constantly aerated and pumped through the biomass
Moreover, the other parameters required by the Italian regulation on
compartment. Finally, during the 0.5 h drawing phase, 60 L of treated
reuse were monitored three times during the experimental campaign.
wastewater were discharged from the aerator (by opening a motorised
These additional parameters include: heavy metals, cyanides, sulfur, chlo-
valve) into the top of the sand filter. Taking into account the working
rines, fluorides, animal and vegetable fats and oils, mineral oils, phenols,
volume of the plant (i.e., 300 L), a hydraulic residence time of 30 h can
chlorinated solvents, trihalomethanes, aromatic organic solvents, organic
be obtained. The reactor operation was controlled by using a programma-
nitrogenous solvents, surfactants and pesticides. The analysis of these
ble logic controller (PLC) which allowed flexible operating times of the
compounds was conducted according to the standard methods.
filling pump, the recirculation pump, the blower and the drawing valve.
The disinfection efficiency of the proposed treatment was evaluated
The sand filter consisted of a plastic cylindrical unit with a volume of
by monitoring Escherichia coli, somatic coliphages, Clostridium
about 120 L (diameter: 0.35 m), partially filled with river sand support-
perfringens, Salmonella, Cryptosporidium parvum oocysts, Giardia lamblia
ed by gravel as follows: 10 cm of gravel (irregular shape; larger diame-
cysts, enteroviruses and adenoviruses in the raw wastewater, after
ter: 5–15 mm), 40 cm of sand (diameter: 0.3–0.8 mm), 5 cm of gravel on
SBBGR treatment and in the final effluent (i.e., after sand filtration).
the filter top to promote uniform wastewater distribution. The flow rate
Ten sampling campaigns were performed for all the microbiological pa-
through the filter was checked a minimum of two times a week. Due to
rameters with the exception of enteroviruses and adenoviruses (five
the filter clogging, a washing step was carried out when the flow rate
sampling campaigns). The disinfection efficiency of the system was
decreased under 10 L/h.
expressed in terms of log units removed.
Main features and operating conditions of SBBGR and sand filter are
E. coli abundance was assessed by using IDEXX Colilert-18 and
summarized in Table 1.
Quanti-Trays/2000 according to the company instructions (IDEXX
The SBBGR plant used during the experimental campaign, which
Laboratories, Inc., 2013). It is an enzyme-based test based on the most
lasted about fifteen months between 2014 and 2015, had already been
probable number (MPN) technique. Each analysis was carried out in du-
in operation for treating domestic wastewater coming from tourist
plicate for each ten-fold serial dilution tested (two). Somatic coliphages
areas since 2011. Starting from 2014 the plant treated wastewater com-
were quantified according to the ISO 10705-2 (2000) method. Each ten-
ing from small villages in the district of Bari which was supplied daily to
fold serial dilution tested (three dilutions for wastewater and SBBGR efflu-
the plant by means of a truck. The composition of wastewater fed to the
ent) was analysed in quadruplicate. For SF effluent, 20 mL of undiluted
plant during the present study is reported in Table 2.
sample were scrutinised. The results are expressed in terms of plaque-
forming units per unit volume (PFU/100 mL). Salmonella was analysed ac-
2.2. Treatment performance evaluation
cording to the ISO 19250 (2010) method in 1 L of the sample. Spores of C.
perfringens were quantified according to the ISO 7937 (2004) method. The
Treatment effectiveness was evaluated in terms of removal of the
analysis was performed in triplicate for each ten-fold serial dilution tested
typical physical and chemical gross parameters and also in terms of mi-
(three), and the results are expressed in terms of colony forming units per
crobial indicators.
unit volume (CFU/100 mL). C. parvum and G. lamblia, were analysed in
The monitored physical and chemical gross parameters included:
250 mL of influent wastewater and 5 L of treated wastewater, respective-
pH, EC, COD, BOD5, TSS, volatile suspended solids (VSS), ammonia
ly, according to the US EPA (2005) method 1623. Briefly, cysts and oocysts
(NH3), nitrites (NO− −
2 ), nitrates (NO3 ), total Kjeldahl nitrogen (TKN),
were concentrated and eluted by IDEXX foam filters and a Filta-Max® elu-
total nitrogen (TN) and total phosphorus (TP). With the exception of
tion station (IDEXX), purified by immuno-separation using dynabeads
pH and conductivity (measured by selective probes) and TKN (obtained
GCcombo (Invitrogen) and detected by immunofluorescent staining by
as the difference between TN and oxidised nitrogen), the other param-
MERIFLUOR® Cryptosporidium/Giardia (Meridian Bioscience Inc.). The
eters were measured according to the standard methods (APHA et al.,
results are expressed in terms of oocycts/L and cysts/L, respectively.
2005). These parameters were analysed in SBBGR influent and effluent
Analyses of enteroviruses and adenoviruses were conducted in 1 L of
three times a week (190 samples) and their removal efficiency was
influent wastewater and 10 L of treated wastewater, respectively. Sam-
expressed in terms of percentage reduction.
ple concentration was performed by means of Virosorb Zeta Plus 1MDS
According to the aim of agricultural reuse of the treated wastewater,
filters (CUNO-3M); filters were eluted in beef extract (3%) and purified
the sodium adsorption ratio (SAR) in some SBBGR influent and effluent
with PEG 6000 as described in Masciopinto et al. (2011). Enteroviruses
were quantified by RT-QPCR as reported in Masciopinto et al. (2011)
Table 1 and adenoviruses were analysed as described in Wyn-Jones et al.
Main features and operating conditions of SBBGR and sand filter. (2011). PCR analysis was conducted in duplicate for each sample.
SBBGR Sand filter

