Water Air Soil Pollut (2019) 230:215

https://doi.org/10.1007/s11270-019-4227-5

Enhanced Bioremediation of Heavy Metal Contaminated

Landfill Soil Using Filamentous Fungi Consortia:
a Demonstration of Bioaugmentation Potential
Auwalu Hassan & Agamuthu Pariatamby &
Aziz Ahmed & Helen Shnada Auta &
Fauziah Shahul Hamid

Received: 31 March 2019 / Accepted: 9 July 2019

# Springer Nature Switzerland AG 2019

Abstract This study aimed to determine the (Paecilomyces lilacinus, Antrodia serialis, and Penicillium
mycoremediative capacity of filamentous fungi consortia cataractum) were used to amend the contaminated soil;
in landfill heavy metal contaminated soil. Streak plate meanwhile, the unamended soil served as control. Maxi-
method was utilized for the isolation of fungi from the mum tolerance index of 1.0 was reported in Cr-, Cu-, and
landfill soil. Isolates were identified using morphological Fe-amended PDA medium. Meanwhile, the maximum
and molecular techniques. Heavy metal tolerance of the heavy metal bioremoval efficiencies were for highly toler-
fungi was determined using radial growth diameter tech- ant fungal consortium treated soil and were recorded as As
nique. Twelve species of landfill indigenous fungi were (62%) > Mn (59%) > Cu (49%) > Cr (42%) > Fe (38%).
used for the bioremediation process. Two categories of Likewise, the maximum metal removal rate constant (K)
fungi consortia namely highly tolerant fungi (Perenniporia and the half-lives (t1/2) were 0.0097/day 71 days, 0.0088/
subtephropora, Daldinia starbaeckii, Phanerochaete day 79 days, 0.0067/day 103 days, 0.0054/day 128 days,
concrescens, Cerrena aurantiopora, Fusarium equiseti, and 0.0048/day 144 days for As, Mn, Cu, Cr, and Fe,
Polyporales sp., Aspergillus niger, Aspergillus fumigatus, respectively, which were all for soil treated with consor-
and Trametes versicolor) and moderately tolerant fungi tium of highly tolerant fungi (P. subtephropora,
D. starbaeckii, P. concrescens, C. aurantiopora,
F. equiseti, Polyporales sp., A. niger, A. fumigatus, and
A. Hassan : A. Pariatamby : A. Ahmed : F. S. Hamid (*) T. versicolor). Spectra analysis revealed a clear distinction
Institute of Biological Sciences, Faculty of Science, University of in the functional groups between the fungal treated and the
Malaya, 50603 Kuala Lumpur, Malaysia untreated soils. Peaks at 874 ± 2 cm−1 and 1425 ± 2 cm−1
e-mail: [email protected]
were only found in fungi amended soil. Physicochemical
A. Hassan : A. Pariatamby : A. Ahmed : F. S. Hamid parameters mainly pH and redox potential played a key
Center for Research in Waste Management, Faculty of Science, role in the bioremediation process, and bioaccumulation
University of Malaya, 50603 Kuala Lumpur, Malaysia was believed to be the favored mechanism for the metal
bioremoval. The data are suitable for assessing the contri-
A. Hassan
Department of Biological Sciences, Faculty of Science, Federal bution of bioaugmentation with consortia of fungi. It is
University, Kashere, Gombe State, Nigeria equally important for assessing the synergistic effect of
fungi on the reduction of extractable heavy metals in
A. Ahmed
contaminated soil.
Faculty of Marine Sciences, Lasbela University of Agriculture,
Water and Marine Sciences, Uthal, Balochistan, Pakistan

H. S. Auta Keywords Bioaugmentation . Bioaccumulation .

Department of Microbiology, Federal University of Technology, Filamentous fungi . Heavy metals . Consortium . Soil
Minna, Nigeria pollution
 215 Page 2 of 20 Water Air Soil Pollut (2019) 230:215

1 Introduction 2011). They are capable of degrading waste substances

and thrive under drastic conditions. In heavy metal con-
Increased industrialization, urbanization, and moderni- taminated environments, some microorganisms adapt to
zation of life have brought about environmental contam- elevated concentrations and toxicity of heavy metals
ination and pollution as a result of the generation of (Singh et al. 2011), and as such, they are utilized as
large amount of municipal solid waste (MSW) (Fazli effective biosorbents for heavy metal contaminants.
et al. 2015). Landfilling process is among the recog- Recently, fungal organisms have effectively been used
nized methods of handling MSW worldwide. However, as biosorbents for the bioremoval of heavy metals from
this technology has brought about excessive release of polluted soil (Iram and Abrar 2015). Fungi perform an
landfill leachate from the decomposition of waste (de important function in the fate of heavy metals in the
Godoy Leme & Miguel 2018; Jayanthi et al. 2017; environment. They help to transform metal contaminants
Pariatamby et al. 2015). Landfill leachate has been to soluble and insoluble forms through several biological
known to contain various environmental contaminants mechanisms (Mandal et al., 2016). These mechanisms are
including organics, e.g., alcohols, acids, alkanes, integral part of the biogeochemical cycling of substances
hydroxybenzene, aromatic hydrocarbons, amides, al- and are important for both ex situ and in situ bioremedia-
kenes, esters, as well as inorganic substances such as tion processes (Miransari, 2011). Fungi have both ecolog-
ammonia and heavy metals (Fauziah et al. 2013). ical adaptability and biochemical capability to decompose
The migratory nature of landfill leachate leads to pollutants and reduce the threat of these pollutants by either
dissemination of pollutants in the environment (de chemically modifying their chemical structure or by affect-
Freitas et al. 2018; Madejón et al. 2018). Among the ing their bioavailability (Mandal et al. 2016). Formation of
contaminants that are disseminated in the environment mycelia network as well as broad spectrum activity of their
by landfill leachate, heavy metals are of significant degradative enzymes also contributed to their suitability in
concern because of their cumulative and toxic nature. bioremediation technology (Nagajyoti et al. 2010). They
Heavy metals such as Cu, Cd, Pb, Zn, Hg, Ni, and Mn also possess negative charges on their cell surface due to
constantly accumulate and contaminate ground and sur- the anionic functional groups which form the binding sites
face water bodies. Metal contaminants also accumulate for the heavy metal cations. Some of the negatively
in soil and exert potential lethal effects on humans, charged anionic groups that are involved in the biosorption
plants and animals. Therefore, as a consequence of the of the metals are amine, alcohol, hydroxyl, ester, carboxyl,
toxic effects of heavy metals on the environment and thiol, sulfhydryl, sulfonate, phosphoryl, as well as thioester
their subsequent accumulation in the food chain groups (Abdi and Kazemi 2015; Ahemad and Kibret
(Emenike et al. 2016), global sustainability tilted to- 2013). Extra- and intracellular precipitation, active metal
wards alternatives that are environmentally friendly uptake, and valence transformation are other mechanisms
and inexpensive for the decontamination of heavy metal through which fungi deal with heavy metal contaminants
polluted soil. Among the technologies that are employed (Dhankhar and Hooda 2011).
for the decontamination of heavy metal polluted envi- Quite a number of fungi are currently been studied as
ronments is bioremediation. Bioremediation is a feasi- possible biosorbents for metal bioremoval, and significant
ble, inexpensive, and environmentally friendly technol- successes have been recorded (Fawzy et al. 2017; Igiehon
ogy that involves the use of living organisms including and Babalola 2019). However, optimization of the impact
microorganisms to decontaminate polluted sites of fungal organisms on the bioremoval of heavy metal
(Benaisa et al. 2019; Chakraborty et al. 2012; Oladipo from contaminated soil is still necessary (Emenike et al.
et al. 2016; Ullah et al. 2019). 2017; Rocco et al. 2018). Therefore, the present research
Soil contains a diverse group of microorganisms, and focuses on bioaugmentation. The concept of bioaugmen-
their functions in the ecosystems are also as diverse as the tation or supplementation of culture of microorganisms
microorganisms themselves. Microorganisms are adapted into engineered or natural environment has been in exis-
to their microenvironments and live together in association tence and has been applied in wastewater treatment pro-
as consortia, and often live with other part of the soil cesses, agriculture, and bioremediation of polluted sites
inhabitants. Microorganisms play significant functions (Singh et al. 2011). The efficiency of potential bioaugmen-
and sustain the natural ecosystem through biodegradation tation may be enhanced by using soil containing popula-
and biogeochemical cycling of nutrients (Singh et al. tions of autochthonous degrader microorganisms which
Water Air Soil Pollut (2019) 230:215 Page 3 of 20 215

