Bioremediation of Explosive TNT by Trichoderma viride

Article abstract of DOI:10.3390/molecules25061393

Nitroaromatic and nitroamine compounds such as 2,4,6-trinitrotoluene (TNT) are teratogenic, cytotoxic, and may cause cellular mutations in humans, animals, plants, and microorganisms. Microbial-based bioremediation technologies have been shown to offer several advantages against the cellular toxicity of nitro-organic compounds. Thus, the current study was designed to evaluate the ability of Trichoderma viride to degrade nitrogenous explosives, such as TNT, by microbiological assay and Gas chromatography–mass spectrometry (GC–MS) analysis. In this study, T. viride fungus was shown to have the ability to decompose, and TNT explosives were used at doses of 50 and 100 ppm on the respective growth media as a nitrogenous source needed for normal growth. The GC/MS analysis confirmed the biodegradable efficiency of TNT, whereas the initial retention peak of the TNT compounds disappeared, and another two peaks appeared at the retention times of 9.31 and 13.14 min. Mass spectrum analysis identified 5-(hydroxymethyl)-2-furancarboxaldehyde with the molecular formula C₆H₆O₃ and a molecular weight of 126 g·mol^?1 as the major compound, and 4-propyl benzaldehyde with a formula of C₁₀H₁₂O and a molecular weight of 148 g mol^?1 as the minor compound, both resulting from the biodegradation of TNT by T. viride. In conclusion, T. viride could be used in microbial-based bioremediation technologies as a biological agent to eradicate the toxicity of the TNT explosive. In addition, future molecular-based studies should be conducted to clearly identify the enzymes and the corresponding genes that give T. viride the ability to degrade and remediate TNT explosives. This could help in the eradication of soils contaminated with explosives or other toxic biohazards.

Full text of DOI:10.3390/molecules25061393

molecules
Article
Bioremediation of Explosive TNT by
Trichoderma viride
1
,
2
2,
2
Zeid A. Alothman * , Ali H. Bahkali , Abdallah M. Elgorban *, Mohammed S. Al-Otaibi ,
Ayman A. Ghfar 1 , Sami A. Gabr 3 , Saikh M. Wabaidur , Mohamed A. Habila and
Ahmed Yacine Badjah Hadj Ahmed 1
1
1
1
Chemistry Department, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia,
P.O. Box 2455, Riyadh 11451, Saudi Arabia; aghafr@ksu.edu.sa (A.A.G.); swabaidur@ksu.edu.sa (S.M.W.);
mhabila@ksu.edu.sa (M.A.H.); ybadjah@ksu.edu.sa (A.Y.B.H.A.)
Botany and Microbiology Department, College of Science, King Saud University, P.O. Box 2455,
2
Riyadh 11451, Saudi Arabia; abahkali@ksu.edu.sa (A.H.B.); motaibi@yahoo.com (M.S.A.-O.)
College of Applied Medical Sciences, King Saud University, Riyadh 11451, Saudi Arabia;
3
dr.samigabr@gmail.com
*
Correspondence: zaothman@ksu.edu.sa (Z.A.A.); elgorban@yahoo.com (A.M.E.);
Tel.: +966-5-0050-5570 (Z.A.A.); +966-5-3453-6548 (A.M.E.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 26 January 2020; Accepted: 13 March 2020; Published: 19 March 2020
Abstract: Nitroaromatic and nitroamine compounds such as 2,4,6-trinitrotoluene (TNT)
are teratogenic, cytotoxic, and may cause cellular mutations in humans, animals, plants,
and microorganisms. Microbial-based bioremediation technologies have been shown to offer several
advantages against the cellular toxicity of nitro-organic compounds. Thus, the current study was
designed to evaluate the ability of Trichoderma viride to degrade nitrogenous explosives, such as TNT,
by microbiological assay and Gas chromatography–mass spectrometry (GC–MS) analysis. In this study,
T. viride fungus was shown to have the ability to decompose, and TNT explosives were used at doses
of 50 and 100 ppm on the respective growth media as a nitrogenous source needed for normal growth.