Volume (biofilter + aerator) (L) 300 Volume 120 3. Results and discussion
Wastewater recirculation (L/h) 120 Sand bed high (cm) 55
Wastewater up-flow velocity into biofilter (m/h) 3 Flow rate (L/h) ≥10
Aeration (m3/h) 3.5 3.1. Treatment performance evaluation in terms of physical and chemical
Cycle length (h) 6 parameter removal
Hydraulic loading rate (L/d) 240
Hydraulic retention time (h) 30 The performances of the SBBGR plant recorded during fifteen
Organic loading rate (KgCOD/m3biofilter d) 1.1
months of operation are shown in Figs.s 2, 3 and 4. Fig. 2 shows the
20 M. De Sanctis et al. / Science of the Total Environment 580 (2017) 17–25

Fig. 2. EC, SAR and pH in wastewater (influent) and SBBGR effluent. The error bars represent the standard deviation interval (n. samples: 10 for SAR and 190 for pH and EC).

average values of pH, EC and SAR measured in the influent and effluent of biofilms in irrigation pipe systems, resulting in their clogging. The re-
of SBBGR. These parameters acquire great relevance in case of agricul- sults shown in Fig. 3 clearly indicate that this risk can be considered neg-
tural reuse of the treated wastewater. A pH near to neutrality is required ligible with the effluent produced by SBBGR. In fact, an average TSS
in almost all regulations about water reuse (i.e. between 6.5 and 8.5 in removal efficiency of 96% (almost always higher than 90%) was obtain-
Cyprus, Greece and Portugal; Sanz and Gawlik, 2014). Thus the pH of ed with a residual concentration in the effluent of 10 mg/L. Effluent
SBBGR effluent (on average 7.8, Fig. 2) is compatible with its reuse. A values resulted almost independent from influent value even at the
study by Rietz and Haynes (2003) clearly highlights that high values highest concentrations (i.e., 650 mg/L). Furthermore, sand filtration
of EC and SAR in the irrigation water have multiple negative effects. In allowed to further reduce suspended solids concentration constantly
particular, they cause the deterioration of soil properties and reduce below 5 mg/L.
the growth of crops as well as soil microbial biomass. Indeed, several Also BOD5 concentration in SBBGR effluent was not affected by
countries (such as Italy, Spain, Greece, Portugal; Sanz and Gawlik, the wastewater composition. In spite of a wide variation in BOD5
2014; Paranychianakis et al., 2015) have set limits for these parameters concentration in the raw wastewater, this parameter was always
in case of wastewater reuse in agriculture (i.e. Italy has set the maxi- removed with an efficiency approaching 100% (Fig. 3). In particu-
mum values of 3 mS/cm and 10 for EC and SAR respectively). The data lar, BOD5 concentration in SBBGR effluent was always lower than
in Fig. 2 clearly show that there is no concern if SBBGR effluent is used 5 mg/L. In terms of COD, an average removal efficiency of 93%
for crop irrigation since EC and SAR values were always lower than (COD removal was higher than 90% in 85% of the samples and always
1000 mS/cm and 3.8, respectively. It is worth noting that SBBGR treat- higher than 85%) was obtained with a residual concentration of
ment is able to significantly reduce EC value from 1.22 (average influent 38 mg/L in the effluent.
value) to 0.88 mS/cm (average effluent value). This can be ascribed to The stable and efficient removal of TSS, BOD5 and COD by the SBBGR
the high absorption capability of SBBGR biomass as also confirmed by derives from the particular structure of the biomass confined in the fill-
its low VSS/TSS ratio (lower than 0.5; see below). ing material of the biofilter. In this section, the biomass generates a
Fig. 3 shows the concentrations of COD, BOD5 and TSS measured in dense and compact matrix (composed by a mixture of biofilm and gran-
the influent and effluent of the SBBGR unit with relative removal effi- ules, see De Sanctis et al., 2010). In particular, a biomass concentration
ciencies. The TSS and BOD5 concentrations are other two important fea- as high as 75 gTSS/Lbiofilter was recorded. When wastewater flows
tures of an irrigation water. In particular they acquire more relevance in through the biofilter, most of the suspended solids are entrapped in
case of the use of drip irrigation systems. Indeed, TSS tend to accumulate the biological matrix. Successively they are subjected to degradation
in irrigation tubing systems and water diffusers, causing their gradual by the lytic enzymes produced by the biomass and then used by the bio-
obstruction. Similarly, high presence of BOD5 favours the development mass itself for its growth.

Fig. 3. COD, BOD5 and TSS concentrations in wastewater (influent) and SBBGR effluent and their relative removal efficiencies. The error bars represent the standard deviation interval (n.
samples: 190).
 M. De Sanctis et al. / Science of the Total Environment 580 (2017) 17–25 21

Fig. 4. Nitrogen and phosphorous concentrations in wastewater (influent) and SBBGR effluent and their relative removal efficiencies. The error bars represent the standard deviation
interval (n. samples: 190).