are previously exposed to pollutants. The inoculation of carried out on a hot plate until dense fumes diminishes
naturally occurring consortia of microorganisms may be and a clear solution was observed. The digested samples
more successful than inoculation of single isolated strains were allowed to cool and then filtered through a
applied as pure cultures (Singh et al. 2011). However, Millipore filter (0.45 μm), diluted to 50 mL with dis-
majority of the bioaugmentation researches have been tilled water. Filtration was carried out carefully to avoid
conducted using bacteria, belonging to species like contamination of the samples by the filter. Elemental
Flavobacterium, Pseudomonas, Sphingomonas, analyses were carried out using atomic absorption spec-
Lysinibacillus sp., Alcaligenes, Rhodococcus, trometry (PG instruments AA500 model) (USEPA
Achromobacter, Bacillus, and Mycobacterium (Emenike 1996). Blank sample was also prepared for quality as-
et al. 2016, 2017; Singh et al. 2011). Meanwhile, on the surance, and all analyses were carried out in triplicate to
other hand, little information is surfaced to date about minimize errors.
bioaugmentation using fungal consortia to decontaminate
heavy metal polluted soil. Therefore, this study is aimed to 2.3 Isolation and Morphological Characterization
study the mycoremediative capacity of filamentous fungi of Fungi
consortia in landfill leachate contaminated soil. For this,
two groups of filamentous fungi consortia were used for Serial dilution of the soil samples with distilled water
the bioremediation of the heavy metal contaminated soil. was carried out for the isolation of the fungal colonies.
Suspensions were made by dissolving 1 g of freshly
collected landfill soil in 10 mL of sterile distilled water
2 Materials and Methods and agitated for about 10 min. One milliliter suspension
was diluted up to 10−7, after which 0.1 mL of the 10−7
2.1 Description of the Sampling Site and Sample dilution was pipetted onto Potato Dextrose Agar (PDA)
Collection medium (Friendemann Schmidt, Parkwood, WA, Aus-
tralia) and spread using sterile spreader and incubated at
Taman Beringin landfill, which is a non-engineered, 28 °C for 6 days. To obtain pure cultures, isolates were
stabilized, and closed (MSW) landfill, was selected for subcultured on PDA slants and preserved. Macroscopic
the research on the basis of its grade and status. It is characterization was performed to observe some fea-
situated in Jinjang Utara, Kuala Lumpur (3° 13.78′ N; tures such as the colony color, presence or absence of
101° 39.72′ E) (Jayanthi et al. 2016), where it was in aerial mycelia, presence of wrinkles and furrows, and
operation between 1992 and 2005. It received about pigment production. Wet mount preparations of the
1800–2000 t of household, industrial, and commercial fungal isolates using lactophenol cotton blue were car-
wastes daily. The generated leachate is treated using ried out, and the slides were viewed under microscope
biological and physical methods while the landfill gas (× 40) to identify the isolates for the presence of spore,
is released passively to the atmosphere (Jayanthi et al. columella, phialides nature, conidiospore color, etc.
2016). Triplicate surface (0–30 cm) soil samples were (Yin et al. 2017).
collected within the landfill from different areas (Fig. 1)
using coring device according to EPA (2000). Soil sam- 2.4 Molecular Identification and Characterization
ples collected were placed in sterilized plastic con- of Fungi
tainers, labeled, stored in a cooler at 4 °C, and
transported to the laboratory for analysis (EPA 2000). Fungal isolates were cultivated on malt extract agar for
7 days at 30 °C. Genomic DNA extraction was carried
2.2 Analytical Procedure out, and examination of the extracted DNA was per-
formed through electrophoresis using 0.8% agarose gel.
The collected soil samples were subjected to analyses Red Safe DNA Dye (Sinaclon, Karaj, Iran) was used to
for pH, conductivity, and oxidation-reduction potential stain the DNA for effective visualization. The internal
using a multiprobe meter (YSI Professional Plus, OH, transcribed spacer region was amplified using primers
USA). For the analysis of heavy metals, 1 g of soil ITS-1F (5′-CTT GGT CAT TTA GAG GAA GTA A-3′)
sample was treated with 10 mL of aqua regia (25% and ITS-4 (5′-TCC TCC GCT TAT TGA TAT GC-3′).
HNO3; 75% HCl). Digestion of the soil samples was Mixture of PCR containing PCR buffer, MgCl2, Taq-
 215 Page 4 of 20 Water Air Soil Pollut (2019) 230:215

