Journal of Environmental Management 255 (2020) 109821
Contents lists available at ScienceDirect
Journal of Environmental Management
journal homepage: http://www.elsevier.com/locate/jenvman
Research article
Bioremediation of landfill leachate by Aspergillus flavus in submerged
culture: Evaluation of the process efficiency by physicochemical methods
and 3D fluorescence spectroscopy
Yassine Zegzouti a, b, Aziz Boutafda a, Amine Ezzariai a, Loubna El Fels a, c, Miloud El Hadek b,
Lalla Amina Idrissi Hassani d, Mohamed Hafidi a, e, *
a
Laboratory of Microbial Biotechnologies, Agrosciences and Environment, Faculty of Science Semlalia, Cadi Ayyad University, Marrakesh, 40000, Morocco
Laboratory of Process Engineering Faculty of Sciences, Ibn Zohr University, Agadir, 80000, Morocco
c
Higher Institute of Nursing Professions and Health Technics, Marrakech-Safi, Morocco
d
Laboratory of Plant Biotechnology, Department of Biology, Faculty of Sciences of Agadir, Ibn Zohr University, BP 8106, 80000, Agadir, Morocco
e
Agrobiosciences Program, Mohammed VI Polytechnic University (UM6P), Benguerir, 43150, Morocco
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bioremediation
Landfill leachate
Aspergillus flavus
3D fluorescence spectroscopy
The present study investigates the ability of Aspergillus flavus (A. flavus) for organic and nitrogen matter removal
from landfill leachate. Experiments were carried out with different types of leachate, (Young (YL), Intermediate
(IL) and Old (OL)) used at different concentrations of the leachate up to 100%. The organic fraction of landfill
leachate was measured by biological oxygen demand (BOD5) and chemical oxygen demand (COD) then it was
qualitatively assessed using three dimensional excitation emission matrix (3D-EEM). The nitrogen fraction was
þ
measured by ammonium (NHþ
4 ) and nitrate (NO3 ). The experiments revealed that, BOD5, COD and NH4 removal
rates after 4 weeks of treatment in flasks were within the ranges of 47.90–81.63%, 12.91–48.50% and
70.84–98.81%, respectively and that affected the reduction of the phytotoxicity in a positive way. A. flavus with
25% concentration of YL recorded the best results in reducing COD and BOD5 with maximum removal rates of
around 48.50% and 81.63%, respectively. However, the highest NHþ
4 removal rate of 98.81% was found in 25%
concentration of OL. The 3D-EEM results showed that the intensities of the fluorescent peaks for the three treated
leachates have decreased sharply after treatment. This was confirmed by the increase of the organic matter
complexity index for different treatments (from 0.55 to 0.87). Therefore, A. flavus may be potentially useful in
the treatment of landfill leachate at a concentration of less than or equal to 50% as it was able to remove organic
and nitrogen compounds, particularly in the treatment of YL leachate at a concentration of 25%.
1. Introduction
Leachate generation is an important environmental problem that is
caused by municipal solid waste (MSW) landfills (Zegzouti et al., 2019).
The pollutant load of leachate can be divided into four major groups of
contaminants, namely, dissolved organic matter, inorganic macro
components, heavy metals, and xenobiotic organic compounds (Renoua
et al., 2008). Improper management of landfill leachate lead to an
adverse impact on the environment as well as on the human health
(Chofqi et al., 2004). The Landfill leachate is a high-strength wastewater
with considerable variations in both chemical composition and
volumetric flow (Renoua et al., 2008). Although leachate composition
may vary widely within different stages of refuse decomposition, three
types of leachate can be defined according to landfill age, namely,
young, intermediate and old landfill leachate (YL, IL and OL) (Chian and
DeWalle, 1977). A wide range of treatment processes have been
explored for leachate including physico-chemical, and biological
methods with their own advantages and limitations. Physico-chemical
methods of leachate treatment are considered efficient. However, their
disadvantages like sludge generation and high cost cannot be ignored
(Kumari et al., 2016). Biological treatment is often preferred due to its
reliability, low operating costs and simplicity and provides many
* Corresponding author. Laboratory of Microbial Biotechnologies, Agrosciences and Environment, Faculty of Science Semlalia, Cadi Ayyad University, Marrakesh,
40 000, Morocco.
E-mail address: hafidi.ucam@gmail.com (M. Hafidi).
https://doi.org/10.1016/j.jenvman.2019.109821
Received 23 September 2019; Received in revised form 1 November 2019; Accepted 3 November 2019
Available online 25 November 2019
0301-4797/© 2019 Elsevier Ltd. All rights reserved.
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
advantages in terms of biodegradable matter and nitrogen compounds
removal. Various biological methods such as activated sludge (AS),
sequencing batch reactors (SBR) and aerated lagoons (AL) have been
used for leachate treatment (Alijani Ardeshir et al., 2016). The microbial
bioremediation has also recently gained interest in the field of landfill
leachate treatment as a means to remove contaminants (Morris et al.,
2018). Studies have reported that some bacteria showed great ability to
be resistance to a wide range of compounds found within leachate and
potential application for its treatment (Morris et al., 2018).
However, in bacterial systems, if the organic contaminant is avail­
able at low concentration, a degradation will no longer take place as its
concentration will not be sufficient for a growth of bacteria (Singh,
2006a). In other words, bacterial cells must expend energy to induce the
catabolic process used in biodegradation and if the organic contami­
nants, which can be used as a source of carbon and energy, are not
available in adequate quantities, induction will not occur (Maier, 2000).
On the contrary, fungi have a great ability to adapt to extreme growth
conditions which could be found in the landfill leachate such as high
levels of metals, polychlorinated hydrocarbons, polyaromatic hydro­
carbons, and ammonia, while the process of degradation can still be
initiated (Singh et al., 2015). Such extreme conditions result in the in­
duction of wide variety of degradative enzymes (Singh, 2006b). The
major advantage lies in the fact that the production of these enzymes
does not depend on the organic contaminant or its concentration.
. Only a few studies have been carried out to treat leachate using
fungi (Ellouze et al., 2008a; Tigini et al., 2013; Razarinah et al., 2015;
Bardi et al., 2017; Smaoui et al., 2018; Spina et al., 2018) may be due to
the difficulty of finding fungal strains that are able to thrive in leachate
extreme environment while removing contaminants from landfill
leachate. These studies were conducted at laboratory-scale in micro­
cosms involving flasks, at bench-scale involving box or pan reactors, or
at pilot-scale in the filed involving one or more small-scale treatments’
plots where they have been used for the bioremediation of pollutants
including organic matter contaminants quantified by COD and BOD5, as
well as nitrogen matter such as ammonium (NHþ
4 ). In this context, these
authors reported the ability of many selected fungi, particularly
white-rot (ligninolytic) fungi such as Bjerkandera adusta MUT 2295,
Ganoderm australe, Phanerochaete chrysosporium, for their bioremedia­
tion ability for landfill leachate effluent. However, in most of these
studies, the treatment of leachate was applied using fungi without
addressing the type of leachate and its concentration. Variation in these
two parameters may affect the treatment efficiency by these fungi due to
the various changes that may occur periodically in the leachate
composition according to its associated type, as well as the rainfall
dilution effect. In addition, they have primarily involved white-rot
fungi, owing to their production of multiple extracellular
lignin-modifying enzymes, which might also play an important role in
the biotransformation of organic compounds (Rajendran et al., 2017).
However, some non-ligninolytic fungi have demonstrated their capacity
to remove environmental pollutants, thus overcoming some of the lim­
itations observed in white-rot fungi such as sensitivity to stress condi­
tions, low growth in neutral pH and high oxygen concentrations
requirements (Marco-Urrea et al., 2015). Among the non-ligninolytic
fungi, A. flavus have been well known for their potential application in
bioremediation of effluents with different strengths from different in­
dustries and sewage (Mukherjee, 2016). Somewhat, surprisingly, there
have been no studies of leachate treatment using sporal inoculum re­
ported in the literature. There have, however, been numerous studies
that address the treatment of landfill leachate by free mycelia and fun­
gus mycelia immobilized within a matrix (Ellouze et al. 2008a; Tigini
et al., 2013; Razak et al., 2016; Bardi et al., 2017). Therefore, the aims of
the current study were; (1) to investigate A. flavus capability as a
non-ligninolytic fungus through an acclimated sporal inoculum of this
strain in removal of leachate organic and nitrogen matter in three types
of leachate (i.e., YL,IL and OL); to (2) determine the type of leachate
which is more suited for bioremediation using this strain; and (3) to
determine the leachate concentration level that gives the highest effi­
ciency in leachate organic and nitrogen matter removal and toxicity
reduction.