The plant resulted effective in nitrogen removal also. Fig. 4 shows that Table 3 shows the concentration of heavy metals and other addition-
the SBBGR plant removed about 79% of the total nitrogen with an average al parameters (i.e. required by the Italian regulation on reuse) into
effluent concentration of 15 mg/L. Therefore, SBBGR effluent could comply wastewater and SBBGR effluent. According to the domestic origin of
with the current TN limits for wastewater reuse in agriculture in European the wastewater they were present at low concentrations.
countries which ranges from 15 to 30 mg/L for Italy and Greece respective-
ly (Table S.1; Sanz and Gawlik, 2014; Paranychianakis et al., 2015). This 3.2. Treatment performance evaluation in terms of microbial indicators
good result is ascribable to the presence of a stable simultaneous nitrifica- removal
tion-denitrification process in the system which leads to a final concentra-
tion in the effluent of NH4-N and NO3-N of 2 and 11 mg/L, respectively. The disinfection efficiency of the proposed treatment was evaluated
This ability of SBBGR biomass derives from high biomass concentration monitoring the following microbial indicators and pathogens after each
in the biofilter which allows the coexistence of nitrifying and denitrifying treatment phase: Escherichia coli and Salmonella (representative of bac-
bacteria (De Sanctis et al., 2010). teria), Clostridium perfringens spores (representative of spore-forming
Fig. 4 also indicates that a relevant phosphorous removal efficiency bacteria), somatic coliphages, human enteroviruses and adenoviruses
(on average, 62%) with a residual effluent concentration of 4 mg/L was (representative of viruses), Giardia lamblia and Cryptosporidium parvum
obtained by SBBGR. This removal can be considered quite high, taking (representative of protozoa). The results obtained during ten sampling
into account the low sludge production observed (0.15 KgTSS/ rounds are reported in Table 4. In particular, the table shows the con-
KgCODremoved instead of the value of 0.5–0.6 KgTSS/KgCODremoved com- centration of the monitored microorganisms measured in raw waste-
monly reported for activated sludge processes). Furthermore, the excess water, SBBGR effluent and in the final effluent (i.e., after sand
sludge was characterised by a P/VSS ratio of about 10% which is much filtration). The removal efficiency (expressed in terms of log units re-
higher than that one previously observed in SBBGR biomass (b1%; Di moved) after each treatment is also reported. More details on the con-
Iaconi et al., 2011). centration of the indicators and pathogens in SBBGR and sand filter
This result would suggest the presence of a biological phosphorous effluent are presented as Supplementary material in Tables S.2–3.
removal by phosphorous accumulating organisms (PAO). Nevertheless,
these microorganisms require a specific sequence of environmental 3.2.1. Effect on bacteria
conditions in the reactor (i.e., an anaerobic phase followed by an aerobic The first parameter reported in Table 4 is Escherichia coli, which is
one: Adav et al., 2008). Considering that in SBBGR plant there is no one of the most commonly used microbial indicator of faecal contami-
planned anaerobic phase, the presence of a chemical or physical phos- nation. An efficient removal of E. coli is an essential requirement for
phorous removal processes cannot be excluded. For instance, due to wastewater reuse in several EU countries (Sanz and Gawlik, 2014;
the very high biomass concentration in the biofilter (i.e. 75 gTSS/ Paranychianakis et al., 2015), even if a common limit for E. coli concen-
Lbiofilter) phosphorous present in particulate form can be retained by tration in water has not been fixed yet. Biological treatment by SBBGR
the biomass. resulted in effective E. coli removal, allowing 3–4 log units to be re-
In addition to the reduced sludge production, the latter was moved with a residual effluent concentration lower than 1000 MPN/
characterised by an average VSS/TSS ratio of 46%, which is much lower 100 mL. This performance is higher than that observed in conventional
than the typical value reported for waste activated sludge (i.e. about WWTPs, where a 2–3 log units removal is usually observed with a resid-
80%; Bougrier et al., 2008). Therefore, the traditional stabilisation step ual E. coli concentration in the range of 104–105 MPN/100 mL (Lebaron
usually carried out in traditional wastewater treatment plants is no lon- et al., 2011). Similar results (i.e., E. coli effluent concentrations in the
ger required. The high degree of sludge stabilisation in SBBGR is an ef- range of 104–105 MPN/100 mL) have been reported also by other au-
fect of the high biomass age. This aspect represents an additional thors (De Luca et al., 2013; Carducci et al., 2008). The performance of
benefit of SBBGR technology, in particular in case of small wastewater SBBGR should be considered as particularly interesting, all the more
treatment installations. Indeed, usually small plants are not provided so, since it was obtained by a single step compared to the entire water
with a sludge stabilisation unit and the excess sludge needs to be deliv- treatment line of conventional WWTP.
ered to larger plants were it can be properly stabilised. The E. coli concentration in the SBBGR effluent acquires more rele-
Furthermore, these characteristics of SBBGR sludge (high vance considering the quality criteria indicated by WHO guidelines
stabilisation and phosphorous content) would suggest its possible use (World Health Organization – WHO, 2006), which suggested a limit of
as fertiliser. 103 CFU/100 mL in case of wastewater reuse in agriculture. Indeed,
22 M. De Sanctis et al. / Science of the Total Environment 580 (2017) 17–25

Table 3
Average concentration of heavy metals and additional parameters in plant influent and effluent (n. samples: 3).