Fig. 1 Location of the study site and sampling points

DNA polymerase, and dNTPs from Denmerk Ampliqon (266.436 g/mol) (Aldrich), and HAsNa2O 4·7H2O
Company (Stenhuggervej 22, 5230 Odense, Denmark) (312.01 g/mol) (Aldrich)) into distilled water in conical
was used. The volume of the reaction mixture was 25 μl flasks, followed by subsequent serial dilutions to obtain
containing 10 ng of gDNA and (10 pmol/μl) of each various standard concentrations (mg/L). Specific volumes
forward and reverse primers in the Thermocycler (MJ- of the standard concentrations were used for the prepara-
PTC 200 model, Marshall Scientific, Hampshire, USA). tion of the media for tolerance tests. The purified fungal
The optimal PCR conditions involved initial dena- isolates from soil samples were subcultured in triplicate
turation of the extracted DNA for 90 s at 95 °C, 35 cycles onto PDA and incubated at 28 °C for 4 days to obtain fresh
involving denaturation for 30s at 95 °C, annealing for isolates. Fresh isolates were then transferred onto PDA
30s at 52 °C, extension for 30s at 72 °C, and last containing different metal concentrations (10, 20, 30, and
extension at 72 °C for 6 min. The sequences were 40 mg/L), followed by subsequent incubation for 6 days at
obtained using Sanger dideoxy sequencing technology. 28 °C. After incubation, radial growth diameters of the
The obtained sequences were compared with those of fungi were measured and compared with control plates
the already existing species using Basic Local Align- (without any amendment). Several measurements were
ment Search Tool (BLAST) with GenBank database taken at right angle to each other, and averages were taken
(Khamesy et al. 2016), and the phylogenetic tree was and recorded. Tolerance index was calculated as the ratio
constructed using MEGA X. of the radial growth of the metal amended media to that of
the radial growth of the untreated control using (Eq. 1).
2.5 Determination of Metal Tolerance of Fungi Higher tolerance index was considered as greater resis-
tance.
The procedure employed by Fazli et al. (2015) was used 
for the determination of the tolerance capacity of the fungi. Ti ¼ Dt Du
ð1Þ
Stock concentrations of the heavy metals were prepared by
dissolving metal salts (MnSO4·H2O (169.02 g/mol) where Ti is the tolerance index, Dt is the radial diameter (in
(Friendemann), CuSO4 (159.60 g/mol) (Bendosen), cm) of the treated plates, and Du is the radial diameter (in
FeSO4·7H2O (278.02 g/mol) (Bendosen), Cl3CrH12O6 cm) of the untreated plates.
Water Air Soil Pollut (2019) 230:215 Page 5 of 20 215

2.6 Bioaugmentation Table 1 Consortia of fungi used in the bioremediation of the

landfill leachate contaminated soil

Blended fungal isolates were used for the mycoremediation, Fungal consortia (treatments)
and the formula was used based on three major criteria: (1)
the fact that the microorganisms are native to the contami- Highly heavy metal tolerant Moderately heavy metal tolerant
fungi fungi
nated soil may give an advantage in the change of chemical
form of the metals over the non-native microorganisms; (2) Perenniporia subtephropora Paecilomyces lilacinus
the isolation of the organisms from the soil implied the Daldinia starbaeckii Antrodia serialis
presence of an active metabolism; (3) tolerance of the Phanerochaete concrescens Penicillium cataractum
isolated fungi to the heavy metal contaminants suggests Cerrena aurantiopora
the likelihood bioremoval capacity of the fungi (Lebeau Fusarium equiseti
2011; Sprocati et al. 2012). Polyporales sp.
Aspergillus niger
Aspergillus fumigatus
2.7 Microbial Formulation Trametes versicolor

Fungal isolates that showed tolerance to the metal con-

centrations were used in the bioaugmentation experi- 2.9 Heavy Metal Bioremoval
ment. Individual strains were grown as pure cultures
on PDA plates for 4 days at 28 °C before being inocu- The metal bioremoval efficiency was determined ac-
lated in PDB and incubated at 28 °C and 150 rpm on a cording to (Eq. 2) as proposed by Emenike et al. (2017).
rotary shaker. Equal volume of the inoculums contain-
ing about 3 × 109 spores g−1 of the individual fungus %of heavy metal removal
from the broth culture was drawn and combined to set C0ðxÞ−CFðxÞ
up the inoculums of consortium of fungi for bioaugmen- ¼  100% ð2Þ
C0ðxÞ
tation. Two kilograms of the landfill soil was amended
with 10% v/w of the prepared inoculums. where
C0(x) = initial concentration of metal “x” (As, Cr, Cu,
Fe, or Mn) in the soil at the beginning of the experiment.
2.8 Experimental Design for Soil Mycoremediation CF(x) = final concentration of metal “x” (As, Cr, Cu,
Fe, or Mn) in the soil at the end of the experiment.
Different treatments with fungi were set up for the
bioremediation of the soil. The treatments with the
fungal consortium involved; highly tolerant fungi, 2.10 Bioremediation Kinetics
moderately tolerant fungi, and the control (soil with-
out fungal augmentation) (Table 1). The fungal for- The rate at which metal is removed by the fungi per day
mulations were introduced into the designated mi- and their corresponding half-life were calculated using
crocosm’s soil and that mark the beginning of the Eqs. 3 and 4 as proposed by Emenike et al. (2017).
bioremediation. Each treatment was in triplicate and  
1 Cf
was kept for 100 days in poly bags containing pores K¼− ln ð3Þ
t C0
for the draining of excess water and supply of at-
mospheric air into the microcosm. Soil moisture where K = first-order rate constant for metal uptake
content of 60–65% was maintained in the micro- per day, t = time in days, Cf = residual concentration of
cosm through regular watering with distilled water, metal in soil (mg/kg), C0 = initial concentration of metal
which helps to keep an active metabolism. Soil in soil (mg/kg)
subsamples were collected at day 20, day 60, and
In ð2Þ
day 100 and analyzed for pH, electrical conductivi- Half −life t1=2 ¼ ð4Þ
ty, redox potentials, residual metal concentrations, K
and total fungal count (Emenike et al. 2016).
 215 Page 6 of 20 Water Air Soil Pollut (2019) 230:215

