Environmental Pollution 252 (2019) 8e13

Contents lists available at ScienceDirect

Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Household-based biodigesters promote reduction of enteric virus and

bacteria in vulnerable and poverty rural area*
Maria Ce
lia da Silva Lanna a, Aline Viancelli b, Wiliam Michelon b,
Sergio Vinícius Castro Carvalho a, Deyse de Almeida dos Reis a,
Luiz Anto ^ nio Fernandez de Salles a, Iago Hashimoto Sant’Anna a,
Letícia Teresinha Resende a, Clovis de Souza Ferreira a, Igor Aparecido das Chagas a,
Marta Herna
zaro d, Gislaine Fongaro f, *

ndez c, d, Helen Treichel e, David Rodríguez-La
a
Laboratory of Microbiology and Technological Bioprospection, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil
b
Universidade do Contestado e UNC, PMPECSA, Conco
rdia, SC, Brazil
c
Laboratory of Molecular Biology and Microbiology, Instituto Tecnolo
gico Agrario de Castilla y Leo
n, Valladolid, Spain
d
Department of Biotechnology and Food Science, University of Burgos, Burgos, Spain
e
Laboratory of Microbiology and Bioprocesses, Universidade Federal da Fronteira Sul (UFFS), Erechim, RS, Brazil
f
polis, SC, Brazil
Laboratory of Applied Virology, Universidade Federal de Santa Catarina, Floriano

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

Article history: The present study evaluated the river water quality improvement by implementation of household-
Received 21 January 2019 based biodigesters in vulnerability and poverty rural area, in Minas Gerais State-Brazil. For that, 78
Received in revised form household-based biodigesters were installed for domestic wastewater treatment. Wastewater was
20 May 2019
collected before and after treatment and the physicochemical parameters and pathogens removal (hu-
Accepted 20 May 2019
Available online 21 May 2019
man adenovirus (HAdV), hepatitis A (HAV) virus, Salmonella sp. and Escherichia coli) were evaluated;
Additionally, river water was sampled before and after the household-based biodigesters implementa-
tion, to verify the contamination reduction and the positive impact of domestic wastewater treatment on
Keywords:
Decentralized sanitation
waterborne pathogen reduction, considering HAdV, HAV, Salmonella sp. and E. coli quantification. The
Pathogen removal applicability in real-scale of decentralized treatment systems using household-based biodigesters pro-
Viruses and bacteria reduction moted reduction of 90, 99, 99.99 and 99.999% from HAV, Salmonella sp., E. coli and HAdV from domestic
Public health wastewater, respectively; The river water quality improvement before the wastewater treatment
Water management application was highlight in the present study, considering that the reduction of waterborne pathogens
in this water in 90, 99.99 and 99.999% of E. coli, HAV and HAdV, respectively (Salmonella sp. was not
detected in river water). In general, this is an important study for encouraging the decentralized sani-
tation in vulnerable and poverty area, as well in rural sites, considering the positive impact of this
implementation on public health.
© 2019 Published by Elsevier Ltd.

1. Introduction management policies, human and animal health. In this context,

According to the World Health Organization, more than 2.6
billion people worldwide do not have access to sanitation, and 884
million people do not have access to good quality water for con-
sumption (WHO, 2015). Basic sanitation is precarious in developing
countries, as Brazil, and this fact is important to environmental
*
This paper has been recommended for acceptance by Prof. Dr. Klaus Kümmerer.
* Corresponding author.
E-mail address: gislaine.fongaro@ufsc.br (G. Fongaro).
the One Health approach has been discussed to improve the life
quality.
In this sense, official data indicate that only 58% of the popula-
tion has sewage collected and from this, only 42.6% have been
treated (WHO, 2015). Collection, treatment and discharge of do-
mestic sewage can be done in centralized or decentralized systems,
once that in rural areas the wastewater changes greatly in quality,
quantity, and spatial distribution, where the decentralized treat-
ment provide a good choice as it avoids plumbing works for waste
collection (Jorsaraei et al., 2014).