Parameter Influent SBBGR effluent

Aluminum [mg/L] 0.8717 ± 1.3972 0.0620 ± 0.0234

Arsenic [mg/L] 0.0022 ± 0.0025 0.0039 ± 0.0034
Barium [mg/L] 0.1312 ± 0.1697 0.0215 ± 0.0138
Beryllium [mg/L] 0.0006 ± 0.0001 0.0006 ± 0.0001
Boron [mg/L] 0.0600 ± 0.0115 0.0651 ± 0.0237
Cadmium [mg/L] 0.0005 ± 0.0000 0.0005 ± 0.0000
Cobalt [mg/L] 0.0005 ± 0.0001 0.0011 ± 0.0008
Total chromium [mg/L] 0.0034 ± 0.0033 0.0005 ± 0.0000
Iron [mg/L] 2.8443 ± 4.1220 0.0569 ± 0.0962
Manganese [mg/L] 0.1247 ± 0.0909 0.0680 ± 0.1024
Mercury [mg/L] 0.0034 ± 0.0057 0.0010 ± 0.0017
Nickel [mg/L] 0.0048 ± 0.0030 0.0050 ± 0.0020
Lead [mg/L] 0.0048 ± 0.0071 0.0005 ± 0.0000
Copper [mg/L] 0.0346 ± 0.0558 0.0113 ± 0.0093
Selenium [mg/L] 0.0005 ± 0.0000 0.0005 ± 0.0000
Tin [mg/L] 0.0077 ± 0.0124 0.0005 ± 0.0000
Thallium [mg/L] 0.0005 ± 0.0000 0.0005 ± 0.0000
Vanadium [mg/L] 0.0042 ± 0.0045 0.0077 ± 0.0058
Zinc [mg/L] 0.1850 ± 0.1611 0.0253 ± 0.0215
Total cyanides (as CN) [mg/L] N.D.a N.D.a
Sulphides [mg/L] N.D.a N.D.a
Sulphites [mg/L] N.D.a N.D.a
Sulphates [mg/L] 20 ± 11 25 ± 7
Active chlorine [mg/L] 0.18 ± 4 0.05 ± 2
Chlorides [mg/L] 70 ± 4 72 ± 3
Fluorides [mg/L] 0.268 ± 0.151 0.457 ± 0.113
Animal/vegetable fats and oils [mg/L] 0.10 ± 0.06 0.09 ± 0.05
Mineral oils [mg/L] N.D.a N.D.a
Total phenols [mg/L] 0.820 ± 0.313 0.094 ± 0.067
Pentachlorophenol [mg/L] N.D.a N.D.a
Total aldehydes [mg/L] 0.51 ± 0.08 N.D.a
Tetrachlorethylene + trichlorethylene [mg/L] N.D.a N.D.a
Total chlorinated solvents [mg/L] N.D.a N.D.a
Trihalomethanes [mg/L] 0.0004 ± 0.0003 N.D.a
Total aromatic organic solvents [mg/L] N.D.a N.D.a
Benzene [mg/L] N.D.a N.D.a
Benzopyrene [mg/L] N.D.a N.D.a
Total nitrogenous organic solvents [mg/L] N.D.a N.D.a
Total surfactants [mg/L] 4.1 ± 1.2 0.6 ± 0.5
Chlorinated pesticides [mg/L] N.D.a N.D.a
Phosphorus pesticides [mg/L] N.D.a N.D.a
Other pesticides [mg/L] N.D.a N.D.a
a
N.D.: never detected.

SBBGR effluent exceeded this value only in one sample during the Organization – WHO, 2006) and already compatible with its unrestrict-
experimentation. ed reuse in some European countries (Sanz and Gawlik, 2014;
The quality of SBBGR effluent further increased after the sand filtra- Paranychianakis et al., 2015) such as Spain (100 CFU/100 mL; Iglesias
tion step. In fact, the sand filter removed on average 1 log unit of E. coli, et al., 2010) and France (250 CFU/100 mL; Paranychianakis et al.,
thus leading to an overall disinfection efficiency higher than 4 log units. 2015). In particular, E. coli concentration exceeded 10 MPN/100 mL
The corresponding E. coli concentration in the effluent after sand filtra- (i.e., Italian limit for reuse) only in 50% of the samples and was always
tion was on average as low as 60 MPN/100 mL. Thus, referring to the pa- lower than 270 MPN/100 mL. Referring to wastewater reuse in agricul-
rameter E. coli, the quality of the plant effluent was much higher than ture, another important bacterial parameter is represented by Salmonella.
that suggested by WHO guidelines (103 CFU/100 mL; World Health This bacterium is not only an indicator of faecal contamination, but