2.11 FTIR Spectroscopy Analysis of Bioremediated 3.2 Determination of Metal Tolerance of Fungi
Soil
Fungal isolates were defined according to their tol-
The structural changes in the bioremediated soil were erance behaviors against the tested heavy metals.
subsequently determined using FTIR spectroscopy The tolerance rating index proposed by Oladipo
(Perkin Elmer 400FTIR/FTFIR version 10.4.2) in the et al. (2018) was utilized for the categorization of
frequency range of 500–4000 cm−1. the isolates. Fungal isolates were named as very low
tolerance (0.00–0.39), low tolerance (0.40–0.59),
moderate tolerance (0.60–0.79), high tolerance
2.12 Statistical Analysis
(0.80–0.99), and very high tolerance (1 and above).
Figure 3 showed the tolerance index of the identified
Data obtained were subjected to statistical analyses to
fungi, and it was seen that the tolerance of the fungi
determine the occurrence of significant difference and
towards the metals varied depending on the metal.
relationships between the variables. Descriptive statistics
Based on the averages of the tolerance index, Fu-
was carried out for mean and standard deviation of the
sarium chlamydosporum did not meet the criteria to
parameters. Two-way and one-way ANOVAs were used,
be selected for the bioremediation experiment; as
and multiple comparison between groups using Tukey was
such, it was not included.
also performed to measure the differences or otherwise
Aspergillus fumigatus was the most tolerant to As as
between the variables. The relationship between the con-
it had the highest tolerance index of 1.0. Other fungi of
centrations of the heavy metals and some of the physico-
high tolerance were P. subtephrophora, A. niger, and
chemical parameters was analyzed using Pearson’s corre-
C. aurantiopora measuring between 0.8 and 0.9. There
lation coefficient. The strength of the correlation coefficient
was a statistically significant tolerance difference be-
was determined on a scale of + 1.00 to − 1.00. All statis-
tween the fungi towards As exposure (F(12, 26) =
tical analyses were carried out using SPSS software (ver-
35.866, P = 0.000). Meanwhile, Tukey post hoc test
sion 23) at 95% confidence limit. All graphical work was
revealed that A. fumigatus was statistically tolerant than
carried out using Excel (version 16.0) and Origin Pro 2015
F. chlamydosporum (P = 0.000), D. starbaekii (P =
SR2 version b9.2.272.
0.000), P. concrescens (P = 0.000), Polyporales sp.
(P = 0.000), A. lilacinus (P = 0.002), A. serialis (P =
0.000), P. cataractum (P = 0.001), and T. versicolor
3 Results and Discussion (P = 0.000). The tolerance demonstrated towards As
might be connected to the intracellular accumulation of
3.1 Fungal Organisms As in the cell vacuoles which was shown to play an
important role in As detoxification (Cánovas and De
A total of thirteen species of fungi were identified Lorenzo 2007). Likewise, biomethylation/volatilization
(Fig. 2). These fungi include Perenniporia also helps through excretion via volatilization of meth-
subtephropora (MK209003), Daldinia starbaeckii ylated arsenic species (Srivastava et al. 2011). On the
( M K 2 0 9 0 0 4 ) , P h a n e ro c h a e t e c o n c re s c e n s other hand, A. serialis (0.0), D. starbaeckii (0.1),
(MK209005), Cerrena aurantiopora (MK209006), Fu- T. versicolor (0.3), and P. concresens (0.4) were severely
sarium equiseti (MK209007), Polyporales sp. affected by As toxic effects. The damage caused can be
(MH541016), Aspergillus niger (MH541017), supported by the fact that As has a long history of
Paecilomyces lilacinus (MH541018), Antrodia serialis toxicity and can cause damage to a variety of living
(MH541019), Aspergillus fumigatus (MK534500), Pen- systems including microbial lives which leads to affect-
icillium cataractum (MK534497), Trametes versicolor ing the microbial interactions with other life forms
(MK534498), and Fusarium chlamydosporum (Hettick et al. 2015). Furthermore, the inhibition might
(MK534502). These results are supported by several be attributed to the lack of effective defense mechanisms
researchers that isolated fungal organisms in heavy met- against the toxicity. It has been revealed that damages
al contaminated environments (Abdel-Azeem et al. caused by As can be related to the lack of glutathione
2015; Akhtar et al. 2013; Datta 2015; Igiehon and (GSH) which is a strong antioxidant against metal stress
Babalola 2019; Khamesy et al. 2016). (Muneer et al. 2016).
Water Air Soil Pollut (2019) 230:215 Page 7 of 20 215

Fig. 2 The evolutionary history was inferred using the neighbor- involved 52 nucleotide sequences. All ambiguous positions were
joining method. The optimal tree with the sum of branch length = removed for each sequence pair (pairwise deletion option). There
2.03508204 is shown. The evolutionary distances were computed were total of 3782 positions in the final dataset. Evolutionary
using the maximum composite likelihood method and are in the analyses were conducted in MEGA X
units of the number of base substitutions per site. The analysis

All isolates had tremendous tolerance against Cr, and 0.000). The high tolerance to Cr might be the result of
all were in the range of 0.7–1.0 tolerance index. Mean- the GSH and other non-protein thiol production (Viti
while, one-way ANOVA revealed that Polyporales sp., et al. 2014; Wang and Chen 2014). Efflux of chromate
A. fumigatus, and C. aurantiopora were significantly ions and extracellular reduction of Cr (VI) to Cr (III) are
tolerant to Cr than other fungi (F(12, 26) = 5.042, P = other mechanisms employed by fungi against toxic
 215 Page 8 of 20 Water Air Soil Pollut (2019) 230:215