2. Materials and methods
2.1. Site description
This study was conducted in Morocco in three different landfills: two
are located in Marrakech city (Marrakesh Closed Landfill) (MCL), Al
Mnabha village (Marrakesh New Landfill) (MNL) and one in Agadir city
(Greater Agadir landfill) (GAL), (Table 1). These locations were selected
to be representative of landfills of different ages and types of waste. One
of the three landfills (MCL) was non-controlled (i.e., without liners and
leachate collection system), while the two others (MNL and GAL) were
controlled (i.e., with liners and leachate collection systems). Among
these three landfills, two were operational (MNL and GAL) while the
third one was closed (MCL) but it is, currently, under re-planting
operations.
2.2. Sampling and physico-chemical characterization of landfill leachate
The raw landfill leachate samples (i.e., YL,IL and OL) were collected
from the three above-mentioned landfill sites as described by Zegzouti
et al. (2019). The main physico-chamical charcteristics of landfill
leachate originating from MNL, MCL and GAL were previously analyzed
as shown by Zegzouti et al. (2019) (Table 2).
2.3. Subculture and preparation of spore suspension
The fungal culture used in this study was A. flavus strain isolated
form leachate contaminated soil provided by the Ecology and Environ­
ment laboratory of the Biology Department (L2E), the Faculty of Sci­
ences Semlalia at Cadi Ayyad University. Spore suspensions of A. flavus
were prepared following Aneja(2007). The fungal spores were initially
harvested by aseptically and gently scraping the aerial mycelium from
the medium surface of the plate with 200 ml saline solution containing
0.85% (w/v) with the aid of sterile loop, followed by vigorous agitation
to break up clumps of spores. The spores suspension solution was then
filtered through several layers of chees-cloth to remove most of the
fungal mycelium and permits passage of only of fungal isolates spores.
The concentration of spores in the suspension was measured by a
cell-counting hemocytometer chamber (Neubauer Germany) Aneja
(2007), and giving an average count of 1.12 � 106 spores/mL.
Table 1
General conditions of the landfill sites included in the study.
Condition class
Marrakesh New
Landfill (MNL)
Marrakesh Closed
landfill (MCL)
Greater Agadir
landfill (GAL)
Landfill type
Sanitary
(operational)
2016-date
Non-sanitary
(closed)
1980–2016
Sanitary
(operational)
2010-date
Young
Old
Intermediate
900 to 1000
703
756
Household
Household,
commercial and
industrial
Physical
Household
No facility
No facility
No facility
31� 540 47.6"N
8� 040 54.1"W
31� 420 09.8"N
8� 030 59.7"W
30� 260 27.1"N
9� 300 47.2"W
Period of
landfilling
Age of
classification
Daily average of
waste disposed
(tonnage)
Waste type
Form of the
leachate
treatment
Fate of generated
landfill gas
Coordinates at the
landfill sites
2
Physical and
Biological
Physical and
Biological
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
Table 2
Characteristics of raw leachate at Marrakesh (new and closed) and Greater
Agadir landfills (MNL MCL and GAL). Values represent means � SD (n ¼ 3)
(Zegzouti et al., 2019).
No
1
2
3
4
5
6
7
8
9
10
11
12
Parameter
pH
Electrical conductivity
(mS/cm)
Suspended solids (mg/L)
COD (mg/L)
BOD5 (mg/L)
BOD5/COD
Total Kjeldahl Nitrogen
(TKN)
Ammonium (mg/L NHþ
4N)
3Nitrate-N (mg/L NO -N)
Temperature (� C)
Total Phosphorus
C:N:P
Marrakesh landfills
Greater Agadir
landfill
New
Old
Medium aged
Average
Average
Average
6.8
66.76
9 .1
78.9
8.1
55.43
1926.93
19 523.33
11600
0.6
6335.21
1293.6
9517.4
1756.8
0.18
4026.37
430.05
14560.7
5300.15
0.36
6149
5931.25
3112.68
5226.13
35.03
25.25
65.07
100:32:0.3
12.22
25.1
12.4
100:3 :
0.13
27.47
24.89
33.6
100:35:0.23
Table 3
Leachate treatment applications used the in experiment.
Treatments
Details of treatments
Y-RL
Y-100%RLI
Y-50%
DRLI
Y-25%
DRLI
I-RL
I-100%RLI
I-50%DRLI
100% Raw Young Leachate
100% Raw Young Leachate with 10% inoculum of spores
50% Raw Young Leachate with 10% inoculum of spores þ50%
distilled water
25% Raw Young Leachate with 10% inoculum of spores þ75%
distilled water
100% Raw Intermediate Leachate
100% Raw Intermediate Leachate with 10% inoculum of spores
50% Raw Intermediate Leachate with 10% inoculum of spores þ50%
distilled water
25% Raw Intermediate Leachate with 10% inoculum of spores þ75%
distilled water
100% Raw Old Leachate
100% Raw Old Leachate with 10% inoculum of spores
50% Raw Old Leachate with 10% inoculum of spores þ50% distilled
water
25% Raw Old Leachate with 10% inoculum of spores þ75% distilled
water
I-25%DRLI
0-RL
0-100%RLI
0-50%DRLI
0-25%DRLI
2.6. Fluorescence spectroscopy analysis
Fluorescence is the light emission caused by the excitation of a
molecule. This usually occurs due to the absorption of a photon, fol­
lowed by a spontaneous emission. 3D Fluorescence spectra of landfill
leachate and treated leachate samples were obtained with a PerkinElmer
LS55. Excitation wavelengths varied from 200 to 600 nm with in­
crements of 10 nm. The slit width of emission and excitation in the
monochromator was fixed at 10 nm. The scanning speed of a mono­
chromator was set at 1200 nm s 1. Fluorescence emission spectra were
recorded every 0.5 nm between 200 and 600 nm. According to the work
of Chen et al. (2003), Muller et al. (2014), and Jimenez et al.(2015), the
obtained spectra were decomposed on seven zones (zone I to VII) cor­
responding to the fluorescence of each biochemical molecules. The
zones I, II and III represent essentially protein-like materials, and the IV
can be associated with fulvic-like materials. The zone V is often due to an
inner filter area proteins fluorescence is absorbed by aromatic molecules
which reemit fluorescence inside this zone). The zones VI and VII are
related to glycated protein-like materials and humic-like and
lipofuscin-like materials, respectively. In attempt to assess the OM
complexity during the different leachate treatments, complexity index
was calculated according to the method described by Muller et al.
(2014), and Jimenez et al.(2015).
2.4. Spore development in liquid medium (germination)
10% (v/v) spore suspension obtained as described above were
initially germinated and induced to sporulate in a submerged culture
using a series of 250 ml of sterilized Erlenmeyer flasks containing 200 ml
of inoculum development medium. The medium contained (in g/l
distilled water): glucose, 30 g; NaNO3,3 g; K2HP04, 1 g;
MgS04.7H2O,0.5 g; KCl,0.5 g; 1 mL of trace element solution was used
per liter of medium, pH adjusted to 5.5 with glacial acetic acid (V�
ezina
et al., 1965). The medium was sterilized by autoclaving at 121 � C for 20
min. During inoculation, the spore suspension was constantly agitated to
overcome variation in inoculum size owing to sedimentation. Three
fungal inoculums for leachate bioremediation were then prepared
separately by supplementing flasks with 2 ml of sterile raw YL, IL and OL
samples, respectively in the way that the inoculum development me­
dium were sufficiently similar to the leachate-based culture medium to
minimize any period of adaptation of the fungal culture to the landfill
leachate as stated by Lincoln (1960), thus reducing the length of the lag
phase. The inoculated flasks were incubated at 28 � C for 3 days in an
electric shaker (Sanyo-Gallemkemp, UK) with constant shaking at a 150
rpm/min. Subsequently, the number of harvested spores from each flask
was increased and was adjusted to give a final spore concentrations of 1
� 107 spores/mL.
