Treatment of sewage sludge contaminated with bacteria by using anolyte water

Enas Mortada1,Waheed Emam2, A.A. Melegy3, Hala Ahmed Hegazi1 and Mohamed A. El-
Said1

1- Housing and Building Research Center, Egypt

2- Zoology Department, Faculty of Science, Ain Shams University,

3- Geological Sciences Department, National Research Centre,

Abstract

The main aim of this study is to evaluate the disinfection effect of anolyte water on some
species of bacteria detected in sewage sludge, by using different concentrations of anolyte water
and different incubation periods. The results showed that tested bacterial sp. showed different
reduction in their numbers by increasing incubation periods. Also, the obtained results showed
that anolyte water in concentration of 1 ml produced 100% inactivation for E. coli,
Staphylococcus sp., Bacillus sp., and Enterococcus sp. after 30min exposure time. While 2ml
anolyte water produced complete inactivation for E. coli, Staphylococcus sp., Bacillus sp. and
Enterococcus sp. after 20 min exposure time. In conclusion anolyte water can be used safely for
inactivation of some pathogens found in sewage sludge.

Key words: Sewage sludge, treatment, anolyte water, bacteria.

Introduction

Sludge is the solid material remaining after sewage treatment facilities purify wastewater
produced from homes, business and industries. Obtaining cleaner water from treatment facilities
means producing more sludge. The sludge produced from municipal wastewater treatment plants
seems to be a problem in some countries, while in other countries is recognized as an
environment friendly source of power rather than being a burden on the environment. Whether
the sludge is used or disposed of, it is important to avoid creating additional environmental
problems and to keep costs down. In the past the sewage sludge was disposal in the least

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troublesome, most affordable ways possible. It was dumped at sea, buried in landfills or burned
in incinerator (Haller, 1999).

It has a various contents such as inorganic substances and agriculture value components
(organic substances, nitrogen, phosphorous and potassium) and lesser amount of calcium,
sulphur and magnesium but the sludge must first undergo treatment of various natures .It may
also contain pollutants such as heavy metals and pathogens (Rulkens, 2008).

Due to the increase in the cost of landfill and more strict environmental standards and
increased public concern about air, land and water, new methods for sludge treatment was
developed (Haller, 1999 and Jun Yin and Xuejun Tan, 2005). The produced sludge from
developing countries usually differs from which generated in developed ones due to divergent
industrialization and public health levels. In developing countries, the metal and toxic content is
usually much lower, while pathogens content is much higher (Rulkens, 2008 and Zaini et al.,
2006). A lot of diseases spread due to the use of sludge from wastewater treatment plants without
further treatment, which cause harmful effects to human, animals and plants.

Sludge contains many different types of pathogens that are secreted by humans and animals
through the feces. Feces originate from wastewater from households and from storm water,
which can contain feces from birds, cats, dogs, etc. (Naturvårdsverket, 2002).

Sludge utilization in agriculture has been increased in recent years (Stone et al., 1998). It
is therefore, very important to control pathogens present in sewage sludge for protection of
humans, animals and plants (Lepeuple et al., 2004).There detection of pathogens in sludge is
usually influenced by the level of treatment methods (Carrington, 2001).

The present disposal routes of sewage sludge represent a critical environmental issue in
Egypt. Recently, there has been an increasing concern about sewage sludge management due to
the environmental risks, which resulted from the fast expansion of wastewater treatment plants
without equal attention in dealing with the produced sludge. Therefore, the pressing needs are to
develop appropriate low cost methods to treat the sewage sludge to be safe and suitable for reuse
in agriculture (Ghazy et al., 2009).

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 Anolyte water was used as alternative of chlorine to avoid contamination of wastewater
with pathogens (Perçin and Esen, 2009). Anolyte water was produced from electrolyzing salt
water through double chamber membrane electrolytic cell.
Anolyte water is obtained from electrolyzing salt water moreover; electrolyzed water
consists of HOCl, ClO2-, Clo3-Cl- dissolved oxygen, superoxide radicals and others.

The anolyte water was colorless liquid with pH value of 2-3 and contains reactive ions and
free radicals, which contribute to its powerful oxidizing properties. Also, anolyte water is
nontoxic and harmless to human (Leonov, 1999).

The current study is investigated using anolyte water in treatment of sewage sludge from
different forms of pathogens.

MATERIALS AND METHODS

1-Anolyte water:

Anolyte water was prepared according to Wolf, (2012) CEO W.P.C- Estonia and it obtained
from Egypt Envirolyte Ltd.