Fig. 3 Tolerance index of the

isolated fungi As Cr Cu Fe Mn

1.2

Tolerance index
1
0.8
0.6
0.4
0.2
0
-0.2

Isolates

effect of Cr (Viti et al. 2014). Similar tolerance behavior Penicillium sp., Trichosporon montevideense,
was observed for Cu with exception of P. lilacinus Trichosporon sporotrichoides, Trichosporon otae,
which measured only 0.5. With regard to Fe, Galactomyces geotrichum, and Rhodotorula
P. lilacinus was also the least tolerant with 0.6 tolerance mucilaginosa on Zn, Pb, and Ag.
index, while others such as D. starbaeckii and
C. aurantiopora had the highest tolerance index of 3.3 Physicochemical Parameters
1.0, with statistical significance at (F(12, 26) = 2.764,
P = 0.015). For Mn, P. subtephropora (0.5), P. lilacinus 3.3.1 Specific Conductivity
(0.6), and A. niger (0.6) had the least tolerance, and the
highest index was 0.9 for C. aurantiopora and was It was clear that the values of soil specific conductivities
found statistically significant than P. subtephropora were higher at the commencement of the bioremediation
(P = 0.006), P. lilacinus (P = 0.021), and P. cataractum (Fig. 4). The highest value at initial level was 487.73 S/
(P = 0.039) using Tukey post hoc comparison. It was m for both treated soils set up. However, as the duration
noticed that P. lilacinus responded weakly to almost all of the mycoremediation increased, the level of the spe-
the heavy metals; meanwhile, C. aurantiopora demon- cific conductivity continued to decelerate up to the last
strated a strong resistance against all the heavy metals. day (day 100). For soil amended with the highly tolerant
The most plausible explanation for the resistance of fungal consortium, the lowest value was 186.34 S/m;
C. aurantiopora is that its tolerance is largely related meanwhile, 189.78 S/m was the least recorded and was
to the possession of laccase enzyme which is a broad- for moderately tolerant fungi-treated soil. On contrast,
spectrum activity enzyme that confers resistance to dif- both treated soils had relatively lower specific conduc-
ferent heavy metal exposure (Xu et al. 2018). It was tivity than the untreated control soil. However, two-way
similarly reported by Xu et al. (2018) that Cerrena sp. ANOVA pairwise comparison showed insignificant dif-
HYB07 produced an efficient laccase enzyme that has ference in specific conductivity between the days and
the potential to cleanup various environments contami- between the treatments (F(6,24) = 0.129, P = 0.991). On
nated with pollutants. the other hand, the decreasing trend observed was in line
The results of tolerance index recorded in this re- with the concentrations of the metals recovered. On the
search are in line with those of other findings: other hand, results obtained in the current research were
Mohammadian et al. (2017) showed some fungal spe- in agreement with those obtained by Goswami and
cies such as Fusarium verticillioides, Alternaria Sarma (2008) from contaminated soil of a municipal
chlamydosporigena, Penicillium simplicissimum, solid waste dumping site. However, contrastingly, the
Acremonium persicinum, Trichoderma harzianum, and results were lower (3209.57 ± 0.05) and (3089.24 ±
Seimatosporium pistaciae which were tolerant to differ- 0.04) than those reported by Hanif et al. (2005) from
ent concentrations of Cu, Zn, Pb, and Cd. Similarly, industrial effluent. The likely reasons for the disparity
Munoz et al. (2012) reported tolerance indices of between the present results and that of Hanif et al.
Water Air Soil Pollut (2019) 230:215 Page 9 of 20 215

Fig. 4 Specific conductivity for

in situ mycoremediation of metal- Day 0 Day 20 Day 60 Day 100
contaminated soil of Taman
Beringin landfill 600

Specific conducvity of soil

500
400
300

(S/m)
200
100
0
Highly tolerant Moderately Control
fungi tolerant fungi
Fungal Treatment

(2005) might be the difference in the contaminated dropped to 6.4 at day 100. Similar occurrences were
medium which is the effluent, and also the source observed for moderately tolerant fungal consortium
(industries) of the contaminants might have contributed treated soil with the highest pH of 7.9 at initial phase
for high values of conductivity as compared to the and least of 6.4 at day 100. Likewise, for the control
current research. setup, the highest pH at initial level was similar to that of
Statistical determination of the relationship between the consortium amended microcosm; however, at the
specific conductivity and the metal concentrations in the end of the experiment, the pH was a bit higher (6.7)
contaminated soil revealed that all the treatments had than those of the treated experiments. Post hoc compar-
correlations ranging from strong to very strong positive. ison of the two-way ANOVA revealed significant pH
This suggested strong relationships between the metal difference between the days (F(6,24) = 4.372, P =
concentrations and the specific conductivity. The stron- 0.004), with exception of between day 0 and day 20
gest correlation recorded was r = .94 for Fe for soil (P = 0.622). Even though, there was continued decrease
treated with moderately tolerant fungal consortium, in pH in the control, which might be from the action of
followed by r = .92 for Cr and Mn. On the other hand, the already existing microbes inhabiting the soil; how-
r = .80 for Cu for moderately tolerant fungi-treated soil ever, there was slight pH difference at the end of the
was the least correlation coefficient recorded. The real- bioremediation as compared to the amended soil.
ization of a strong positive relationship is supported by It was understood that throughout the experimental
the findings of Sharma and Raju (2013) who opined that period, there was continuous acidification of the con-
the solubility of metal ions in soil is governed by factors taminated soil. This might be connected to the action of
such as conductivity, pH, and moisture content. the bioaugmented fungal organisms, and also fungal
Likewise, Alam et al. (2017) stressed that the amount metabolism, this can be from the stand point that the
of specific conductivity serves as an index of the amount fungi might have released certain acidic compounds
of dissolved inorganic compounds present in a medium. especially low molecular weight organic acids which
will ultimately decrease the solution pH of the soil
3.3.2 pH (Wang et al. 2015). It was equally highlighted by
Boonchan et al. (2000) that a change in the solution
In Fig. 5, it was noted that the landfill leachate contam- pH from the initial level of bioremediation could be
inated soil had a pH range between 6.4 and 7.9. The pH attributed to the production of acidic and alkaline me-
of all the setup including the control followed a decreas- tabolites during biodegradation of the contaminants.
ing trend from the initial date of the experiment to the Equally, it was reported that when the pH of a medium
last date. The pH of the highly tolerant fungi-treated soil decreased, the concentration of dissolved metal in-
started at 7.9, which was maintained up to day 20. creased; this is for the fact that acid pH enhanced the
However, at day 60, the pH dropped to 6.9 which further dissolution of metals, thereby making them more
 215 Page 10 of 20 Water Air Soil Pollut (2019) 230:215

Fig. 5 pH for in situ

bioremediated soil Day 0 Day 20 Day 60 Day 100

10
8

Soil pH
6
4
2
0
Highly tolerant fungi Moderately tolerant Control
fungi
Fungal Treatment

bioavailable, a factor that enabled the bioaccumulation This can be explained by the fact that, at the initial
of the metal into the microbial cells (Wang et al. 2015). phase, all the experimental soil including the control
In view of the current pH, it can be asserted that bioac- had positive redox values; however, as the duration
cumulation might be the likely favored mechanism for increased, the redox potential begun to dropped to
the metal bioremoval. Bioaccumulation occurs within the negative values. At the initial stage, 50.11 mV
the cell, and the contaminants are taking up through the was measured for all the treatments; however, from
cell barriers across plasma membrane into cytoplasm, day 20 onward to day 100, a range of − 19.07 to −
and the intracellular accumulation (heavy metal trans- 147.03 mV was recorded for all the treatments in-
port through cell membrane) may be brought about by cluding control.
similar mechanism that move metabolically important The frequent occurrence of the reduced redox poten-
ions like Mg, K, and Na (Hansda and Kumar 2016). tials in all the treatments might likely be associated with
the reduction of the oxygen content which might be
3.3.3 Redox Potentials connected to the microbial action within the microcosm.
Furthermore, the decline of the redox potential can be
It was observed that the redox potential undergone a justified by the values of the pH and the residual metal
change in state from oxidized at the beginning to a concentrations recorded. This is for the fact that it was
reduced state at the end of the experiment (Fig. 6). reported that pH and redox potential are interrelated in

Fig. 6 Soil redox potential for in

situ mycoremediation of metal- Day 0 Day 20 Day 60 Day 100
contaminated soil of Taman
Beringin landfill 100.00
Soil redox potenal (MV)