2.7. Germination bioassay
The Lepidium sativum L (cress) and Lactuca sativa L. (lettuces) seeds
were chosen on account to test their sensitivity to MSW leachate toxicity
�
�
_ 2008). 20 seeds of cress and lettuce were
(Zaltauskait
e_ and Cypait
e,
washed ten times in sterile deionized water, then placed on a filter paper
in petri dishes. Seven different concentrations of each type of raw
leachates 100%, 80%, 50%, 25%, 10%, 5%, 1% were prepared in order
to select the concentrations at which the seed germination was not
inhibited. Afterwards, on the basis of the selected concentrations a
comparison was made between each type of raw leachate and its cor­
responding treatment. The petri dishes were then watered with 5 mL of
each concentration of the selected dilution series with three replicate
samples and placed in darkness at 25 � C. The germination index (GI) was
calculated using the following equation (Zucconi, 1981):
� �
ðNSe � ​ LReÞ
GI % ¼
� 100
ðNSw ​ � ​ LRw ​ Þ
2.5. Shake flask study for leachate treatment
The raw landfill leachates were subjected to twelve treatments as
detailed in Table 3. Each treatment was conducted in 250 ml Erlenmeyer
flasks containing 150 mL of culture medium. The liquid leachate-based
culture medium of each type of leachate samples was prepared at
different concentrations, 100%, 50% and 25% of landfill leachate ((v/v)
in distilled water). Each flask was inoculated in triplicate with 10% v/v
(Lincoln, 1960) of the sporal inoculum, respectively. A set of raw
leachate flasks were similarly prepared and were not inoculated to serve
as controls. All flasks of leachate medium were autoclaved before
inoculating with spores suspension. Incubation was performed on an
electric shaker (Sanyo-Gallemkemp, UK) with constant shaking at a 150
rpm/min at temperature (28 � 2 � C) for a period of 30 days (Wan et al.
0.2016). Samples were collected and measured every week (for four
weeks) for BOD5, COD, NHþ
4 , NO3 and pH.
NSe, NSw are number of seeds germinated in leachate samples and
distilled water, respectively (after 24 h); LRe and LRw are the length of
roots in soluble extracts and distilled water, respectively (after 72 h).
3
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
2.8. Statistical analysis of the data
launched without having to consider the status of electron acceptor,
especially oxygen which is the most common electron acceptor used to
enhance aerobic biodegradation. The amount of oxygen required to
oxidize the organic carbon present in a wastewater could be quantified
by BOD5. In this study, the average BOD5 values for leachate at MNL,
MCL and GAL were 11 600 mg/L, 1756.8 mg/L and 5300.15 mg/L,
respectively. These results showed that the three types of leachate YL, IL
and OL require a high oxygen amount for the metabolism process of
organic matter through A. flavus. Therefore, according to these results,
oxygen have to be provided to the three types of leachate. The tem­
perature is also an important abiotic factor as it may affect the microbial
survival and growth in leachate. According to Ahmed et al. (2016), the
optimum temperature for growth of A. flavus is between and around 25
�
C and 30 � C. The temperature for the three leachates fall within the
previously mentioned range. This range of temperatures that supports
active microbial growth and makes this isolate suitable for use in
contaminated sites located in semi-arid climates. Considering all previ­
ous results obtained, the physico-chemical properties of YL, IL and OL
might allow A. flavus to thrive in it and may be degrade leachate organic
compounds. This last point is discussed in details in the following
sections.
The statistical analysis was done with repeated measures analysis of
variance (ANOVA) to evaluate the significant differences between
treatments. In this study, two-way ANOVA was selected and conducted
to test the significant differences in BOD5, COD and NHþ
4 removal rates
and germination index (GI) for the different treatments described above,
at 0.05 of significant level (p < 0.05). The ANOVA was performed with
leachate level of concentration and types of leachate as factors and
fungal and removal rates of organic and nitrogen matter and germina­
tion index as the dependent variables.
3. Results and discussions
3.1. Factors affecting the microbial degradation of organic and nitrogen
matter in leachate
The major abiotic factors (pH, nutrient availability, aeration and
temperature) that might affect the survival and persistence of A. flavus in
the landfill leachate environment are summarized in Table 2. This list is
not exhaustive, but it adequately gives an indication of the applicability
of bioremediation by this strain to a landfill leachate without the
adjustment of environmental conditions to improve biodegradation ac­
tivity. The average values of pH for leachate originating from MNL, MCL
and GAL landfills were 6.8, 9.1 and 8.1, respectively. This range of pH is
suitable for the survival alkali-tolerant microorganisms, such as
A. flavus, which was reported to grow over a wide pH range 4–9 (Frisvad,
2002). Thus, it could be able to thrive over the three landfill leachate
range of pH. Other fungi such as Trametes trogii and Ganoderma australe
have shown their ability to adapt to landfill leachate alkaline conditions
(Razarinah et al., 2014; Smaoui et al., 2018). While the pH gives an
indication of the applicability of bioremediation to a landfill leachate
alone it is not a sufficiently detailed measure. For this reason, it is
necessary also to assess the treatability of leachate by checking the ratio
of carbon, measured as BOD5 to nitrogen and phosphorus (C:N:P). In the
present study, the BOD5:N:P ratio for MNL, MCL and GAL were 100:32:
0.3, 100:32:0.13 and 100:35:0.23, respectively. The present ratio sug­
gests that nutrient requirements for aerobic heterotrophic growth in the
three landfill leachates are not adequate with what is usually reported in
the literature for C:N:P ratio of 100:5:1 for aerobic treatment of such
wastewater (Metcalf & Eddy, 1991). This differences in C:N:P ratios is
due to the high ammonium range (3112,68–5931,25 mg/L) present in
the three type of leachate, which represents 80–90% of the total nitrogen
contained in the landfill leachate (Ellouze et al., 2009). Fungi, like other
organisms, require substantial amounts of nitrogen for synthesis of
proteins and other cell constituents. Therefore, the naturally ammonium
present in the leachate NHþ
4 -N can be used as a cheap nitrogen source for
fungi (Meti et al., 2011), thereby facilitating treatment of landfill
leachate. However, high-strength ammonium exert inhibitor effects on
fungal growth and enzymes secretion during the landfill leachate
treatment (Ellouze et al., 2009). Moreover, it causes also the inhibition
of ammonia-oxidizing bacteria and the nitrite-oxidizing bacteria that are
responsible for autotrophic nitrification process (Yusof et al., 2010).
Under such stressful conditions for autotrophic nitrification, heterotro­
phic processes could be advantageous. Indeed, various groups of het­
erotrophic fungi can perform nitrification with the potential to oxidize
both inorganic nitrogen and organic compounds (Hayatsu et al., 2008).