The GC/MS analysis confirmed the biodegradable efficiency of TNT, whereas the initial retention
peak of the TNT compounds disappeared, and another two peaks appeared at the retention times of
9
.31 and 13.14 min. Mass spectrum analysis identified 5-(hydroxymethyl)-2-furancarboxaldehyde
−1
with the molecular formula C H O and a molecular weight of 126 g
and 4-propyl benzaldehyde with a formula of C H O and a molecular weight of 148 g mol as the
·
mol as the major compound,
6
6
3
−1
10
12
minor compound, both resulting from the biodegradation of TNT by T. viride. In conclusion, T. viride
could be used in microbial-based bioremediation technologies as a biological agent to eradicate the
toxicity of the TNT explosive. In addition, future molecular-based studies should be conducted to
clearly identify the enzymes and the corresponding genes that give T. viride the ability to degrade and
remediate TNT explosives. This could help in the eradication of soils contaminated with explosives
or other toxic biohazards.
Keywords: bioremediation; explosives; Trichoderma viride; GC/MS; microbial screening
1
. Introduction
Explosives are reactive chemical substances present in compounds or mixtures that contain
a great amount of potential energy that can produce an explosion if released suddenly. This is
usually accompanied by the production of light, heat, sound, and pressure. Nitro-aromatic and
nitramine compounds, such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine
(RDX), and octahydrol-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), are common military explosives
Molecules 2020, 25, 1393; doi:10.3390/molecules25061393
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1393
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that are found in soils at destruction ranges, explosive dumping grounds, industrial production sites,
firing ranges, and ammunition factories [ ]. The United States Environmental Protection Agency
USEPA) has listed Nitro-substituted explosives, including TNT and RDX, as priority pollutants,
1–3
(
whereas RDX, classified as a potential carcinogen that is toxic to organisms, is comparatively mobile
in the soil, has a low rate of degradation in soil, and presents distinct problems for bioremediation.
Environmental pollution comes from nitrite industrial chemicals associated with vehicles, such as nitro
explosives, dyes and polyurethane compounds, herbicides, pesticides, solvents, and others [4–7].
Many studies have discussed the synthesis of nitro-aromatic explosives such as 2,4,6-trinitrotoluene
(
TNT) and 2,4,6-trinitrophenol (picric acid) and their uses for military purposes because of their
highly explosive properties, thermal stability, and their insensitivity to shock and friction [ ].
In civilian industries, these compounds are used as raw materials for the manufacturing of
pesticides, herbicides, pharmaceutical products, dyes, and explosives [10 11]. Thus, the extensive
8,9
,
use of these explosives in military applications requires the implementation of extensive handling
and disposal techniques, whereas their transformation products lead to increased environmental
pollution—particularly in the soil, sediment, surface, and groundwater—to levels that threaten human
health and the environment [12–16]. In addition, animal experimental studies have reported that the
transformation products of TNT are teratogenic, cytotoxic, and may cause cell mutation; however, the
carcinogenic effects of TNT on humans still need to be explored [17,18]. This may be related to the
eco-toxicological effects and persistence of TNT and its transformation products in the environment,
which significantly affects a wide range of ecological receptors, particularly microorganisms, algae,
plants, invertebrates, some vertebrates, and humans [19
and bioremediation technologies [23 26] have been applied to remediate environments polluted by
TNT, only microbial-based bioremediation technologies offer a number of advantages [16 27]. It was
–21]. Although several physico-chemical [8,22]
–
,
reported that microbial-based bioremediation significantly uses the in situ microbial community to
remediate toxic contaminates and return the polluted environment to its original state or at least
minimize the toxicity of the environment toward the normal range [28–32].
Previous studies showed that many fungal species had the ability to decompose xenobiotic alien
vehicles, including nitrogenous explosives, via transformation mechanisms using certain cellular
degrading enzymes [30
bacteria and fungi to neutralize and sustain the effective transformation or degradation of nitroaromatic
pollutants [33 34]. Thus, fungi could be helpful as a biological control in the treatment of TNT
and Composition C4 explosives [35 38]. Basidiomycetes—lignin-decomposing organisms such as
–32]. In addition, several studies have previously reported the ability of
,
–
Phanerochaete chrysosporium—have been shown to be the only organisms capable of mineralizing
TNT [39,40].