50.00

0.00
Highly tolerant Moderately Control
-50.00 fungi tolerant fungi
-100.00

-150.00

-200.00
Fungal Treatment
Water Air Soil Pollut (2019) 230:215 Page 11 of 20 215

determining the solubility, mobility, and bioavailability 1010 CFU/g was recorded for soil treated with moder-
of metals in a medium (Popenda 2014). It was similarly ately tolerant fungi.
asserted by Chuan et al. (1996) and Popenda (2014) that Comparing the growth between the treatments, soil
generally, in acidic and reduced conditions, metal treated with the moderately tolerant fungi had more
solubility and mobility are more favorable. In similar fungal count even though only three species make up
vein, Chuan et al. (1996) realized that when the pH of the consortium as compared with the soil treated with
the setup was kept at 5.0, metal solubility increased highly tolerant fungal consortium which contains nine
drastically by three- to fourfold under continued reduc- species of fungi. This showed that species diversity did
tion of redox potential from 100 to − 100 mV. Similarly, not influence the fungal reproduction within the soil.
when the redox potential was reduced from 330 mV to a This might be attributed to the fact that some of the
moderately oxidizing state (200 mV), the solubilization fungal organisms within the soil treated with nine spe-
of Pb was doubled from 280.8 to 678.6 mg/kg; likewise, cies might have been affected by some factors such as
Zn increased from 110.3 to 189.6 mg/kg (Chuan et al. competition for available resources, inhibition by some
1996). Similarly, Popenda (2014) reported that the con- toxic metabolites released by other members in the
centration of As in highly As-contaminated sediment microcosm, or might have been inhibited by toxicity
was found to increase under low redox potential. of the heavy metals, since the fungal tolerance varied
with the heavy metals as observed in Fig. 3. These
3.4 Fungal Population observations can be buttressed by the assertion that the
growth and performance of microorganisms in any en-
Fungal growth showed considerable variation among vironment is influenced by certain factors such as mois-
the treatments (Fig. 7). For soil treated with highly ture content, nutritional availability, pH, pollutant con-
tolerant fungi, the fungal count recorded at initial stage centration, temperature, and presence of other metabo-
was 3 × 109 CFU/g; this was followed by a significant lites (Mandal and Das 2018).
increase of 1.39 × 1010 CFU/g at day 20. However, as On the other hand, even though there was a decrease
the days further increased, the counts assumed a de- in the fungal counts for both treated soils, both treat-
scending order up to day 100 and recorded 1.1 × ments had higher counts as compared with those of the
109 CFU/g. Similar growth patterns were noticed for control. The lower counts in the control soil showed an
soil treated with moderately tolerant fungi and the con- influence of the bioaugmentation; this is with the view
trol; however, a reduced growth was observed in the that, in control, only the survived inhabitants were able
control soil with the highest count as 1 × 109 CFU/g. to withstand the condition and reproduced. Furthermore,
Meanwhile, a range of between 4.2 × 109 and 1.47 × these survived inhabitants did not receive any nutritional

Fig. 7 Fungal colony count for in

situ mycoremediation of metal- Day 0 Day 20 Day 60 Day 100
contaminated soil of Taman
Beringin landfill 2.5E+10
Fungal colony count (CFU/g)

2E+10

1.5E+10

1E+10

5E+09

0
Highly tolerant Moderately Control
-5E+09 fungi tolerant fungi
Fungal Treatment
 215 Page 12 of 20 Water Air Soil Pollut (2019) 230:215

Fig. 8 As concentration for in

situ mycoremediation of metal- Day 0 Day 20 Day 60 Day 100
contaminated soil of Taman
Beringin landfill 25.00

Soil As concentraon (mg/Kg)

20.00
15.00
10.00
5.00
0.00
Highly tolerant Moderately Control
fungi tolerant fungi
Fungal Treatment

supplement like those in the consortium-treated soil individual isolates isolated from polyaromatic hydrocar-
which received nutritional addition in the form of nutri- bon contaminated soils of different sites (Adayar and
ent media which enhanced the growth of the fungal Guindy) in Chennai, India. The counts are 7.2 CFU/g
organisms. for Rhizopus stolonifer isolated from Adayar soil,
The fungal count recorded in the current research 6.3 CFU/g for Mucor racemosus, 5.3 CFU/g for Peni-
exceeded that found by Mandal and Das (2018), who cillium chrysogenum, and 3.4 CFU/g for Aspergillus
reported highest yeast count as 8.7 × 105 CFU/g dry soil. niger, all isolated from Guindy soil.
The likely cause of the variation between their results
and the current results might be related to the experi- 3.5 Heavy Metal Bioremoval by Fungal Consortia
mental setup, the adaptability, morphology, and physi-
ology of the organisms involved in the bioremediation. The metal removal increased with increasing duration
This is because, in their research, even though a consor- (number of days) of the bioremediation (Fig. 8). Among
tium was also used, there was also involvement of plant the consortium-treated microcosms, the soil treated with
(sun flower) in the consortium, and it has been reported highly tolerant fungi had higher As reduction as com-
that plant may release water, root exudates, and nutrient pared with that treated with moderately tolerant fungi.
fluxes which may affect the microbial populations The As bioremoval and the efficiencies recorded in the
(Butler et al. 2003). Similarly, it has also been reported two treatments are 14.37 mg/kg (62%) and 11.11 mg/kg
that rhizosphere has a complex ecology with many (48%), respectively (Table 2), and contrasting results
feedback loops which influence the microbial popula- showed insignificant difference between them (P >
tions (Toal et al. 2000). In another related research, 0.05) using post hoc comparison of two-way ANOVA.
Balaji et al. (2014) reported fungal colony counts for On the other hand, lower As bioremoval was observed

Table 2 Residual mean concentrations of heavy metals and the heavy metal removal efficiency at 100 days from the bioremediation of
leachate contaminated soil

Heavy metals Highly tolerant fungi Moderately tolerant fungi Control

Residual mean (mg/kg) % removal Residual mean (mg/kg) % removal Residual mean (mg/kg) % removal

As 8.74 62 12.00 48 19.29 17

Cr 378.12 42 419.45 36 527.36 19
Cu 112.33 49 128.54 41 206.40 6
Fe 1449.37 38 1525.81 35 2056.07 12
Mn 11.81 59 16.88 41 26.11 8
Water Air Soil Pollut (2019) 230:215 Page 13 of 20 215

Fig. 9 Cr concentration for in

situ mycoremediation of metal- Day 0 Day 20 Day 60 Day 100
contaminated soil of Taman
Beringin landfill 800.00

Soil Cr concentraon (mg/Kg)