2-Microbial Examination:
Different concentrations of anolyte water (1 and 2ml) were added to microbial species (E. coli,
Enterococcus sp., Baccillus sp. and staphylococcus sp.) to evaluate the biocidal effect of anolyte
water at different incubation periods (zero time, 1min, 5min, 10min, 15min and 20min). These
microbial species were prepared as follow, under sterilization condition one disk was taken from
each microorganism and inoculated in sterile 5 ml tryptice soy broth and incubated at 37°C for
24 hours. After incubation, the inoculated broth was centrifuged at 5000 rpm for 15 minutes, and
then the pellets were transferred to 5 ml sterile saline water. The centrifugation step was repeated
three times. The obtained counts from saline water were adjusted at 95, 102, 110 and 150 cfu/ml
for E. coli., Staphylococcus sp, Bacillus sp. and Enterococcus sp., respectively. By used poured
plate count agar and dilution test tubes. One ml of saline water microbes was separately
inoculated in an autoclaved water (1000 ml) to evaluate the biocidal effect of anolyte water.

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Anolyte water was applied to bacterial strains in concentrations 1 to 1 and 1 to 2 with exposure
time from 1, 5, 10, 20 and 30 minutes .After the exposure to the anolyte water the viability of
microorganisms was estimated to APHA (2017).

The second part of the experiment was to evaluate the effectiveness of anolyte on
untreated sewage sludge which collected from Abo Rawash wastewater treatment plant. The
samples were collected and transferred to the lab in ice box within 4 hours (APHA, 2017).
4 gm of solid sewage sludge was added to 100ml of dist. water (HBRC, 2015).Different volumes
of anolyte water (100, 50, 25,10,5 ml) was added to the sewage sludge at different incubation
periods (zero time, 5, 10, 15 and 20 min).

Results
The effect of anolyte water was evaluated by application on some bacterial strains. The
effect of 1ml of anolyte water to 1ml of the different stains (E. coli, Staphylococcus sp.,
Bacillus sp., and Enterococcus sp.) represented in table (1) ,while table (2) shows the effect of
adding 2ml of anolyte water to 1ml of the different strains of bacteria.

Table (1): The effect of adding 1ml of anolyte water to 1ml of different strains of
bacteria at different incubation periods

Count(CFU/ml)

Time
control 1 min. 5 min. 10 min. 20 min. 30 min.
Bacteria sp.
E. Coli 59 59 95 33 51 0

Staphylococcus sp. 501 55 59 15 59 0

Bacillus sp 550 50 90 0 0 0

Enterococcus sp. 590 95 59 10 0 0

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Table (2) the effect of adding 2ml of anolyte water to 1ml of different strains of bacteria at
different incubation periods

Count(CFU/ml)

Time
control 1 min. 5 min. 10 min 20 min. 30 min.
Bacteria sp.

E.Coli 59 13 51 0 0 0
Staphylococcus sp. 501 10 35 50 0 0
Bacillus sp. 550 10 10 0 0 0
Enterococcus sp. 590 51 35 55 0 0

Fig. 1 The effect of anolyte water with concentration (1/1) on some strains of bacteria at
different incubation periods

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Fig. 2 The effect of anolyte water with concentration (1/2) on some strains of bacteria at
different time incubation periods

Anolyte water was applied to sewage sludge from Abo Rawash treatment plant, results of
total colifrom and fecal coliform represented in table 3 and 4

Table 3: The effect of different concentrations of anolyte water on Total Coliform at

different incubation periods on sewage sludge from Abo Rawash treatment plant

Total coliform Count (MPN/100ml)

Time Zero
Conc. time 5 min 10 min 15 min 20 min
100/100 160000 8000 0 0 0
50/100 160000 33000 14000 0 0
25/100 160000 32000 22000 8000 0
10/100 160000 23000 0 0 0
5/100 160000 16000 16000 11000 0

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 180000
160000
140000
MPN/100l 120000
100/100
100000
80000 100/50
60000
40000 100/25
20000
100/10
0
control 5 min 10 min 15 min 20 min
100/5
Time

Fig. 3: Effect of different concentrations of anolyte water with different incubation periods
on sewage sludge from Abo Rawash treatment plant on Total Coliform

Table 4: The effect of different concentrations of anolyte water on Fecal Coliform at

different incubation periods on sewage sludge from Abo Rawash treatment plant

Fecal coliform Count (MPN/100ml)

Time Zero
Conc. time 5 min 10 min 15 min 20 min
100/100 160000 8000 0 0 0
50/100 160000 9000 7000 0 0
25/100 160000 13000 12000 8000 0
10/100 160000 14000 0 0 0
5/100 160000 23000 16000 1000 0

200000
100/100
MPN/100ml

150000
100/50
100000 100/25
100/10
50000
100/5
0
control 5 min 10 min 15 min 20 min
Time

Fig. 4: Effect of different concentrations of anolyte water with different incubation periods
on sewage sludge from Abo Rawash treatment plant on fecal Coliform

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 Discussion
As shown in Tables and Figures (1) and (2) 1ml of anolyte water reduced 100% of E.coli
and Staphylococcu sp. after 30 min, Enterococcus sp. after 20min and Bacillus sp. after 10min
and adding 2ml of anolyte water reduced 100% of E. coli and Bacillus sp. after 10min,
Staphylococcu sp.and Enterococcus sp. after 20min exposure time (Table 2 and Fig. 2).
From the tables, the results show that anolyte water at concentration (1/1) after 30 min killed
the investigated bacteria; while anolyte water at concentration of (1/2) get rid of the bacteria after
20 min.
Some studied reported that, the efficacy of disinfectants depended on the concentration of
biocide, organic matters, pH and counts of microorganisms as well as types which they play a
role in efficacy of anolyte water (EPA, 2001; WHO, 2001; 2008).