Fungi of different species and habitats have been the subject of TNT biodegradation studies [41,42].
In comparison with other fungal species, Trichoderma species have shown a better ability to remove
TNT, DNT, and their transient intermediates (amines) in pure cultures. These results suggest that fungi
such as Trichoderma may have the potential to be used in biological decontamination systems, while
further research studies should be directed toward TNT and/or DNT biodegradation capacities of these
fungi [40,43]. Also, populations of soil micro fungi were shown to have a high natural variation of
TNT tolerance and biotransformation ability, irrespective of any previous long-term exposure to this
xenobiotic. The Trichoderma species was one of the mitosporic fungi that showed a higher tolerance
and also had a high capacity to biotransform the compounds of TNT [40,43,44].
Based on the aforementioned facts relating to the ability of microorganisms, particularly Trichoderma
species, to degrade and remediate toxicants and nitrogenous explosives [40–43,45], the current study was
designed to evaluate the ability of T. viride to degrade nitrogenous TNT explosives by microbiological
assays and GC/MS analysis. In this study, T. viride was raised on media containing TNT at doses of 50
and 100 ppm, respectively. The ability of T. viride to decompose TNT was evaluated in growth media
using GC-MS analysis.”

Molecules 2020, 25, 1393
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2
. Results and Discussion
In this study, T. viride fungus have the ability to decompose and use TNT explosives at doses of 50
and 100 ppm on its growth media as the nitrogenous source needed for normal growth. In addition,
the GC/MS analysis confirmed the biodegradable efficiency of TNT, whereas the initial retention peak
of TNT compounds disappeared, and another two peaks appeared at the retention times of 9.31 and
1
3.14 min.
2
.1. Screening of Fungal Growth on TNT-Containing Media
In order to measure the ability of the T. viride fungus to decompose and use TNT as a source for
the nitrogen needed during growth, T. viride colonies were cultivated on malt extract agar medium
and Sabouraud dextrose agar medium, as shown in Table 1 and Figures 1 and 2. The experiment was
repeated in triplicate, and the standard deviation (SD) was found to be below 2.1 for both concentrations.
Table 1. Presents the radial growth measurements of the studied fungi at 50 and 100 ppm
2
,4,6-trinitrotoluene (TNT) as the sole nitrogen source with malt agar medium.
After 4 Days
After 8 Days
After 11 Days
Fungi
Control
TNT ± SD
Control
TNT ± SD
Control
TNT ± SD
C1
80.0
C2
50 ppm
100 ppm
C1
85.0
C2
50 ppm
100 ppm
C1
85.0
C2
50 ppm
100 ppm
T. viride
85.0
35.0 ± 2.0
32.5 ± 1.8
85.0
85.0 ± 1.9
85.0 ± 1.3
85.0
85.0 ± 2.1
85.0 ± 2.0
The growth of T. viride expressed by diameter of the mean colony (mm). C1 = Control 1 (malt extract agar medium)
and C2 = Control 2 (sabouraud dextrose agar medium).
Figure 1. T.viride growth on malt agar suspended with TNT as a sole source of nitrogen at 50 ppm
(
A, C) and at 100 ppm (B, D). The growth of T.viride is indicated according to the size of the colony
in mm.

Molecules 2020, 25, 1393
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As shown in Table 1, Figure 1a–d, and Figure 2, at TNT doses of 50 and 100 ppm, the results show
that colony diameters significantly increase from 35.5 and 32.5 to record higher colony formation with
a colony diameter of 85.0 mm at 8 and 11 days of growth. This confirms the biological use of TNT as a
source of nitrogen, which is assimilated by T. viride on both the malt extract and Sabouraud dextrose
agar medium (Figure 2a,b). The data obtained suggests that the fungus T. viride is significantly capable
of decomposing TNT explosives, and uses them as a nitrogenous source for normal growth. It may be
of interest to use this fungus as a good model in microbial-based bioremediation technologies against
toxicological agents.