https://doi.org/10.1016/j.envpol.2019.05.104
0269-7491/© 2019 Published by Elsevier Ltd.
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elia da Silva Lanna et al. / Environmental Pollution 252 (2019) 8e13
The solution for sanitation problems in rural communities is
based on the use of social and innovative technologies consistent
with population economic reality. In this sense, the use of low-cost
wastewater and sewage treatment systems, such as the evapo-
transpiration tank, biodigestion septic tank, filtering gardens, and
biodigesters have been studied with successful. Anaerobic bio-
digestion is a promising technology in countries with tropical cli-
mates, and it could also recover nutrients and reuse water in the
treated final effluent (Gallia et al., 2015).
The release of untreated domestic wastewater and sewage into
springs poses risks to human, animal and environmental health, as
they can cause various diseases. On outbreaks caused by pathogens
carried by water (waterborne pathogens), the most frequently re-
ported are those caused by viruses, specially human adenovirus
(HAdV), norovirus (NoV) hepatitis A virus (HAV), rotavirus A (RVA)
and others (Fong and Lipp, 2008) or bacteria as pathogenic E.coli,
Salmonella sp., and others enterobacteria generus (Frickmann et al.,
2015).
Enteric pathogens are excreted in high concentrations in feces of
contaminated people and animals, persisting for long periods of
time in residues, and can reach water sources leading to contami-
nation (Frickmann et al., 2015; Fong and Lipp, 2008). The long-term
stability of enteric viruses in aquatic environments is due to its
undeveloped structure as well as to its adsorption capacity in
organic particles (Bosch et al., 2006). In Brazil many studies
monitored the enteric viruses in lagoon water (Elmahdy et al.,
2016), in reuse water (Fongaro et al., 2014), in a dairy farm (Spilki
et al. al. 2013), in river water (Rigotto et al., 2010) and, as well the
pathogens removal from wastewater in Brazilian treatment sys-
tems (Villar et al., 2007; Victoria et al., 2010; Prado et al., 2011).
According to Technology at the Health Service System (Brazil,
2018), like most small Brazilian communities, Miguel Burnier (Ouro
Preto city - Brazil) has precarious sanitation conditions, where a
great part of its population throw the sewage directly into the rivers
and the waterborne diseases are frequent in this site, as gastroen-
teritis, schistosomiasis, and enteric hepatitis.
Although the region is an important geological-
geomorphological domain and has anomalous concentrations of
metals, and is used by major companies for iron ore mining
(Hartmann et al., 2006) and the local population is poor and
vulnerable, often unassisted aspects of health and education.
Fig. 1. (A) Implementation area of household-based biodigester in Miguel Burnier-MG, Brazil and evaluated site of river in this area. (B) Groups of evaluated household-based
biodigester according to spatial distribution.
9
In this sense, the present study evaluated the environmental
positive impacts after implementation of household-based bio-
digesters in the vulnerable rural area, in Minas Gerais State-Brazil,
considering the microbial reduction in domestic wastewater and in
river water used for human activities and animal consumption.
2. Material and methods
2.1. Studied area and household-based biodigesters
The study was performed in Miguel Burnier located in Minas
Gerais State (Latitude 20
26 ‘03 00 South and Longitude 43
460 3500
West). This site have 900 habitants and is inserted in the geomor-
phological unit “Quadrila
tero Ferrífero”, and its economy is based on
mining, especially iron ore, which has been exploited since the 19th
century. This site was historically characterized by the absence of
sanitation, and discharge of wastewater in a local river.
This area is supplied by the Miguel Burnier River, used for
fishing, irrigation of vegetables and ornamental plants, as well as
animal consumption (swine, poultry and livestock for human food).
In this area 78 household-based biodigesters (Acqualimp®) with a
300 L volumetric capacity were implemented in 2016, considering
that was first treatment of domestic wastewater/sewage in Miguel
Burnier-MG 9 (Fig. 1).
2.2. Sampling events
Wastewater and river water from studied area were evaluated
before and after household-based biodigesters installation were
sampled during one year (2016).
Wastewaters were collected, considering the (affluent) and after
(effluent), from household-based biodigesters. Twenty percent
household-based biodigesters (12/78) were segregated in five
groups (according to spacial distribution in the evaluated area in
Miguel Burnier) and randomly evaluated. For this, 2 L of affluent
and effluent samples were collected during four collect campaigns
divided in five groups, to cover five different plots of the studied
area (G1, G2, G3, G4 and G5). In this same period, 5 L of river water
were collected from 5 different sites utilized for human activities
and animal consumption (Fig. 1).
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elia da Silva Lanna et al. / Environmental Pollution 252 (2019) 8e13

2.3. Physicalchemical analysis 3. Results and discussion

Wastewater was evaluated as the physicalchemical character- 3.1. Physicalchemical profile

istic according APHA (2012). Biochemical oxygen demand (BOD),
chemical oxygen demand (COD), nitrate (NO 
3 ), nitrite (NO2 ), total Table 1 shows the physicalchemical profile from the wastewater
ammoniacal nitrogen (TAN), pH, and temperature (T) were samples. pH ranged from 6.0 to 7.0 in both the affluent and the
evaluated. effluent. Temperature ranged from 22.0 to 23.0
C in the affluent
and from 23.8 to 28.4
C in the effluent. All other evaluated pa-
rameters were not removed efficiently. This occurs because the
2.4. Enteric virus quantification