700.00
600.00
500.00
400.00
300.00
200.00
100.00
0.00
Highly tolerant Moderately Control
fungi tolerant fungi
Fungal Treatment

for control setup as compared with those for highly bioremoval despite the fact that they are tolerant to its
tolerant (P = 0.002) and moderately tolerant (P = toxicity. This behavior is in line with the fact that mi-
0.020) consortia-treated soil using multiple comparison. croorganisms possess different mechanisms for toler-
This signifies the likely function of the bioaugmented ance and biosorption and/or bioaccumulation of con-
fungal organisms which provided an edge over the taminants from the polluted sites (Dhankhar and
untreated soil. Hooda 2011). This assertion is further supported by
On the other hand, the lack of reasonable difference the fact that biomethylation and volatilization have been
between the fungal amended soils, even though there regarded as part of the mechanisms for the removal of
was variation in the species diversity, seems to be sur- As by fungi in contaminated environments (Su et al.
prising considering the fact that it can easily be hypoth- 2011); meanwhile, the mechanisms responsible for the
esized that the microcosm containing the highest num- tolerance against toxic effects of As include sequestra-
ber of fungal diversity may perform better than the one tion, intracellular compartmentalization, and complexa-
with less microbial diversity (looking at the fact that tion (Singh et al. 2015; Srivastava et al. 2011).
enhanced metabolic activities are achieved through the Furthermore, it was reported that fungi have devel-
increase in microbial diversity) (Emenike et al. 2016). oped biochemical mechanisms for the exploitation of As
This suggested the likelihood that some of the fungal oxyanions, either in the form of arsenate (electron ac-
isolates within the microcosm did not perform in As ceptor) for anaerobic respiration, or in the form of

Fig. 10 Cu concentration for in

situ mycoremediation of metal- Day 0 Day 20 Day 60 Day 100
contaminated soil of Taman
Beringin landfill 250.00
Soil Cu concentraon (mg/Kg)

200.00
150.00
100.00
50.00
0.00
Highly tolerant Moderately Control
fungi tolerant fungi
Fungal Treatment
 215 Page 14 of 20 Water Air Soil Pollut (2019) 230:215

arsenite which is an electron donor for the fixation of Table 3 Removal rate constant (K) of heavy metals for bioreme-
diation of soil from Taman Beringin landfill
carbon dioxide into cell carbon (Wang and Zhao 2009).
It was also highlighted that the uptake of As takes place Metal Removal rate constant (K) day−1
through various transporters which include glycerol,
phosphate, and hexose transporters, and the sorption of Highly tolerant fungi Moderately tolerant fungi Control
As onto the fungal cell wall and the subsequent bioac-
As 0.0097 0.0066 0.0019
cumulation into the cell are also involved in the fungal
Cr 0.0054 0.0044 0.0021
responses to As (Su et al. 2012).
Cu 0.0067 0.0053 0.0006
As presented in Fig. 9, the residual concentration of
Fe 0.0048 0.0043 0.0013
Cr followed a decreasing order from day 0 onward up to
Mn 0.0088 0.0052 0.0009
day 100. The residual Cr level at the end of the exper-
iment ranged from 378.12 to 527.36 mg/kg. On the
other hand, a maximum Cr bioreduction of nature (Akgul and Akgul, 2018). It was equally reported
273.55 mg/kg (42%) was witnessed in soil amended that removal of Cu by fungal organisms is attributed to
with highly tolerant fungal consortium; meanwhile, the presence of certain specific proteins which have
124.31 mg/kg (19%) was reported as the least Cr re- particular functions that are associated with the regula-
moval in the control (P < 0.05). However, statistical tion of Cu concentration. These proteins are Cu-
consideration using post hoc multiple comparison of transporting ATPase pumps and Cu-homeostasis
two-way ANOVA revealed insignificant Cr removal (CutC) gene (Tang et al. 2013).
difference between the consortium-treated microcosms The bioremoval efficiency of Fe and Mn in fungi-
(P = 0.722). The decreased Cr concentration witnessed amended soil ranged from 35 to 59% (Figs. 11 and 12);
in the current research can be attributed to the reduced however, only 8–12% was achieved in the control soil
redox condition of the soil observed during bioremedi- and recorded a significant difference of P < 0.05 using
ation. Similarly, these findings can be supported by the multiple comparison. The bioremoval of Fe might be
fact that, in bioremediation technique, Cr is rendered related to some metabolites that were likely released by
harmless through the reduction of toxic Cr(VI) in the the fungi, typical among them are siderophores. This is
soil to Cr(III) which is less harmful, and the generated based on the assertion that, in soil, iron exists as Fe(III)
Cr(III) is immobilized in the soil matrix (Ali et al. 2019; and forms insoluble oxyhydroxides and hydroxides, and
Jeyasingh and Philip 2005). In comparison, Achal et al. as such, is not readily assessable by fungi (Ahemad and
(2011) reported 94% reduction of Cr from Cr- Kibret 2014). Therefore, under such conditions, fungi
contaminated soil using brown rot fungi. Similarly, obtained iron through the secretion of siderophores
these results were lower than those of Fukuda et al. (Schalk et al. 2011), which are iron chelators and have
(2008). Different experimental setup, which involved a high affinity for Fe(III). Siderophores help to trans-
difference in the inoculum concentrations, soil sample form heavy metals that are not available into available
size, concentration of the metal contaminants, and through solubilizing them, which improve the bioaccu-
nutritional supplement, might have contributed to the mulation capacity of the microorganisms (Ahemad
disparity in the Cr bioremediation efficiency between
the current research and those of Achal et al. (2011) and
Table 4 Half-life value for bioremediation of soil from Taman
Fukuda et al. (2008). Beringin landfill
For the bioremediation of Cu, similar trend was ob-
served as in the removal of As and Cr, and the maximum Metal Half-life t1/2 (days)
bioremoval efficiency was 106.41 mg/kg (49%) follow- Highly tolerant fungi Moderately tolerant fungi Control
ed by 90.2 mg/kg (41%) (Fig. 10; Table 2). The removal
of Cu by the fungal consortia can be supported by the As 71 105 365
assertion that some species of fungi including Cu- Cr 128 158 330
resistant are capable of utilizing Cu resources, bind Cu Cu 103 131 1155
with a metabolite specifically oxalate which is readily Fe 144 161 533
produced during decay; this metabolic activity results in Mn 79 133 770
the production of Cu oxalate crystals which are of inert
Water Air Soil Pollut (2019) 230:215 Page 15 of 20 215

Fig. 11 Fe concentration for

in situ mycoremediation of metal- Day 0 Day 20 Day 60 Day 100
contaminated soil of Taman
Beringin landfill 3000.00

Soil Fe concentraon (mg/Kg)