Figure 2. Differential growth of T. viride on dextrose agar (A) and malt agar media (B), treated with
TNT at doses of 50 and 100 ppm respectively.
Previous studies showed that 2,4,6-TNT is a man-made substance that is released into the
environment and represents a potential hazard due to toxicity, originating either from 2,4,6-TNT
substances, or its transformed metabolites during the manufacturing process, or in the process of
incomplete combustion [
higher persistence in the environment [
products of TNT are teratogenic, cytotoxic, and may cause cell mutations in animal experimental
models [17 18]. However, the carcinogenic effects of TNT on humans still need to be explored [17 18].
1]. These compounds have been shown to have a low biodegradability and
2,3]. In addition, it was reported that the transformation
,
,
This may be related to the eco-toxicological effects and persistence of TNT and its transformation
products in the environment, which significantly affect a wide range of ecological receptors, particularly
microorganisms, algae, plants, invertebrates, some vertebrates, and humans [19–21].
It was reported that microbial-based bioremediation significantly uses the in situ microbial
community to remediate toxic contaminates and return the polluted environment to its original state, or
at least minimize the toxicity of the environment toward the normal range [28
showed that bioremediation through transformation mechanisms has no or little benefit, and may lead
to transformation into chemically unstable toxic organic compounds [46 47]. However, many fungal
–32]. Previous research
,

Molecules 2020, 25, 1393
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species have the ability to decompose xenobiotic alien vehicles, including nitrogenous explosives, by
using certain cellular degrading enzymes [30–32].
2
.2. Determination of TNT and Metabolites by GC/MS Analysis
To further elucidate the biological activity of T. viride to decompose TNT, GC/MS analysis was
conducted to search the presence of TNT in growth media before and after growth at two doses
50 and 100 ppm). At the initial time of growth (zero time), GC/MS analysis showed a visible peak
of 2-methyl-1,3,5-trinitrobenzene (TNT) on the chromatogram at the retention time of 13.57 min
(
(
Figure 3). The structure was evaluated by mass spectroscopy, and the results show a significant
−1
molecular structure formula (C H N O ), with a molecular weight of 227 g
compounds are visible on the chromatogram except for some very small peaks, which are considered
as impurities (Figure 4).
·mol (Figure 4). No other
7
5
3
6
Figure 3. TNT sample determination by GC-MS. The chromatogram shows the peak of a TNT reference
dissolved in acetone (1 mL acetone containing 50 ppm or 100 ppm TNT).
Figure 4. This spectrum corresponds to 2-methyl-1,3,5-trinitrobenzene (TNT), which has the following
−1
formula: C H N O , and a molecular weight of 227 g·mol
.
7
5
3
6

Molecules 2020, 25, 1393
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When TNT was used at doses of 50 and 100 ppm in the growth media of T. viride, the analyzed media
solution following growth for 8–11 days, showed no detectable peaks for TNT on the chromatogram
chart (Figure 5). This indicates that all TNT compounds were degraded in the medium by the biological
decomposing action of T.viride at doses of 50 and 100 ppm.
Figure 5. Chromatogram for 50 ppm TNT with a T. viride culture.
Figure 6 shows new peaks of the analyzed media following the growth of T. viride. Two main
peaks can be observed: one peak appears at the retention time of 9.31 min, for a compound that was
identified as 5-(hydroxymethyl)-2-furancarboxaldehyde with the molecular formula C H O and a
6
6
3
−1
molecular weight of 126 g
media, the detected compound C H O was shown to be the major degradation product of TNT.
·
mol , as shown in Figure 7. As T.viride was grown on respective growth
6
6
3
However, another peak was observed by GC/MS analysis at the retention time of 13.14 min, which was
−
1
identified as 4-propyl benzaldehyde, with a formula of C H O and a molecular weight of 148 g
·
mol
,
10
12
as shown in Figure 8. The detected compound C H O is the minor degradation product of TNT
10
12
decomposition, obtained under the conditions required for the growth of the T. viride.