Table 4
Performances of the proposed treatment in terms of concentrations of the selected microbial indicators measured in wastewater, SBBGR effluent and after sand filtration (SF effluent).
Removal efficiency expressed in terms of log units removed is also reported after each treatment step. All results are given as mean value ± standard deviation interval (n. samples: 10).

Parameter Wastewater SBBGR effluent SF effluent

7 3
E. coli Concentration (MPN/100 mL) 1.0 ± 2.9 × 10 0.9 ± 2.1 × 10 6.0 ± 9.1 × 10
Log unit removed 3.2 ± 1.1 4.2 ± 1.5
5
C. perfringens Concentration (CFU/100 mL) 5.2 ± 5.2 × 10 4.9 ± 6.5 × 104 1.8 ± 2.1 × 103
Log unit removed 1.1 ± 0.4 2.7 ± 0.8
Somatic coliphages Concentration (PFU/100 mL) 2.8 ± 3.3 × 105 1.5 ± 1.7 × 104 2.9 ± 4.7 × 102
Log unit removed 1.4 ± 0.3 3.2 ± 0.4
G. lamblia cysts Concentration (cysts/L) 1.3 ± 1.6 × 103 2.9 ± 3.6 × 10 0.7 ± 0.7
Log unit removed 1.5 ± 0.9 2.9 ± 0.6
C. parvum oocysts Concentration (oocysts/L) 4.7 ± 4.7 × 101 0.7 ± 0.5 NDa
Log unit removed 1.8 ± 0.3 2.4b
a
ND: never detected.
b
The log units removed were calculated considering the detection limit.
 M. De Sanctis et al. / Science of the Total Environment 580 (2017) 17–25 23