2500.00
2000.00
1500.00
1000.00
500.00
0.00
Highly tolerant Moderately Control
fungi tolerant fungi
Fungal Treatment

2015; Schalk et al. 2011), and this is in line with our faster than the corresponding other metals; as such, it
obtained pH values which also enhanced the solubility had only 71 days as its half-life which when compared
and bioaccumulation of metals. with that of Fe, it will be reduced twice as faster than Fe
The calculated results for the metal removal rate which had 144 days half-life. This highlighted that the
constant (K) and the half-life (t1/2) are presented in bioremoval of As was the prioritized potential of the
Tables 3 and 4, respectively. The highest values for the fungal organisms. However, comprehensive interpreta-
rate of removal constant were all for soil treated with tion of the reasons behind the higher removal of As,
highly tolerant consortium for all the five heavy metals; nevertheless, is not simple since a group of fungi is
on the other hand, control setup had the highest half-life. considered as a consortium; similarly, many factors
The represented values as recorded are 0.0097/day may play different role in the metal bioaccumulation
71 days, 0.0088/day 79 days, 0.0067/day 103 days, mechanisms, both active and passive (Sprocati et al.
0.0054/day 128 days, and 0.0048/day 144 days for As, 2006). However, it was opined by Gola et al. (2016)
Mn, Cu, Cr, and Fe, respectively, in their decreasing that in multiple metal bioremediation experiment, the
order of rate constant and increasing order of half-life. properties of the metals such as ionic or atomic radius,
It was noted that the rate constant for As was the atomic mass, and electronegativity affect the overall
highest achieved, this showed that it will be reduced efficiency of the metal removal. It was also reported that

Fig. 12 Mn concentration for

in-situ mycoremediation of metal Day 0 Day 20 Day 60 Day 100
contaminated soil of Taman
Beringin landfill 35.00
Soil Mn concentraon (mg/Kg)

30.00
25.00
20.00
15.00
10.00
5.00
0.00
Highly tolerant Moderately Control
fungi tolerant fungi
Fungal Treatment
 215 Page 16 of 20 Water Air Soil Pollut (2019) 230:215

a b

Fig. 13 a FTIR spectrum of soil treated with highly tolerant fungal consortium, b soil treated with moderately tolerance fungal consortium,
c soil without fungal amendment (control)

fungi possess various mechanisms to deal with metal contaminant to reduce and is a function of the
contaminants, which include active metal uptake, intra- bioremoval rate constant (Emenike et al. 2017). There-
cellular precipitation and accumulation, as well as va- fore, considering the fact that in the control set up, there
lence transformation, all of which are utilized by fungi was no microbial amendment; as such, the metal reduc-
in dealing with metal pollutants, and all of which varies tion will ultimately take longer time as compared to the
with the nature of the metal contaminants (Dhankhar bioaugmented setup and might even be attributed to a
and Hooda 2011). bioattenuation phenomenon which is natural. The cal-
The high K values observed in the consortium- culated K and t1/2 values obtained in the current study
treated soil signifies an interaction between the rate of were in line with those of Emenike et al. (2016, 2017).
metal removal and the metal concentration in the con- Contrastingly, these results were above those reported
taminated soil; however, in the case of control, which by Namkoong et al. (2002) (0.015–0.188/day and 2.6–
had lower rate of removal constants and subsequently 19.2 days) for the decontamination of diesel-
higher half-life, it might be from the decreased activity contaminated soil using composting technology. Differ-
of the soil microbial flora in the contaminated soil ent approaches employed coupled with the difference in
(Adesodun and Mbagwu 2008). This can be related to the contaminating agents might be the result in the
the fact that half-life is the time to take for half of a variation of the results.
Water Air Soil Pollut (2019) 230:215 Page 17 of 20 215

3.6 FTIR Spectroscopy Analysis of Bioremediated Soil influence on the bioremoval of the heavy metals in the
bioremediated soil. Heavy metal tolerance of the fungi,
FTIR spectra analysis of soil after bioremediation n a m e l y, P. s u b t e p h ro p o r a , D . s t a r b a e c k i i ,
was carried out to determine the presence and dis- P. concrescens, C. aurantiopora, F. equiseti,
appearance of any functional groups. The spectrum Polyporales sp., A. niger, P. lilacinus, A. serialis,
was assessed by comparing the absorption peaks on A. fumigatus, P. cataractum, T. versicolor, and
fungi amended soil with that of the unamended F. chlamydosporum had equally played a cogent role
control. Any changes in the fingerprint region that in the process and resulted to higher metal reduction in
is below or equals to 1500 cm−1 and the functional soil treated with highly tolerant fungal consortium. The
group region (above 1500 cm−1) were noted. Each research has demonstrated the contribution of the fungal
spectrum was studied thoroughly by comparing the bioaugmentation in the bioremediation of heavy metals
peak values with the standard FTIR charts to iden- contaminated soil; therefore, blending of native filamen-
tify the represented functional groups (Damodaran tous fungi for heavy metal bioremediation could serve
et al. 2013; Ivanova et al. 2008). A vibrational mode as potential tool for the decontamination of heavy metal
right around 692 ± 2 cm−1 representing the presence polluted sites.
of broad cis-C-H out-of-plane bending was ob-
served. Similarly, also bending at 778 ± 2 cm−1 was Funding Information This work was funded by the University
recorded (Fig. 13a–c) which is attributable to meth- of Malaya with the research grants RP011A-14SUS and PG070-
ylene rocking vibration which indicated a long-chain 2014B.
linear aliphatic structure. These assignments were
Compliance with Ethical Standards
consistent with those of Coates (2000). A sharp
bending of high intensity was equally monitored at Conflict of Interest The authors declare that they have no
1025–1005 ± 2 cm−1; this corroborated with the cy- conflict of interest.
clohexane ring vibrations.
On the other hand, our results were supported by
a clear distinction observed in the functional groups
between the fungal treated and the untreated soil. It
was clear that peaks at 874 ± 2 cm−1 and 1425 ± References
2 cm−1 were only found in fungi amended soil.
The peak at 874 ± 2 cm−1 denoted the presence of Abdel-Azeem, A., El-Morsy, E., Nour El-Dein, M., & Rashad, H.
C–O–O–C which corresponded to the presence of (2015). Occurrence and diversity of mycobiota in heavy
peroxides stretch; meanwhile, 1425 ± 2 cm−1 is an metal contaminated sediments of Mediterranean coastal la-
goon El-Manzala, Egypt. Mycosphere, 6, 228–240.
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This signifies the presence of phenol or tertiary heavy metals and comparison between different biosorbents.
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In the functional group region, a stretching of N–H 1399.
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bond which suggested the presence of primary or second-
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