RT: 0.00 - 22.00
9
.31
NL:
9.19E8
1
00
TIC MS
Viridi100
9
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
13.14
1
3.58
1
3.93
14.86 15.78
17.19
1
1
2.80
7.82
1
8.59
19.54
1
2.35
20.60
1
1.66
1
0.99
4
.14 4.39
6.16 6.76 7.95 8.46
0
1
2
3
4
5
6
7
8
9
10
11
Time (min)
12
13
14
15
16
17
18
19
20
21
Figure 6. Chromatogram of 100 ppm TNT after treatment with T. viride.

Molecules 2020, 25, 1393
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Viridi100 #1050 RT: 9.34 AV: 1 NL: 2.65E7
T: + c EI Q1MS [30.000-1000.000]
4
1
1
00
9
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
3
9
31
9
7
69
3
8
5
1
5
0
3
7
4
2
1
26
4
9
6
66
8
81
91
5
5
98
125
81
109
8
6
119
127
147
1
57 165 176
160 180
m/z
189
207
223
220
251
4
0
60
80
100
120
140
200
240
260
280
300
Figure 7. Mass spectrum of main peak, 5-(hydroxymethyl)-2-furancarboxaldehyde.
Viridi100 #1791-1802 RT: 13.10-13.16 AV: 12 SB: 83 12.71-12.90 , 13.43-13.65 NL: 1.32E7
T: + c EI Q1MS [30.000-1000.000]
9
1
1
00
9
9
8
8
7
7
6
6
5
5
4
4
3
3
2
2
1
1
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
5
0
6
5
1
19
148
39
41 45
51
6
9
77
147
7
3
92
103
6
3
89
3
1
52 58 61
79
36
50
50
100
120
146 149
145
66
107
117
7
0
84
128 131
130
9
3
109
135
156 163 165
173 178
160 170 180
3
0
40
60
70
80
90
100
110
120
140
150
m/z
Figure 8. Mass spectrum of the minor component, 4-propyl benzaldehyde.
In this study, as shown in Figures 5–8, TNT was degraded by T. viride to other new
compounds, as identified by the GC/MS analysis. The results showed that the initial peak
corresponding to TNT compounds disappeared, and another two peaks appeared, which represent
new compounds at the retention times of 9.31 and 13.14 min. Mass spectrum analysis identified
5
-(hydroxymethyl)-2-furancarboxaldehyde with the molecular formula C H O and molecular weight
6 6 3
−1
of 126 g
·
mol as the major compound, and 4-propyl benzaldehyde with the formula of C H O and a
10
12
−1
molecular weight of 148 g mol as a minor compound, both resulting from the biodegradation of TNT
·
following the growth process of T. viride for 8–11 days. The formation of these possible degradation
products was confirmed by matching their mass spectra with the library, which showed a probability
of 90% for both products. In addition, in the chromatogram after TNT degradation, no peaks other
than these two degradation compounds were noticed. This may confirm the degradation of TNT
compound via cellular enzymatic action.
Even though TNT microbial degradation was studied over the years, the major challenge in this
area is the resistance and refraction of these types of compounds to biological degradation, chemical

Molecules 2020, 25, 1393
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oxidation, and hydrolysis. The resistance shown towards the biodegradation methods could be due to
the symmetrical arrangement of three nitro-groups and a methyl group on the aromatic ring, coupled
with strong electron-withdrawing properties of the nitro group, which limits the attack of the aryl
group by dioxygenase enzymes. These structural arrangements prevent the aromatic ring with an
electron shortage from acting as an electrophilic oxygenation mechanism, hindering its mineralization
and removal from the contaminated sites [7,8,12,22,48]. In addition, the resistance of TNT to complete
mineralization is also due to the easy reduction of nitro groups into amino groups, and the ultimate
chemical misrouting reactions of its intermediates, in particular, triaminotoluene (TAT) [49,50].