For enteric viruses analysis, 200 mL of wastewater was clarified

and concentrated using the glycine buffer method and poly-
ethylene glycol precipitation, eluated in phosphate buffer pH 9.0
and reconcentrated by centrifugation step using the Centriprep YM
50 (Millipore) filtration system (Schlindwein et al., 2010 and Pina
et al., 1998). Subsequently, nucleic acid extraction was performed
using a QIAmp MinElute Virus Spin Kit (Qiagen) following manu-
facturer instructions; For enteric viruses analyses in river, 2 L of
water samples were concentrated using negatively-charged
membrane, as describe by Katayama et al. (2005).
From concentrated samples, the extraction of viral nucleic acid
was performed using a commercial RTP DNA/RNA Virus® II Mini Kit
(Invitek), according to the manufacturer's instructions. Subse-
quently, HAdV and HAV were quantified by qPCR, according to
Hernroth et al. (2002) and Jothikumar et al. (2016), respectively,
using the TaqMan assay methods, in a StepOne Plus® Real-Time PCR
System (Applied Biosystems). Each sample was carried out in
duplicate, and on each plate four serial dilutions of standard were
run in triplicate for each assay. Ultrapure water was used as non-
template control for each assay. The genome copies (GC) were
defined as the mean of the data obtained and results were
expressed in GC L-1.

2.5. Enterobacteria quantification

E. coli was enumerated using the MPN IDEXX Quanti-Tray sys-

tem with Colilert reagents according to the manufacturer's in-
structions, using 100 mL of water or wastewater. Results were
expressed as Most Probable Number (MPN 100 mL-1).
Salmonella spp. quantification was performed using 25 mL of
sample added in 225 mL buffered peptone-NaCl solution and
incubated at 37
C for 24 h. The colonies forming unity (CFU) were Fig. 2. Average concentration of enteric pathogens (Log10) in the (a): affluent and
quantified in xylose-lysine-tergitol-4 (XLT4), according to ISO 6579 effluent from household-based biodigesters; (b): river water (before and after
(2002). Results were expressed in CFU mL-1. household-based biodigesters implementation for domestic wastewater treatment).

Table 1
Physical-chemical parameters evaluated on affluent and effluent from the biodigestors.

Samples TAN (mg$L-1) Nitrite (mg$L-1) Nitrate (mg$L-1) BOD (mg$L-1) COD (mg$L-1)