One of these heterotrophic fungi is A. flavus which was reported with
capacity to accomplish the ammonia oxidation (Stein, 2011). On the
other hand, the BOD5/N ratio of landfill leachate has been studied by
many researchers. For example, Trabelsi et al. (2000) reported that
BOD5/TKN presents the nutrient ratio of organic matter to nitrogen
(similar to C/N) in landfill leachate and generally decreases as the
landfill age increase. The results of this study are in line with this trend
and revealed that the ratio BOD5/TKN decreased from 1.83 in YL to 0.43
in OL. On the other hand, the bioremediation experiment cannot be
3.2. Effect of leachate types and concentrations on removal of leachate
organic matter: BOD5 and COD
Removal rates of BOD5 and COD in the twelve treatment assays after
over 4 weeks using A. flavus are shown in Fig. 1. The fungus A. flavus, has
proven according to many previous studies a pronounced ability for
dismantling some of the most hazardous and toxic compounds like those
present in landfill leachate such as aromatic and xenobiotic compounds
and the associated lignocellulosic materials (Gomaa and El Nour, 2014;
Barapatre and Jha, 2016). In addition, it has recently been reported to
produce extracellular ligninolytic and cellulolytic modifying enzyme
such as laccase and endoglucanases which are known for their ability to
degrade a large variety of environmental pollutants (Pant and Adholeya,
2007; Anita et al. 2013). The results indicated that the removal rate of
BOD5 increased gradually every week in treated leachate up to final days
of treatment by this strain in the different treatments. For instance, after
7 days of incubation, 45.48%, 48.41% and 65.50% of BOD5 removal was
obtained, and it’s followed by 53.67%, 58.64% and 73.65% after 14
days, and after that time 62.45%, 65.81% and 80.34% were achieved in
21 days and after 4 weeks of operation the removal rate of BOD5 was
65.63%, 71.29% and 81.63% respectively in Y-100%RLI, Y-50%DRLI
and Y-25% DRLI, respectively. Similarly, removal rates of COD in YL
were important and showed variations across the different treatments
compared to the BOD5 removal rates. In Y-100%RLI, Y-50%DRLI and
Y-25%DRLI treatments, the results showed that the removal rate of COD
was increased markedly to 17.57%, 27.34% and 39.56% after 14 days of
treatment, respectively. After 21 days of incubation the increasing trend
in COD removal rate was slightly reversed and dropped to 15.72%,
24.71% and 36.84% which could be related to cell lysis and release of
intracellular organic matter which contributes to the COD of the effluent
(Zheng et al., 2019). At the end of 4 weeks of treatment, the removal rate
of COD was slightly increased to 21.11%, 30.12% and 48.50%. On the
other hand, the removal rates of BOD5 in the two other types of leachate,
intermediate and old during 4 weeks of treatment were within the
ranges of 34.27–60.94% and 25.46–47.90%, respectively. Whilst the
removal rates of COD were within the ranges of 10.56–32.67% and
9.26–20.05%, respectively, as shown in Fig. 1. On comparing the effect
of each type of leachate on the removal rate of leachate organics, it was
observed that removal rate of BOD5 was increased from 55.76% to
46.63% in I-50%DRLI and O-50%DRLI, respectively to 71.29% in Y-50%
DRLI over the duration of this experiment. Based on these results, the
removal rate of BOD5 and COD in YL was generally higher than those of
OL and IL after 4 weeks. This could probably because of the different
characteristics and quality of leachate in each type. Indeed, researchers,
4
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
Fig. 1. Removal rate of 100%, 50% and 25% leachate BOD5 (A) and COD (B) by A. flavus after 4 weeks treatment in submerged culture incubated at temperature (28
� 2 � C), shaking at 150 rmp. Error lines represent � standard deviation of the mean.
� et al. (2012) have also reported that
Amaral et al. (2009) and Kal�cíkova
YL contains a high concentration of easily hydrolyzed pollutant organic
compounds which would be more bioavailable. Moreover, these authors
have concluded that as landfill age increased, the amount of refractory
compounds in landfill leachate tended to increase (Amaral et al., 2009;
� et al., 2012). The results in the present study showed also that
Kal�cíkova
Y-25%DRLI treatment of YL with A. flavus demonstrated higher BOD5
removal than in Y-100%RLI, where in Y-25%DRLI, 81.63% of BOD5 was
removed compared to 65.63% for Y-100%RLI. In the same way, higher
BOD5 removal was demonstrated in I-25%DRLI and O-25%DRLI in
5
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
comparison to those obtained in I-100%RLI and O-100%RLI. This in­
dicates the effect of leachate organic concentrations on the rate of BOD5
removal in YL, OL and IL, the same effect was recorded in COD removal
rates in the three types of leachate. This effect of leachate concentration
on the rate of BOD5 removal rate in YL landfill leachate was also
recorded in COD removal. In general, the differences in both BOD5 and
COD removal rates values among the different treatments could be
associated to the leachate concentration and types of leachate. The
two-way analysis of variance (ANOVA) confirmed these findings and
revealed a significance of type of leachate and leachate level of con­
centration effects on BOD 5 and COD removal rates (p < 0.05). Indeed,
Ellouze et al.(2009) has demonstrated the inhibitory and toxic effect of
non-diluted landfill leachate on fungal metabolism which was attributed
to the high ammonia concentrations. Similar effect of high concentra­
tion of liquid pollutant against microorganisms was observed in both
aerobic treatment of landfill leachate and olive mill wastewater
(OMWW) (Fadil et al., 2003; Ellouze et al., 2008b). Apart from
ammonia, heavy metals could also cause inhibition of the fungal growth.
However, A. flavus has been reported to exhibit a rapid growth as well as
the ability to accumulate toxic heavy metals such as Ni, Zn, Cd, Pb, Cr
and Cu (Thippeswamy et al., 2012; Gomaa and Azab, 2013; Kumar and
Dwivedi, 2019). On the other hand, it must be mentioned that our
previous study has reported that the three leachates studied in this work
contained heavy metals at very low concentrations (0.06–3.78 mg/L)
(Zegzouti et al., 2019). These findings seem to suggest that heavy metals
present in the these three leachates at these trace levels don’t affect
probably the activity of A. flavus during leachate treatment. As stated
previously, most of the studies on fungal leachate treatment have been
focused only on free mycelia and fungus mycelia immobilized within a
matrix as a forms of inocula and no one has evaluated the effectiveness
of using sporal inoculum. For example, Razak et al. (2016) reported that
using immobilized fungal mycelium G austral on Ecomat for treating
landfill leachate displayed the best results in the removal of BOD5 and
COD leachate after 4 for weeks of treatment in flask with 93.09% and
17.84% removal rate BOD5 and COD, respectively. In another study,
48% removal of COD was obtained in raw leachate using B. adusta in
batch tests, with biomass cultivated in encapsulated form on poly­
urethane foam cubes (PUFs) (Bardi et al., 2017). On the other hand,
other studies have been published in support of the concept of using
enzyme or immobilized enzymes/with suspended or pellet forms pro­
duced by fungi. A case in point, is the reduction of COD by 80% in
landfill leachate using Trichoderma harzianum by Awasthi et al. (2017)
and the enhancement of the enzymatic activity through immobilization
of fungi on polyurethane foam (PUF) in attempt to treat and decolorize
landfill leachate Spina et al.(2018). The results of this work in terms of
BOD5 and COD reducing efficiency by sporal inoculum are generally
consistent with those found by these previous workers on the landfill
leachate. This confirms also that the treatment removal efficiency of
using sporal inoculum is no less efficient than the immobilized cell and
enzymes methods in treating landfill leachate. Contrarily to this finding,
another study revealed that the mycelial treatment was more effective
than the sporal treatment of Palm Oil Mill Effluent using Trichoderma
viride in reducing the COD content (Karim and Kamil, 1989). While in
this current study, during the development of the inoculum, fungal
spores were subjected to a process of germination and adaptation to
leachate-based culture medium, as pointed out in the previous section.
This acclimatizing phase allowed to reduce the lag phase and the
treatment duration (Ellouze et al. 2008a). Furthermore, in this study the
use of sporal inoculum was not arbitrary but rather was based on a
number of advantages that sporal inoculum has over mycelial inoculum.
In this light, it’s noteworthy to mention that fungus spores often are
more resistant than vegetative mycelium forms to the extremes of
temperature (Ainsworth and Sussman, 2013). Another advantage of a
spore inoculum is the possibility of obtaining a uniform standard and
quantitative inoculum that allow the adjustment of the inoculum size
according to the leachate treatment objectives. However, the major
problem of using vegetative mycelium as inoculum in submerged cul­
ture is the high shear stress that could occur during the agitation which
causes damage to mycelia (Papagianni, 2004; Cai et al., 2014). It has
been reported also that mycelial inoculum can give rise to an extremely
viscous broth which may be very difficult to aerate adequately and thus
affects the treatment efficiency (Stanbury et al., 2013). The efficiency of
using spores as inoculum in bioremediation was reported by many other
studies. Sembries and Crawford (1997) reported all spore formulations
showed good viability and ability to biodegrade the target compound, 2,
4,6-trinitrotoluene (TNT), after 4 months of storage (Sembries and
Crawford, 1997). All this confirms that leachate bioremediation using
fungal spores of Aspergillus flavus proved to be an effective and practical
method for removing organic compounds such as BOD5 and COD present
in the three different types of leachate likely to be produced.