also a serious human pathogen. Therefore, it should be removed from are viruses pathogenic for bacteria but not for humans and considered
the treated water to ensure its safe reuse. Moreover, some regulations a suitable indicator of virus fate in treatment processes. They were cho-
about wastewater reuse require its complete removal from the treated sen due to their wide diffusion in municipal and domestic wastewaters
wastewater (i.e., the Italian and Spanish laws). According to this, Sal- and their cultivability in laboratory conditions. Adenovirus and entero-
monella was monitored following a presence/absence approach and virus were chosen as they are human pathogens and their presence was
the removal efficiency could not be expressed as per log units removal. monitored by qPCR.
Salmonella was found only in some samples of raw wastewater. Con- Table 4 reports somatic coliphages concentration in raw wastewater,
versely, it was never detected in the samples of treated wastewater. after SBBGR and in the sand filter effluent. The data clearly show that co-
The efficient removal of Salmonella by SBBGR treatment is in line liphages were always present in high concentrations in raw wastewater
with the results reported in a study by Koivunen et al. (2003) for (i.e., from about 104 up to 106 PFU/100 mL) and that they were quite
four Finnish municipal WWTPs based on the activated sludge process. sensitive to SBBGR treatment and sand filtration. In particular, SBBGR
In this study, the authors report removal efficiencies of Salmonella systems removed on average 1.4 log units of these viruses. This removal
higher than 98% for all the WWTPs. These results indicate that Salmo- was lower than that obtained for E. coli (i.e., 3.2 log units), confirming
nella is efficiently removed either in conventional WWTPs or in the the higher surviving capacity of viruses in WWTPs with respect to
SBBGR system. The absence of Salmonella in few samples of raw E. coli. Nevertheless, SBBGR performances are comparable to that usual-
wastewater could derive from the wastewater origin (i.e., small com- ly reported for conventional WWTPs. Lucena et al. (2004) have reported
munity of Bari) which cannot ensure the presence of this bacteria in removals of 1.5 log units of coliphages for four conventional WWTPs
all the samples. located in Argentina, Colombia, France and Spain. Similarly, another
study by Jebri et al. (2012), carried out in three conventional WWTPs
3.2.2. Effect on spore-forming bacteria located in Tunisia, displayed coliphages removals ranging from 1.1 up
The data reported in Table 4 shows that SBBGR is able to ensure a to 2.0 log units. Therefore, as already observed for E. coli and
stable removal of about 1 log unit of C. perfringens. This microorganism C. perfringens, SBBGR seems to ensure at least the same removal efficien-
has been widely used for the evaluation of water quality due to its abil- cy of conventional WWTPs obtained, however, by preliminary, primary
ity to generate spores in the presence of adverse growing conditions and secondary treatments.
(Lucena et al., 2004; Ottoson et al., 2006; Briancesco and Bonadonna, Sand filtration provided an effective and easy strategy for a further
2005; Shannon et al., 2007). This ability makes C. perfringens extremely reduction of coliphages in SBBGR effluent. Indeed, SF removed at least
resistant to biological, chemical and physical disinfection strategies. The 1.3 log units of coliphages, with an average removal of 1.9 log units,
high surviving ability of C. perfringens in WWTPs has been well thus leading to an overall removal higher than 3 log units. As already ob-
highlighted by Lucena et al. (2004) in a study carried out in six munici- served for C. perfringens, the reduction of coliphages is currently normed
pal treatment plants. All these plants removed only about 1 log unit of in France which requires a removal between 2 and 4 log units depend-
this bacteria. Therefore, the performance of SBBGR in removing ing on the type of agricultural reuse (Paranychianakis et al., 2015; Sanz
C. perfringens is in line with those recorded in primary and secondary and Gawlik, 2014).
treatments of conventional WWTPs. Conversely, the pilot SBBGR result- According to the literature (Skraber et al., 2004; Ballester et al.,