G1 Affluent 3269 1.4 1.2 230 450

Effluent 2684 2.3 0.015 110 395
Removal (%) 17.9 0 98.8 52.2 12.2
G2 Affluent 3323 2.3 1.7 240 430
Effluent 2840 3.1 0.013 112 356
Removal (%) 14.5 0 99.2 53.3 17.2
G3 Affluent 3421 4.3 1.14 356 423
Effluent 2789 2.3 0.019 219 396
Removal (%) 18.5 46.5 98.3 38.5 6.4
G4 Affluent 3419 5.2 1.6 234 487
Effluent 2384 2.4 0.015 183 321
Removal (%) 30.3 53.8 99.1 21.8 34.1
G5 Affluent 3487 2.6 0.99 211 456
Effluent 2823 3.1 0.018 178 312
Removal (%) 19 0 98.2 15.6 31.6
Fig. 3. Log10 reduction of each enteric pathogen evaluated in treated wastewater (a); and in river water (b).
12 M. C
elia da Silva Lanna et al. / Environmental Pollution 252 (2019) 8e13
operation time is still short (about 35 days of operation), however,
it is possible to observe that G1 and G2 already present a removal
efficiency above 50% of BOD.
Removal pathogen efficiency above 90% and biogas production
depends of hydraulic retention time, inoculum quality and sub-
strate, been necessary months or even years, however, the suc-
cessful utilization of this treatment process have been reported all
around the world (Capson-Tojo et al., 2016).
3.2. Enteric pathogens profile
Average concentration of enteric pathogens in domestic
wastewater and in the river water are presented on Fig. 2. In
wastewater (affluent) it is possible to verify high amounts from all
pathogens evaluated, with an average of 106 MPN$100 mL-1 of
E. coli, 103 CFU mL-1 of Salmonella sp., and 108 and 104 (GC L-1) of
HAdV and HAV, respectively. However, when evaluating the
effluent microbial characteristics (Fig. 2a), it is possible to see the
reduction of 90, 99, 99.99 and 99.999% from HAV, Salmonella sp.,
E. coli and HAdV, respectively (Fig. 3a). Environmental positive
impact was demonstrated after implementation of household-
based biodigesters as decentralized sanitation system. River water
quality, that before the wastewater treatment application pre-
sented high concentrations of enteric pathogen, considering 103
MPN$100 mL-1 of E. coli; 105 GC L-1 of HAdV and 105 GC L-1 of HAV
(Fig. 2b), and after the wastewater treatment the removal efficiency
of enteric pathogens contamination was of 90, 99.99 and 99.999% of
E. coli, HAV and HAdV, respectively (Fig. 3b). E. coli concentration
was reduced in the household-based biodigesters effluent (Fig. 1a),
however, the amount observed in river water it is higher than the
observed in the effluent, this could be explained by the fact that this
bacteria is not exclusive of humans, but it is excreted by all warm
blood animals.
Studies conducted by Gallia et al. (2015), evaluating only E. coli,
demonstrated the removal of 99% of these fecal indicators in do-
mestic wastewater by household-based biodigesters system in real-
scale. In important to highlight that virulent E. coli strains, as
enteropathogenic (EPEC), enterotoxigenic (ETEC), enterohemorragic
(EHEC), enteroinvasive (EIEC), and enteroaggregative (EAEC), can be
harmful to humans, typically being the cause of diarrhea, often le-
thal to children (Al-Badaii and Shuhaimi-Othman, 2015; European
Food Safety Authority, 2011).
In environmental conditions (limited nutrient availability, os-
motic stress, large variations in temperature and pH), Salmonella sp.
can enter a viable but non cultivable state in which bacterial cells
are metabolically active but cannot be cultured by known labora-
tory methods (Winfield and Groisman, 2003; Ravel et al., 1995). In
the present study all river water samples were negative for Sal-
monella sp (Figura 1b). Low positivity of Salmonella sp. has been
observed in other rivers, as the located above the Guarani Aquifer in
southern Brazil (Suruvi and Caçador Rivers) (Viancelli et al., 2015;
Pastore et al., 2018).
The virus concentration in river water are lower that the
observed in the affluent, representing the importance of this social
technology for environmental quality and consequently public
health, once that viruses are considered to be the major cause of
acute diarrhea in young children, as described by Bosch et al.
(2008), and in Brazil by Miagostovich et al. (2014) and Barreira
et al. (2010).
Enteric viruses, mainly HAdV, have high stability in aquatic
environments, remaining viable and infectious for more than 90
days (Bosch et al., 2008). In this regard, the monitoring of enteric
viruses is relevant for predicting public health risks and monitoring
the sanitary quality of watersheds (Miagostovich et al., 2014).
However, developing countries, such as Brazil, have not yet
established a standard for monitoring viral indicators in waters
intended for human activities.
The severity of the disease caused by HAV is age-dependent, as
young children are usually asymptomatic transmitters of the dis-
ease is still a neglected health problem in the world. In Brazil, HAV
seroprevalence studies showed that 80% of the general population
has antibodies against HAV in the country (Mantovani et al., 2015),
considering the HAV positivity in recreational marine waters (Dias
et al., 2018), drinking water (Elmahdy et al., 2016), and in foods of
animal origin (Pereira et al., 2018).
Use of household-based biodigesters promoted the reduction of
enteric virus and bacteria in a vulnerable and poverty rural area.
Household-based biodigesters have low-cost installation, take up
little space and meet decentralized sanitation demands in vulner-
able areas such as rural areas in developing countries, improving
human, animal and environmental health. We results demon-
strated that household-based biodigesters could be an
environment-friendly alternative to decentralized sanitation in
vulnerable areas of waterborne diseases. In future studies in this
area, considering a long time, during different year station and
natural optimization of anaerobic microorganisms, we expected to
get a better effect on pathogens inactivation and to prospect future
improve biogas yield for domiciliary uses, considering the tech-
nology of household-based biodigesters.
4. Conclusion
The present study evaluated and demonstrated the efficiency of
anaerobic biodigesters on the reduction of human enteric patho-
gens (>90%), showing that the technology is an alternative to
decentralized sanitation in vulnerable areas of waterborne dis-
eases, prioritizing and protecting the human, animal and environ-
mental health.
Acknowledgments
Coordination of Improvement of Higher Level Personnel
(CAPES), National Council for Scientific and Technological Devel-
opment (CNPq), Gerdau Group, and Ouro Preto Economic and So-
cial Development Agency (ADOP) from Brazil.
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