3.3. EEMs fluorescence spectra: Organic matter complexity revealed
In the recent years, three dimensional excitation emission matrix
(3D-EEM) fluorescence spectroscopy has been widely used for qualita­
tive characterization of dissolved organic matter (DOM) in landfill
leachate samples (Zhang et al., 2013). This was attributed to the fact that
this method is easy to implement, fast and non-expensive compared to
several analytical methods techniques that use large volumes of solvents
and long sample processing times, introducing the possibility for
chemical alteration and interference of the initial material. Evolution of
fluorescence spectra for each raw leachate and its corresponding treat­
ments are highlighted by Fig. 2. The general trend showed that the more
aged was the raw leachate, the more peaks in all complex fluorescence
regions. Indeed, higher number of peaks were located in complex fluo­
rescence zones for O-RL and I-RL than Y-RL. Indeed, the old leachate has
been reported to contain more recalcitrant compounds as mentioned
previously (Shouliang et al., 2008). That means fluorescence had a real
potential to be linked with biodegradability of landfill leachate. These
findings corroborate well the results of another study that compared the
characteristics of DOM in raw leachate with different landfill ages
through spectroscopic analysis (Shouliang et al., 2008). Focusing on
Y-RL, the poorest fluorescence zones IV,V and VI was observed. How­
ever, fluorescence zones I, II and III were the main peaks. These zones
are well known to be characteristic of protein-like compounds (Muller
et al., 2011). This fluorescence fingerprint was reported as fresh organic
matter which is rather easily biodegradable (Reynolds and Ahmad,
1997). Indeed, YL leachate has been reported in many studies to contain
large amounts of readily biodegradable organic matter (Naveen et al.,
2016). The intensities of the fluorescent peaks in YL has decreased
sharply for treatments Y-100%RLI, Y-50%DRLI and Y-25%DRLI in zones
I, II and III. However, fluorescence was observed to become more diffuse
with important fluorescence intensity in zone IV and III which repre­
sents the fulvic acid-like molecules fluorescence. This was confirmed by
the increase of the complexity index in YL treatment from 0.55 to around
0.87. The formation of these more complex organic matter could have
resulted from the microbial degradation of volatile fatty acid (VFA)
which are known to represent more than 80% of young leachate
(Alvarez-Vazquez et al., 2004). Concerning IL, fluorescence zones I, II
and III associated with the presence of less biodegradable compounds
was also higher in I-RL than in all treated intermediate leachate. On the
other hand, fluorescence was observed to become more distinct in zones
IV,V and VI. However, as the concentration decreased, the fluorescence
intensities decreased in these zones. In OL, no significant difference was
found between the fluorescence intensities of the raw leachate and
treated leachate spectra in all fluorescence zones. Especially in the zones
referring to complex and refractory DOM such VI. Indeed, the fluores­
cence zone VI is usually associated to lignocellulose like compound
(Muller et al., 2011), humic acid-like (Chen et al., 2003) or
melanoidin-like compound fluorescence, known to be recalcitrant and
slowly biodegradable (Chandra et al., 2008).
6
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
Fig. 2. 3D-excitation emission matrix fluorescence spectroscopy of dissolved organic matter from raw and treated leachate. The details of each leachate treatment
has been described in detail previously in Table 3. Legend of fluorescence intensity: I, II and III are protein-like materials, IV is fulvic-like materials, V is an inner filter
area, VI is glycated protein-like materials and VII is humic-like and lipofuscin-like materials.
120
100
Removal rate (%)
80
60
40
20
0
Y-100%RLI
Y-50%DRLI
Young Leachate
Y-25%DRLI
I-100%RLI
I-50%DRLI
I-25%DRLI
O-100%RLI
Intermediate Leachate
Day 7
0-50%DRLI
Old Leachate
Day 14
Day 21
0-25%DRLI
Y-RL
I-RL
0-RL
Raw Leachate
Day 28
�
Fig. 3. Removal rate of 100%, 50% and 25% leachate NHþ
4 by A. flavus after 4 weeks treatment in submerged culture incubated at room temperature (28 � 2 C),
shaking at 150 rmp. Error lines represent � standard deviation of the mean.
7
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
3.4. Effect of leachate types and concentrations on removal rate of
nitrogen matter: (NHþ
4 –N)
concentrations of leachate (Molina and Alexander, 1972). This absence
of effect of dilution on the treatment of wastewater was observed in
another study done by Kissi et al. (2001) aiming to treat biologically
olive mill waste water (OMWW) using P. chrysosporium, they found that
no significant differences in enzyme production could be observed be­
tween P. chrysosporium incubation in 20% and 50% (v/v) OMWW.
Similar effect of leachate concentration on NHþ
4 removal rate in a
lab-scale ammonia stripping reactor was reported by Hanira et al.
(2017). These researchers found that the difference in the removal of
NHþ
4 in diluted and raw leachate was insignificant. On the basis of all
these previous findings A. flavus could be considered as a potential strain
þ
capable of removing NHþ
4 in landfill leachate. A high removal NH4 rates
in the range from 90% to 95% were reported in aerobic treatment of
landfill leachate in other studies (Agdag and Sponza 2005; Ellouze et al.
2008a). On the other hand, Yu et al. (2014) reported that Bascillus cereus
and Enterococcus casseliflavus could remove 50.1% and 51.2% of NHþ
4
within 9 days in a mature landfill leachate. This was relatively similar to
our results obtained in O-100%RLI, O-50%DRLI and O-25%DRLI at the
7th day after treatment. However, _Razarinah et al. (2015) reported
when they studied ammonium removal ability of white-rot fungi
þ
T. menziesii to remove NHþ
4 in landfill leachate that no removal of NH4
occurred during a four weeks treatment period. Many factors might
account for theses discrepancies, the most important ones are the
different metabolic pathways leading to removal of NHþ
4 and endurance
abilities of the high ammonium loads with different microorganisms
(Liu et al., 2016). In another study, Ellouze et al.(2008a) demonstrated
that a stirred tank reactor (STR) operated by feeding an acclimatized
consortia consists of nitrifying bacteria was able to remove 92% of NHþ
4
for an ORL of 4.5 g 1 1per day.
As previously proved that the A. flavus was able to remove organic
matter efficiently, the NHþ
4 removal ability of this strain was also eval­
uated within and across the same treatment assays as described under
materials and methods. The results are tabulated in Fig. 3. The results
showed that the maximum NHþ
4 removal rate in IL of 79.89% was
observed up to the 7th day of operation of treatment I-50%DRLI while in
YL and OL, the maximum NHþ
4 removal rates of 57.49% and 55% were
reached in Y-25%DRLI and O-50%DRLI treatments, respectively. The
NHþ
4 removal rates were still maintaining their increase trend in all the
treatment assays until the end of the experiment and reached a
maximum ammonia removal rates of 98.23%, 79.37% and 98.81%
during I-25%DRLI,Y-25%DRLI and O-25%DRLI treatments. Conse­
quently, through previously stated results the NHþ
4 removal rates
increased among all treatment assays with the hydraulic retention time.