Furthermore, it was reported that the non-mineralization of TNT is a direct consequence of
irreversible sorption of this explosive and its transformation products by soil [33]. In our study, the
degradation of TNT compounds by T. viride may proceed via biotransformation mechanisms that
facilitate the elimination of nitrogen and recyclization of the cleaved compounds. Thus, the GC/MS
analysis supports and confirms that T.viride can use TNT as a sole source of nitrogen and has a
cellular enzymatic degradable activity to bioremediate TNT compounds to other compounds, which
are present in the growth media in major (C H O ) and minor (C H O) amounts, following the
6
6
3
10 12
growth process for 8–11 days, as shown in Scheme 1. Previous studies showed that the resistance of
TNT mineralization could be resolved by the activation of lignin peroxidase through the activation
of the nitroreductase enzymes hydroxylaminodinitrotoluenes (HADNT) and aminodinitrotoluenes
(ADNT) [51–61]. The latter enzymes are responsible for nitro group reduction, while the former
catalyzes oxidation and aromatic ring cleavage [51–53].
O
O
N+
N+
-O
O-
N+
-O
O
2-methyl-1,3,5-trinitro-benzene
Trichoderma viride
HO
O
O
O
4-propyl benzaldehyde
5
-(hydroxymethyl)-2-furancarboxaldehyde
Scheme 1. Postulated degradation of the 2-methyl-1,3,5-trinitrobenzene mechanism of T. viride. It is
postulated that TNT biodegradation may occur via biotransformation enzyme mechanisms.

Molecules 2020, 25, 1393
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Finally, microorganisms can be considered a biological tool for removing organic toxic substances
because they are able to carry out biological activities to degrade, concentrate, remove, or even recover
highly toxic chemical substances from contaminated environments [54]. In our study, T. viride was
shown to have a potential biological activity to degrade TNT organic explosives. We postulate that
TNT biodegradation may occur via biotransformation using certain degrading microbial enzymes.
Thus, T. viride could be used as a target agent for microbial-based bioremediation technologies.
Microorganisms have been used as an alternative strategy for conventional treatments against toxic
organic substances and heavy metals [54–57].
3
3
3
. Material and Methods
.1. Chemicals and Biological Materials
.1.1. Chemicals and Preparations
TNT was obtained from the Criminal Investigation Department at the Ministry of Interior, Riyadh,
Saudi Arabia. The purity of TNT was 95%. TNT/acetone stock solution was prepared by dissolving
−
1
4
8 mg of TNT in 50 mL of acetone to give a concentration of 960 ppm (mg L ) of TNT. Malt agar
·
and dextrose agar media were autoclaved for fifteen minutes, then allowed to cool and poured into
Petri plates prior to the addition of TNT, and the whole plates were then ready for the screening of
fungal growth.
3
.1.2. T. viride Specimens
Soil specimens were collected from an explosion area in Riyadh, Saudi Arabia for the isolation of
T. viride. A serial dilution of each sample was prepared in sterilized distilled water. One milliliter of a
diluted specimen was speared on the surface of T. viride selective medium (TSM) [
8
,
58]. All plates were
◦
incubated at 25
±
2 C for 3 days. Colonies were purified in potato dextrose agar (PDA). The purified
◦
isolates were morphologically identified and stored at 4 C and used during this study.
3
.2. Screening of Fungal Growth on TNT Containing Media
Control cultures were screened on both malt agar medium and dextrose agar medium, which
are designed to contain the proper formulation of carbon, protein, and nutrient sources essential
for growth. Dextrose was added to the medium to provide a carbon and energy source for fungi.
Additionally, malt extract agar contains digests of animal tissues (peptones), which provide a nutritious
source of amino acids and nitrogenous compounds for the growth of fungi. In addition, acetone and
Dimethyl Sulfoxide (DMSO) but no TNT were added to culture media (acetone and DMSO did not
inhibit fungal growth).
In order to study the use of TNT as a source of nitrogenous compounds, the growth of T. viride
was screened on both malt agar and dextrose agar growth media devoid of animal tissue (peptones),
which provide a nutritious source of amino acids and nitrogenous compounds. The cultures were
◦
incubated in a shaker at 24 C and 200 rpm. After two days of growth, each received 1 mL of acetone
containing 50 or 100 ppm TNT and was amended with 25 µL of DMSO to help solubilize the TNT.