ed less effective than the bench-scale plant reported in De Sanctis et al. 2005), there is a correlation between coliphages concentration and en-
(2016) which removed about 2 log units of C. perfringens. This result teric viruses (i.e. enterovirus and adenovirus) presence in water sam-
might be a consequence of the process scale-up. ples. In particular, Skraber et al. (2004) found that N 50% of samples
Data reported in Table 4 show, however, that sand filtration of (23 out of 42) containing high concentration of coliphages (N3 log
SBBGR effluent significantly improves C. perfringens removal. In fact, PFU/100 mL) were positive for enterovirus. Conversely, enterovirus ge-
an overall removal of 2.7 log units was obtained with a residual average nome was present in only 1 sample (1 out of 7) when coliphages con-
content of 1.8·103 CFU/100 mL. This removal efficiency is lower than centration was lower than 2 log PFU/100 mL. Thus, the low
that observed for E. coli, but it can still be considered valuable if com- concentration of coliphages in the SF effluent would suggest that en-
pared with that obtained in conventional WWTPs. Similar results have teroviruses were also efficiently removed. This finding was supported
been obtained by Ottoson et al. (2006) for a Swedish WWTP provided by the data obtained from qPCR analysis in our study. SBBGR treatment
with sections for biological nitrogen removal, pre-denitrification, chem- and SF were able to remove about 2–3 log units of adenovirus and en-
ical precipitation and dual media sand filtration. In this case, the whole terovirus, thus showing a removal efficiency similar to that obtained
treatment line removed about 2.4 log units of C. perfringens from waste- for coliphages. However, unlike from somatic coliphages, adenovirus
water. The removal of 2.7 log units, obtained by combining SBBGR treat- and enterovirus were detected only in two wastewater samples. There-
ment and sand filtration, can be regarded even more relevant fore, the limited number of positive samples did not allow a significant
considering that this microorganism is quite resistant to tertiary disin- analysis of the process removal efficiency.
fection treatments (Gehr et al., 2003). Analogous results were obtained The absence of these microorganisms in most of the wastewater
by the authors in a study at laboratory scale aimed at evaluating the samples could derive from the wastewater origin (i.e., small communi-
effectiveness of UV radiation and peracetic acid for disinfecting SBBGR ty) which cannot ensure their constant presence in the analysed sam-
effluent. Indeed, both these treatments removed b0.2 log units of ples. This is confirmed by several studies on municipal wastewaters
C. perfringens (De Sanctis et al., 2016). (Sedmak et al., 2005; Ottoson et al., 2006; Jebri et al., 2012) which report
As the best of the authors' knowledge, currently sulphate-reducing that these viruses were present only in a small fraction of the collected
C. perfringens are cited only in French legislation which requires a re- wastewater samples (about 10–30%).
moval between 2 and 4 log units depending on the type of agricultural
reuse (e.g. vegetables to be eaten uncooked or cooked, fruit tree; 3.2.4. Effect on protozoa
Paranychianakis et al., 2015; Sanz and Gawlik, 2014). Thus, the quality The SBBGR was highly efficient in the removal of C. parvum and
of pilot plant effluent was in line with this reuse criteria. G. lamblia. The average concentration of these pathogenic protozoa in
raw wastewater was 4.7 · 101 oocysts/L and 1.3 · 103 cysts/L, respec-
3.2.3. Effect on viruses tively. With regard to C. parvum, the SBBGR treatment ensured its al-
The presence of viruses represents another important parameter to most complete removal. In fact, an average removal of 1.8 log units
be taken into account for evaluating the suitability of treated wastewa- was obtained, and it was found only in 30% of the effluent samples
ter for agricultural reuse. Three model organisms were chosen for this with a concentration of about 1 oocyst/L. Similarly, the plant removed
study: somatic coliphages, adenovirus and enterovirus. The first ones about 1.5 log units of G. lamblia, but due to its higher presence in the
24 M. De Sanctis et al. / Science of the Total Environment 580 (2017) 17–25