Thus, generally the treatment proved to be effective in reducing the NHþ
4
load, especially that of the NHþ
4 removal rates by using A. flavus were
much higher than those of the control experiments during the whole
treatment process. These results raised the potential of A. flavus to
metabolize the NHþ
4 . Indeed A. flavus has been reported to be one of the
most interesting strains capable of performing heterotrophic nitrifica­
tion because this is one of the few microorganisms able to convert
ammonium to nitrate as stated previously (Marshall and Alexander,
1962; Guest and Smith, 2002). However, with no knowledge of the
favorable and limiting conditions in leachates and the amount of end
products of the heterotrophic nitrification by A. flavus, it is difficult to
speculate with any certainty the cause of decreased NHþ
4 or attribute it to
the heterotrophic nitrification. Among the major conditions in which
heterotrophic nitrification occurs are pH and substrate concentration
(C/N) (Guest and Smith, 2002). These authors reported that fungi
cannot nitrify below pH 6–5. In this study, the pH was maintained above
this pH range during the different treatments without the regulation of
the alkalinity. Indeed, the experiments revealed that the pH of all the
different leachate treatments increased in the range of 6.1–9.52,
7.2–9.58 and 8.3–9.4 for YL, IL and OL, respectively. This might be
associated to the production of alkaline compounds by A. flavus such the
alkaline protease which is directly related to the increasing pH of the
medium (Franco et al., 2017). Concerning the nitrification end products,
NO3 was detected in no significant amounts in the treatments assays
after the 28 incubation period. However, it is interesting to observe that
the concentration of nitrates in leachate increased over all treatment
assays achieving a maximum accumulation of 490.2, 612.4 and 747.575
mg/L at the end of O-25%RLI, Y-25%RLI and I-25%RLI treatments. This
may indicate that the nitrification was occurring, however these values
feel well outside the normal expected for nitrification products, which
raise questions to the other possible fates of NHþ
4 . Guest and Smith
(2007) found that the aerobic fungal nitrogen treatment of wastewater
using Mucor. sp and Penicillium. sp was characterized by a lack of pro­
duction of nitrification end products, which was due to the fact that
NHþ
4 was being assimilated into the cell mass for new cellular material. In
the comparison of NHþ
4 removal rates among the treatments according
the leachate types, the results showed that the maximum NHþ
4 removal
rate of 79.37% was achieved during Y-25%DRLI treatment. While the
maximum NHþ
4 removal rates in OL and IL were achieved 98.81%,
98.23% during. O-25%RLI and I-25%RLI treatments, respectively. This
suggests that the type of leachate had a significant effect on NHþ
4
removal efficiency of this strain (p < 0.05). On the other hand, the
comparison of NHþ
4 removal according to the leachate concentration
indicated that the dilution had a non-significant effect on the NHþ
4
within each type of leachate (YL, OL and IL) (p > 0.05). The reason for
the non-significance of the dilution effect on the NHþ
4 removal was not
clear, however it may have been due to the production of the same
amount of enzyme production such as catalase and peroxidase involved
in the heterotrophic nitrification by A. flavus whatever the
3.5. Phytotoxicity assessment
The results showed that there was no response of germination to raw
leachates exposure observed in lettuce and Cress up to 10% and 1%. No
germination was observed in the remaining leachate concentrations,
100%, 80%, 50% and 25%. The germination index of the lettuce and
cress by varying the concentration of the leachate was indicated as a
percentage with respect to the different treatments applied to the
different types of leachate. The outcomes are presented in Fig. 4. The
results for the raw leachates Y-RL, O-RL and I-RL at a concentration of
10% revealed a considerable inhibitory effect on both investigated
species seeds germination judged by the germination index (GI) which
range from 0% to 1.77%. This inhibitory effect might be associated to
the fact that leachate contains a wide range of inorganic and xenobiotic
organic compounds like ammonium, volatile, aromatic and aliphatic
organic substances, the mixture of which affects the plant growth and
especially the root meristematic tissues (Hou et al., 2017). The same
effect of highly polluted raw leachate on the seed germination process
�
has also been reported in other studies (Del Moro et al., 2014; Vaverkova
et al., 2017). However, the germination index has markedly increased
after treatment in the three types of leachate. In this way, GI of lettuce
increased to 35.28%, 37.14% and 40.18% for the Y-100%RLI, Y-50%
DRLI and Y-25%DRLI treatments, respectively at 10% concentration,
compared with Y-RL (Fig. 4A). The same increasing trend in GI was
observed for the same treatments in YL at a concentration of 10% and
1% using cress with an order of magnitude higher than that obtained for
lettuce seeds at 10% as shown in Fig. 4A. With regard to the effect OL
and IL on seeds germination, the results showed that the germination
index for both investigated species in raw leachate was, again, found
lower that in treated leachates (Fig. 4B and C). For example, IG of cress
for O-100%RLI, O-50%DRLI and O-25%DRLI treatments at a concen­
tration of 10% increased to 57.69%, 98.36% and 74.81%, respectively
compared with O-RL. Thus, these high values of IG showed that treat­
ment using A. flavus in all treatments can effectively reduce the sub­
stances responsible of toxicity in landfill leachate. These results were
expected due to the high removal rate of organic and nitrogen matter
8
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
(A)
140
Germination index ( GI%°)
120
100
80
60
40
20
0
1%
10%
1%
Y-100%RLI
10%
10%
1%
Y-25%DRLI
Cress
(B)
1%
Y-50%DRLI
10%
RL
lettuce
140
Germination index ( GI%°)
120
100
80
60
40
20
0
1%
10%
1%
I-100%RLI
10%
1%
I-50%DRLI
Cress
10%
1%
I-25%DRLI
10%
I-RL
Lettuce
120
(C)
Germination index ( GI%°)
100
80
60
40
20
0
1%
10%
1%
O-100%RLI
10%
1%
0-50%DRLI
Cress
10%
0-25%DRLI
1%
10%
RL
Lettuce
Fig. 4. Effect of treated leachate,Young (A), Intermediate (B) and Old (C) on germination index of lettuce (Lactuca sativa L.) and Cress (Lepidium sativum L.) seeds.
Error lines represent � standard deviation of the mean.
9
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
achieved in all treatments as mentioned in the previous sections. Our
results are consistent with the findings of other workers who also
observed a decrease in seed germination with increase in the concen­
tration of the leachate effluent (Del Moro et al., 2014; Turki and Bouzid,
2017). The osmotic pressure of the effluent also increased at higher
concentrations which retards germination. This may serve as another
explanation for the effect of the leachate concentration on the seed
germination of both cress and lettuce seeds (Ramana et al., 2002). The
germination ability under high osmotic pressure differs with variety as
well as with species (Suliasih et al., 2010). A good example of such effect
is germination index variations of both cress and lettuce across all
treatments. For instance, the GI of cress was greater than that of lettuce
in I-50%DRLI at 10% and at 1%. This suggests a greater sensitivity of
lettuce than cress. The same conclusion on the seeds sensitivity was
reached in another study on landfill leachate (Del Moro et al., 2014). For
all treatments, increase in GI of cress and lettuce seeds was at both
concentration of 1% and 10% higher in comparison with raw leachate at
the same concentrations. But the increase was maximum at 1% con­
centration, corresponding to an GI of 129.38% obtained in I-25%DRLI.
This suggests that treated leachate played fertilizing effect on seed
germination. This positive effect of nutrient value of landfill leachate on
plant growth was also reported in other studies (Cheng and Chu, 2007;
Alaribe and Agamuthu, 2016). On the other hand, of the three types of
leachate, IL had the greatest positive effect on the growth flowed by OL
and YL (p < 0.05). This could be due to the fact that as landfill age in­
crease, the organic fraction in the leachate becomes dominated by humic
substances (Renoua et al., 2008). The latter substances have been re­
� �
�k, 2011).
ported to stimulate the seeds germination (Ser
a and Nova
Anita, B.B., Thatheyus, A.J., Ramya, D., 2013. Biodegradation of carboxymethyl
cellulose using Aspergillus flavus. Sci. Int. 1, 92–97.
Awasthi, A.K., Pandey, A.K., Khan, J., 2017. Potential of fungus Trichoderma harzianum
for toxicity reduction in municipal solid waste leachate. Int. J. Environ. Sci. Technol.
14, 2015–2022.
Barapatre, A., Jha, H., 2016. Decolourization and Biological Treatment of Pulp and Paper
Mill Effluent by Lignin-Degrading Fungus Aspergillus flavus Strain F10. Int. J. Curr.
Microbiol. Appl. Sci. 5, 19–32. https://doi.org/10.20546/ijcmas.2016.505.003.
Bardi, A., Yuan, Q., Tigini, V., et al., 2017. Recalcitrant compounds removal in raw
leachate and synthetic effluents using the white-rot fungus Bjerkandera adusta.
Water (Switzerland) 9. https://doi.org/10.3390/w9110824.
Cai, M., Zhang, Y., Hu, W., et al., 2014. Genetically shaping morphology of the
filamentous fungus Aspergillus glaucus for production of antitumor polyketide
aspergiolide A. Microb. Cell Factories 13, 73.
Chandra, R., Bharagava, R.N., Rai, V., 2008. Melanoidins as major colourant in
sugarcane molasses based distillery effluent and its degradation. Bioresour. Technol.
99, 4648–4660.
Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitation
emission matrix regional integration to quantify spectra for dissolved organic matter.
Environ. Sci. Technol. 37, 5701–5710.
Cheng, C.Y., Chu, L.M., 2007. Phytotoxicity data safeguard the performance of the
recipient plants in leachate irrigation. Environ. Pollut. 145, 195–202.
Chian, E.S.K., DeWalle, F.B., 1977. Sanitary landfill leachates and their treatment.