Cultures were extracted three days after the addition of the TNT. TNT was added to the culture media
either for three days before, or immediately before, extraction. To determine growth, replicate cultures
were filtered, dried, and weighed.
3
.3. Determination of TNT and Metabolites by GC/MS Analysis
Cultures of T. viride were homogenized in an explosion-proof blender with 50 mL of acetone.
This was left to stand for 15 min, followed by filtration through filter paper [58,59]. Filtrates were
extracted in a separating funnel with 100 mL of methylene chloride. Organic fractions evaporated
◦
overnight and were re-suspended in 5 mL of methylene chloride, and stored at
−
20 C. At least four

Molecules 2020, 25, 1393
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replicate cultures per fungus were analyzed. The extraction method used was designed to recover
precipitated TNT, since TNT was added at 50 and 100 ppm [59].
A Thermo Scientific
Quantum Triple Quadrupole Mass Spectrometer (Waltham, MA, USA), equipped with a Phenomenex
Zebron ZB-5MS (5 m 0.25 mm i.d. 0.25 m film thickness or equivalent) column (411 Madrid
™ UltraFast TRACE GC with a Thermo electron-impact ionization (EI) TSQ
™
×
×
µ
Avenue, Torrance, CA, USA), was used in this protocol to separate and quantify TNT and its degradation
products in the concentrated organic fractions. Organic culture extracts were analyzed by GC–MS, as
previously reported [60,61], at different dilutions (10, 25, 50, 100, 250, 500, 1000, and 2000 ng/mL).
4
. Conclusions
In this study, T. viride fungus showed a high natural variation in TNT tolerance, biodegradation,
and biotransformation ability, and was able to use TNT explosives at doses of 50 and 100 ppm
as a nitrogenous source for normal growth. In addition, the biodegradable efficiency of TNT
explosives by T.viride was confirmed by using GC/MS analysis. Mass spectrum analysis identified
5
-(hydroxymethyl)-2-furancarboxaldehyde with the molecular formula C H O and a molecular
6 6 3
−1
weight of 126 g
·
mol (m/z) as the major compound, and 4-propyl benzaldehyde with a formula of
−1
C H O and a molecular weight of 148 g mol (m/z) as the minor compound, both resulting from the
biodegradation of TNT following the growth process of T. viride.
·
10
12
Future molecular-based studies should be conducted to clearly identify the enzymes and
corresponding genes responsible for the ability of T. viride to degrade and remediate TNT explosives.
This could help in the eradication of soils contaminated with explosives or other toxic biohazards.
Author Contributions: Conceptualization, Z.A.A. and A.H.B.; methodology, A.A.G. and S.A.G.; software,
A.A.G.; validation, A.M.E., M.S.A.-O. and S.M.W.; formal analysis, A.A.G., S.A.G., and M.A.H.; investigation,
M.A.H., A.Y.B.H.A.; resources, A.A.G., S.A.G., and A.Y.B.H.A.; data curation, A.A.G., M.A.H., and A.Y.B.H.A.;
writing—original draft preparation, S.A.G. and A.A.G.; writing—review and editing, S.A.G.; visualization, S.A.G.
and A.A.G.; supervision, Z.A.A. and A.H.B.; project administration, Z.A.A. and A.H.B.; funding acquisition,
Z.A.A. All authors have read and agreed to the published version of the manuscript.
Funding: This project was funded by the National Plan for Science, Technology and Innovation (MAARIFAH),
King Abdul Aziz City for Science and Technology, Kingdom of Saudi Arabia, Award number (NAN-1005-02).
Acknowledgments: The authors are grateful to King Abdul Aziz City for Science and Technology, Kingdom
of Saudi Arabia for funding this project through the National Plan for Science, Technology and Innovation
(MAARIFAH).
Conflicts of Interest: The authors declare that they have no competing interests.
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Sample Availability: Samples of the compounds in this study are available from the corresponding author
on request.
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