Samast et al. (2008) reported that anolyte water is more efficient, low cost, and nontoxic
and vide spectrum of usage. In addition it kills bacteria, viruses, fungi and parasites quickly and
can be used for disinfection of hard surfaces and water systems. Moreover, some authors
demonstrated that how it is obtained and how efficient is anolyte water? According to Perçin
and Esen (2009).

Samast et al., 2008; Tallinn, 2008 and Perçin and Esen, (2009) reported that anolyte
water is harmless on human tissue, does not form toxic product and it is also considered to be
safe as well as it kills any microbes during seconds. In addition, some authors (Marais
&Rawhani, 2001 and Mikhailov et al., 2009) concluded that anolyte water is a fair and a
universal germicidal agent alternative of disinfectant can be applied as well as sanitation, pre-
sterilizing treatment in water treatment. Some researchers in Serbia, (Miomir et al., 2014)
showed that water-disinfection appliance from electrolyzing salt can be used in water systems
where it is successful as biocidal in natural water which contains some microbial (Enterobacter,
Citrobacter, Bacillus, Aeromonas, Streptococcus, E. coli, Aeromonas, Pseudomonas aeruginisa
and Sulfate-reducing clostridium. Moreover, in Spain, Sergi et al. (2015) observed that,
oxidizing agent from active chlorine ions which are produced from electrolyzed water (Anolyte

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& Catholyte) play importance role in removal some organic contaminants from wastewater
during treatment.
Among the steps of water treatment is using chlorine in many parts of the world, especially
developing countries, to kill microbes in water moves through the ecosystems until reached
downstream users (WHO, 1984). Lechevallier et al. (1988) found that in water Klebsiella
pneumonia can be resistant to chlorine from 2- to 10-fold of for free chlorine of 0.3 ppm in 30
min contact time. Reasoner et al., (1989) detected in water which were treated with chlorine
collected from distributed systems, some types of bacteria (such as Enterobacter and
Citrobacter) as well as potentially opportunistic pathogens (such as Aeromonas and
Pseudomonas).

Damian and Jeanne (2007) noticed that Mycobacterium. aurum, followed by

Staphylococcus. epidermidis and E. coli. (Log 10 3 to 4 CFU/mL) were survived in water with
initial free chlorine concentrations of 0.2, 0.4, and 0.8 ppm. In addition, USEPA, (2004) WHO,
(2008) reported that, chlorine is to be less effective on viruses and some microbial during the
wastewater treatment, this due to survival microorganisms in effluent and affected on sources of
drinking water. So they refer using ozone and ultraviolet light as disinfectant to killing
microorganisms are recommended.

It was obvious from Table (3) that total coliform count before treatment (160000
MPN/100ml) reduced to (8000, 33000, 32000, 23000, 16000) MPN/100ml with
concentrations (100, 50, 25, 10 and 5ml) of anolyte water after 5min. On the other hand, the
total coliform at concentrations 100/100 and 100/10 was zero after 10min. After 20 min the
total coliform was zero at all concentrations. Table and Figure (4) illustrate that fecal coliform
count before treatment (160000 MPN/100ml) reduced to (8000, 9000, 13000, 14000, 23000)
MPN/100ml with concentrations (100, 50, 25, 10 and 5ml) of anolyte water after 5min.The
count of fecal coliform become zero at concentrations 100/100 and 100/10 after 10min. After
20 min the total coliform was zero at all concentrations.

This result was in agreement with Majumder, (2004), in India, who recorded in samples
collected from different of wastewater treatment Plants, the removal ratio of heterotrophic total

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bacterial count after treatment were ranged from 80 to 90% while were 98 % after treated with
disinfecting material (chlorine or ozone). Park et al., (2008) concluded that present organic
matter reduces the effectiveness of acidic electrolyzed water for reducing microorganisms on the
surfaces of lettuce and spinach. Moreover, Bohra et al., (2012) reported high bacterial content in
wastewater due to the organic matter which is produced from human activity and animal wastes.

Conclusion
 The treatment of sewage sludge with 5ml of anolyte water can make disinfection to fecal
and total coilform after 20min.
 Anolyte water could be concluded that it is better as biocide than other used disinfectants
in sewage sludge treatment and reduce pathogens.

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