J. Environ. Eng. Div. 103, 744–745.
Chofqi, A., Younsi, A., Lhadi, E.K., et al., 2004. Environmental impact of an urban landfill
on a coastal aquifer (El Jadida, Morocco). J. Afr. Earth Sci. 39, 509–516.
Del Moro, G., Barca, E., Cassano, D., et al., 2014. Landfill wall revegetation combined
with leachate recirculation: a convenient procedure for management of closed
landfills. Environ. Sci. Pollut. Res. 21, 9366–9375.
Ellouze, M., Aloui, F., Sayadi, S., 2008. Performance of biological treatment of high-level
ammonia landfill leachate. Environ. Technol. 29, 1169–1178.
Ellouze, M., Aloui, F., Sayadi, S., 2008. Detoxification of Tunisian landfill leachates by
selected fungi. J. Hazard Mater. 150, 642–648. https://doi.org/10.1016/j.
jhazmat.2007.05.013.
Ellouze, M., Aloui, F., Sayadi, S., 2009. Effect of high ammonia concentrations on fungal
treatment of Tunisian landfill leachates. Desalination 246, 468–477. https://doi.org/
10.1016/j.desal.2008.03.068.
Fadil, K., Chahlaoui, A., Ouahbi, A., et al., 2003. Aerobic biodegradation and
detoxification of wastewaters from the olive oil industry. Int. Biodeterior. Biodegrad.
51, 37–41.
Franco, D.G., Spalanzani, R.N., Lima, E.E., et al., 2017. Biochemical properties of a serine
protease from Aspergillus flavus and application in dehairing. Biocatal.
Biotransform. 35, 249–259.
Frisvad, J., 2002. On the safety of Aspergillus Niger – a review. https://doi.org/10.1007/
s00253-002-1032-6.
Gomaa, O.M., Azab, K.S., 2013. Biological indicators, genetic polymorphism and
expression in Aspergillus flavus under copper mediated stress. J Radiat Res Appl Sci
6, 49–55.
Gomaa, O.M., El Nour, S.A., 2014. Aflatoxin inhibition in Aspergillus flavus for
bioremediation purposes. Ann. Microbiol. 64, 975–982.
Guest, R.K., Smith, D.W., 2007. Isolation and screening of fungi to determine potential
for ammonia nitrogen treatment in wastewater. J. Environ. Eng. Sci. 6, 209–217.
Guest, R.K., Smith, D.W., 2002. A potential new role for fungi in a wastewater MBR
biological nitrogen reduction system. J. Environ. Eng. Sci. 1, 433–437.
Hanira, N.M.L., Hasfalina, C.M., Rashid, M., et al., 2017. Effect of dilution and operating
parameters on ammonia removal from scheduled waste landfill leachate in a labscale ammonia stripping reactor. In: IOP Conference Series: Materials Science and
Engineering. IOP Publishing, p. 12076.
Hayatsu, M., Tago, K., Saito, M., 2008. Various players in the nitrogen cycle: diversity
and functions of the microorganisms involved in nitrification and denitrification.
Soil Sci. Plant Nutr. 54, 33–45.
Hou, C., Lu, G., Zhao, L., et al., 2017. Estrogenicity assessment of membrane concentrates
from landfill leachate treated by the UV-Fenton process using a human breast
carcinoma cell line. Chemosphere 180, 192–200.
Jimenez, J., Aemig, Q., Doussiet, N., et al., 2015. A new organic matter fractionation
methodology for organic wastes: bioaccessibility and complexity characterization for
treatment optimization. Bioresour. Technol. 194, 344–353.
� 2012. Variation of landfill
Kal�cíkov�
a, G., Zagorc-Kon�can, J., Zupan�ci�c, M., Gotvajn, A.Z.,
leachate phytotoxicity due to landfill ageing. J. Chem. Technol. Biotechnol. 87,
1349–1353.
Karim, M.I.A., Kamil, A.Q.A., 1989. Biological treatment of palm oil mill effluent using
Trichoderma viride. Biol. Wastes 27, 143–152.
Kissi, M., Mountadar, M., Assobhei, O., et al., 2001. Roles of two white-rot basidiomycete
fungi in decolorisation and detoxification of olive mill waste water. Appl. Microbiol.
Biotechnol. 57, 221–226.
Kumar, V., Dwivedi, S.K., 2019. Hexavalent chromium reduction ability and
bioremediation potential of Aspergillus flavus CR500 isolated from electroplating
wastewater. Chemosphere 237, 124567.
Kumari, M., Ghosh, P., Thakur, I.S., 2016. Landfill leachate treatment using bacto-algal
co-culture: an integrated approach using chemical analyses and toxicological
assessment. Ecotoxicol. Environ. Saf. 128, 44–51.
Lincoln, R.E., 1960. Control of stock culture preservation and inoculum build-up in
bacterial fermentation. J. Biochem. Microbiol. Technol. Eng. 2, 481–500.
Liu, Z., Liu, G., Cai, H., et al., 2016. Paecilomyces variotii: a fungus capable of removing
ammonia nitrogen and inhibiting ammonia emission from manure. PLoS One 11,
e0158089.
4. Conclusion
This investigation revealed the capability of A. flavus fungus to
remove organic and nitrogen matter from landfill leachate while
reducing the toxicity. Moreover, this confirms also that the treatment
removal efficiency of using sporal inoculum is no less efficient than the
other forms of fungal inoculum in treating landfill leachate. Our results
suggested that fluorescence can be used as rapid measurement of
organic matter composition and complexity during the treatment of
leachate using A. flavus. As a conclusion, the aerobic biological treat­
ment using A. flavus, was very efficient for 50% and 25% concentration
of leachate, particularly for the lower concentration 25% with a
maximum BOD5, COD and NHþ
4 removal rate of around 81.63%, 48.50%
and 98.81% recorded in YL and IL, respectively. However, more inves­
tigation needs to be done in order to 1) identify the genes and enzymes
responsible of organic compounds degradation in leachate by A. flavus
2) validate the effects of fungal treated leachate and its impact on plant
growth and development.
References
A�
gda�
g, O.N., Sponza, D.T., 2005. Anaerobic/aerobic treatment of municipal landfill
leachate in sequential two-stage up-flow anaerobic sludge blanket reactor (UASB)/
completely stirred tank reactor (CSTR) systems. Process Biochem. 40, 895–902.
Ahmed, M.M., Fakruddin, M., Hossain, M.N., et al., 2016. Growth response of Aspergillus
flavus IMS1103 isolated from poultry feed. Asian J Med Biol Res 2, 221–228.
Ainsworth, G.C., Sussman, A.S., 2013. The Fungal Population: an Advanced Treatise.
Elsevier.
Alaribe, F.O., Agamuthu, P., 2016. Fertigation of Brassica rapa L. using treated landfill
leachate as a nutrient recycling option. South Afr. J. Sci. 112, 1–8.
Alijani Ardeshir, R., Rastgar, S., Peyravi, M., et al., 2016. A new route of
bioaugmentation by allochthonous and autochthonous through biofilm bacteria for
soluble chemical oxygen demand removal of old leachate. Environ. Technol. 38,
2447–2455. https://doi.org/10.1080/09593330.2016.1264488.
Alvarez-Vazquez, H., Jefferson, B., Judd, S.J., 2004. Membrane bioreactors vs
conventional biological treatment of landfill leachate: a brief review. J. Chem.
Technol. Biotechnol. 79, 1043–1049.
Amaral, M.C.S., Ferreira, C.F.A., Lange, L.C., Aquino, S.F., 2009. Characterization of
landfill leachates by molecular size distribution, biodegradability, and inert chemical
oxygen demand. Water Environ. Res. 81, 499–505.
Aneja, K.R., 2007. Experiments in microbiology, plant pathology and biotechnology.
New Age Int.
10
Y. Zegzouti et al.
Journal of Environmental Management 255 (2020) 109821
Maier, R.M., 2000. Bioavailability and its importance to bioremediation. In:
Bioremediation. Springer, pp. 59–78.
Marco-Urrea, E., Garcia-Romera, I., Aranda, E., 2015. Potential of non-ligninolytic fungi
in bioremediation of chlorinated and polycyclic aromatic hydrocarbons. N. Biotech.
32, 620–628.