wastewater, it was always detected in the plant effluent at an average In particular, the plant completely removed C. parvum and Salmonella
concentration of 29 cysts/L. These pathogenic protozoa survive in the and provided about 4 log units removal of E. coli and 3 log units removal
external environment in the form of cysts which are quite resistant to of C. perfringens, somatic coliphages and G. lamblia.
adverse environmental conditions. The high diffusion and surviving
ability of Giardia and Cryptosporidium in the environment is well docu-
Acknowledgments
mented in the study of Castro-Hermida et al. (2010). In fact, the results
obtained in recreational river areas (28 locations), drinking water and
The authors wish to thank Dr. Maurizio Divizia for his support for the
wastewater treatment plants (52 and 50 plants, respectively) in Spain
analysis of enterovirus and adenovirus.
have shown that both these protozoa were present in about 60% of
This study is part of the Water4Crops – EU project (Grant Agreement
river samples and N30% of effluents from drinking water treatment
N. 311933), which has been financially supported by the EU Commis-
plants. In the case of WWTPs effluent, the percentage of positive sam-
sion within the thematic priority Food, Agriculture and Fisheries, and
ples increased up to 64 and 96% for Cryptosporidium and Giardia, respec-
Biotechnology of the Seventh Framework Programme (Call: FP7-
tively. Moreover, the concentration of Cryptosporidium in the influent
KBBE-2012-6).
and effluent samples of treatment plants was not significantly different,
indicating a removal efficiency near zero. In contrast, a removal of 60%
was observed for Giardia. Similarly, Ajonina et al. (2012), in a study on Appendix A. Supplementary data
three German WWTPs designed for biological nitrogen removal follow-
ed by phosphorous precipitation, found that b60% of Cryptosporidium Supplementary data to this article can be found online at http://dx.
spp. content was removed. A stable removal of Giardia lamblia was re- doi.org/10.1016/j.scitotenv.2016.12.002.
ported by Cacciò et al. (2003) in a study on four Italian municipal
WWTPs. The WWTPs were able to remove between 1 and 2 log units
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