Marshall, K.C., Alexander, M., 1962. Nitrification by Aspergillus flavus. J. Bacteriol. 83,
572–578.
Metcalf and Eddy, I., 1991. Wastewater Engineering Treatment, Disposal, and Reuse, 3rd
Edition. McGraw-Hill, Inc, Singapore.
Meti, R.S., Ambarish, S., Khajure, P.V., 2011. Enzymes of ammonia assimilation in fungi:
an overview. Recent Res. Sci. Technol. 3.
Molina, J.A.E., Alexander, M., 1972. Oxidation of nitrite and hydroxylamine
byAspergillus flavus, peroxidase and catalase. Antonie Leeuwenhoek 38, 505–512.
Morris, S., Cabellos, G., Enright, D., et al., 2018. Bioremediation of landfill leachate using
isolated bacterial strains. International Journal 6 (1), 26–35.
Mukherjee, A., 2016. Role of Aspergillus in bioremediation process. In: New and Future
Developments in Microbial Biotechnology and Bioengineering. Elsevier,
pp. 209–214.
Muller, M., Jimenez, J., Antonini, M., et al., 2014. Combining chemical sequential
extractions with 3D fluorescence spectroscopy to characterize sludge organic matter.
Waste Manag. 34, 2572–2580.
Muller, M., Milori, D.M.B.P., D�el�
eris, S., et al., 2011. Solid-phase fluorescence
spectroscopy to characterize organic wastes. Waste Manag. 31, 1916–1923.
Naveen, B.P., Sivapullaiah, P.V., Sitharam, T.G., 2016. Effect of aging on the leachate
characteristics from municipal solid waste landfill. Japanese Geotech Soc Spec Publ
2, 1940–1945. https://doi.org/10.3208/jgssp.IND-06.
Pant, D., Adholeya, A., 2007. Enhanced production of ligninolytic enzymes and
decolorization of molasses distillery wastewater by fungi under solid state
fermentation. Biodegradation 18, 647–659.
Papagianni, M., 2004. Fungal morphology and metabolite production in submerged
mycelial processes. Biotechnol. Adv. 22, 189–259.
Rajendran, R.K., Lin, C.-C., Huang, S.-L., Kirschner, R., 2017. Enrichment, isolation, and
biodegradation potential of long-branched chain alkylphenol degrading nonligninolytic fungi from wastewater. Mar. Pollut. Bull. 125, 416–425.
Ramana, S., Biswas, A.K., Kundu, S., et al., 2002. Effect of distillery effluent on seed
germination in some vegetable crops. Bioresour. Technol. 82, 273–275.
Razak, W.R.W.A., Mahmood, N.Z., Abdullah, N., 2016. Effect of culture technique of
Ganoderma australe mycelia on percentage removal of leachate organics. Sci. Res. J
13, 131–143.
Razarinah, W., Zalina, M.N., Abdullah, N., 2015. Utilization of the white-rot fungus,
Trametes menziesii for landfill leachate treatment. Sains Malays. 44, 309–316.
Razarinah, W., Zalina, M.N., Abdullah, N., 2014. Treatment of landfill leachate by
immobilized Ganoderma australe and crude enzyme. Sci. Asia 40, 335–339.
Renoua, S., Givaudan, J., Poulain, S., et al., 2008. Landfill leachate treatment: review and
opportunity. J. Hazard Mater. 150, 468–493.
Reynolds, D.M., Ahmad, S.R., 1997. Rapid and direct determination of wastewater BOD
values using a fluorescence technique. Water Res. 31, 2012–2018.
Sembries, S., Crawford, R.L., 1997. Production of Clostridium bifermentans spores as
inoculum for bioremediation of nitroaromatic contaminants. Appl. Environ.
Microbiol. 63, 2100–2104.
� a, B., Nov�
Ser�
ak, F., 2011. The effect of humic substances on germination and early
growth of Lamb’s Quarters (Chenopodium album agg.). Biologia (Bratisl) 66, 470.
Shouliang, H., Beidou, X.I., Haichan, Y.U., et al., 2008. Characteristics of dissolved
organic matter (DOM) in leachate with different landfill ages. J. Environ. Sci. 20,
492–498.
Singh, H., 2006. Fungal Degradation of Polychlorinated Biphenyls and Dioxins.
Mycoremediation John Wiley Sons, Inc, pp. 149–180.
Singh, H., 2006. Mycoremediation: Fungal Bioremediation. John Wiley & Sons.
Singh, S.N., Mishra, S., Jauhari, N., 2015. Degradation of anthroquinone dyes stimulated
by fungi. In: Microbial Degradation of Synthetic Dyes in Wastewaters. Springer,
pp. 333–356.
Smaoui, Y., Fersi, M., Mechichi, T., et al., 2018. A new approach for detoxification of
landfill leachate using Trametes trogii. Environ Eng Res 24, 144–149.
Spina, F., Tigini, V., Romagnolo, A., Varese, G., 2018. Bioremediation of landfill leachate
with fungi: autochthonous vs. Allochthonous strains. Life 8, 27. https://doi.org/
10.3390/life8030027.
Stanbury, P.F., Whitaker, A., Hall, S.J., 2013. Principles of Fermentation Technology.
Elsevier.
Stein, L.Y., 2011. Heterotrophic nitrification and nitrifier denitrification. In:
Nitrification. American Society of Microbiology, pp. 95–114.
Suliasih, B.A., Othman, M.S., Heng, L.Y., Salmijah, S., 2010. Toxicity identification
evaluation of landfill leachate taking a multispecies approach. Waste Manag Environ
311–322.
Thippeswamy, B., Shivakumar, C.K., Krishnappa, M., 2012. Bioaccumulation potential of
Aspergillus Niger and Aspergillus flavus for removal of heavy metals from paper mill
effluent. J. Environ. Biol. 33, 1063.
Tigini, V., Spina, F., Romagnolo, A., et al., 2013. Effective biological treatment of landfill
leachates by means of selected white rot fungi. Chem Eng Trans 32, 265–270.
https://doi.org/10.3303/CET1332045.
Trabelsi, I., Horibe, H., Tanaka, N., Matsuto, T., 2000. Origin of low carbon/nitrogen
ratios in leachate from old municipal solid waste landfills. Waste Manag. Res. 18,
224–234.
Turki, N., Bouzid, J., 2017. Journal of Pollution Effects & Control Effects of Landfill
Leachate application on Crops growth and Properties of a Mediterranean Sandy Soil.
J Pollut Eff Control 5, 1–8. https://doi.org/10.4172/2375-4397.1000186.
Vaverkov�
a, M.D., Zloch, J., Adamcov�
a, D., et al., 2017. Landfill leachate effects on
germination and seedling growth of hemp cultivars (Cannabis Sativa L.). Waste and
Biomass Valorization 1–8.
V�ezina, C., Singh, K., Sehgal, S.N., 1965. Sporulation of filamentous fungi in submerged
culture. Mycologia 57, 722–736.
Yu, D., Yang, J., Teng, F., et al., 2014. Bioaugmentation treatment of mature landfill
leachate by new isolated ammonia nitrogen and humic acid resistant microorganism.
J. Microbiol. Biotechnol. 24, 987–997.
Yusof, N., Hassan, M.A., Phang, L.Y., et al., 2010. Nitrification of ammonium-rich
sanitary landfill leachate. Waste Manag. 30, 100–109.
�
�
_ J., Cypait
_ A., 2008. Assessment of landfill leachate toxicity using higher
Zaltauskait
e,
e,
plants. Environ. Res. Eng. Manag. 46.
Zegzouti, Y., Boutafda, A., El Fels, L., et al., 2019. Quality and quantity of leachate with
different ages and operations in semi-arid climate. Desalin WATER Treat 152,
174–184.
Zhang, Q.-Q., Tian, B.-H., Zhang, X., et al., 2013. Investigation on characteristics of
leachate and concentrated leachate in three landfill leachate treatment plants. Waste
Manag. 33, 2277–2286.
Zheng, M., Li, S., Dong, Q., et al., 2019. Effect of blending landfill leachate with activated
sludge on the domestic wastewater treatment process. Environ Sci Water Res
Technol 5, 268–276.
Zucconi, F., 1981. Evaluating toxicity of immature compost. Biocycle 22, 54–57.
11