applied

sciences
Article
Assessment of Different Spent Mushroom Substrates
to Bioremediate Soils Contaminated with
Petroleum Hydrocarbons
Rafael Antón-Herrero 1 , Carlos García-Delgado 2, * , Natalia Baena 1 , Begoña Mayans 1 ,
Laura Delgado-Moreno 1 and Enrique Eymar 1

1 Department of Agricultural Chemistry and Food Sciences, Universidad Autónoma de Madrid,

28049 Madrid, Spain; [email protected] (R.A.-H.); [email protected] (N.B.);
[email protected] (B.M.); [email protected] (L.D.-M.); [email protected] (E.E.)
2 Department of Geology and Geochemistry, Universidad Autónoma de Madrid, 28049 Madrid, Spain
* Correspondence: [email protected]

Abstract: Bioremediation techniques are being developed as substitutes for physical–chemical

methodologies that are expensive and not sustainable. For example, using the agricultural waste
spent mushroom substrate (SMS) which contains valuable microbiota for soil bioremediation. In this
work, SMSs of four cultivated fungal species, Pleurotus eryngii, Lentinula edodes, Pleurotus ostreatus,
and Agaricus bisporus were evaluated for the bioremediation of soils contaminated by petroleum
hydrocarbons (TPHs). The bioremediation test was carried out by mixing the four different SMSs
with the TPH-contaminated soil in comparison with an unamended soil control to assess its natural
attenuation. To determine the most efficient bioremediation strategy, hydrolase, dehydrogenase, and
Citation: Antón-Herrero, R.;
ligninolytic activities, ergosterol content, and percentage of TPHs degradation (total and by chains)
García-Delgado, C.; Baena, N.;
were determined at the end of the assay at 40 days. The application of SMS significantly improved
Mayans, B.; Delgado-Moreno, L.;
Eymar, E. Assessment of Different
the degradation of TPHs with respect to the control. The most effective spent mushroom substrate to
Spent Mushroom Substrates to degrade TPHs was A. bisporus, followed by L. edodes and P. ostreatus. Similar results were obtained for
Bioremediate Soils Contaminated the removal of aliphatic and aromatic hydrocarbons. The results showed the effectiveness of SMS to
with Petroleum Hydrocarbons. Appl. remove aliphatic and aromatic hydrocarbons from C10 to C35 . This work demonstrates an alternative
Sci. 2022, 12, 7720. https://doi.org/ to valorizing an abundant agricultural waste as SMS to bioremediate contaminated soils.
10.3390/app12157720
Keywords: bioremediation; fungi; soil microbiota; spent mushroom substrate; petroleum hydrocarbons
Academic Editors: José
A. González-Pérez and
Fulvia Chiampo

Received: 30 June 2022 1. Introduction

Accepted: 28 July 2022
Currently, the contamination of soil, water or air by toxic substances, from natural
Published: 31 July 2022
or anthropogenic sources, is considered one of the major environmental problems [1].
Publisher’s Note: MDPI stays neutral Anthropogenic contamination of soils is due to discharges, pesticide use, leaks, extractions,
with regard to jurisdictional claims in and other emissions [2,3]. Among anthropogenic soil contaminants, hydrocarbons represent
published maps and institutional affil- a major environmental threat because of their toxicity, bioaccumulation and persistence. In
iations. addition, their hydrophobicity renders them preferably adsorb to soil organic matter [4],
which makes their removal from contaminated sites more difficult.
Crude oils are extremely complex mixtures, made up mainly of hydrocarbons and to a
lesser extent by metals or nitrogenous, oxygenated, or sulfur derivatives [4]. Petroleum
Copyright: © 2022 by the authors.
hydrocarbons are formed mainly by carbon and hydrogen atoms and are subdivided into:
Licensee MDPI, Basel, Switzerland.
This article is an open access article
saturated hydrocarbons made up of aliphatic carbon chains and aromatic hydrocarbons
distributed under the terms and
made up of one or more benzene rings. In soil, total petroleum hydrocarbons (TPHs)
conditions of the Creative Commons alter the physical–chemical and biological properties of the soil, such as hydrophobicity,
Attribution (CC BY) license (https:// cation exchange capacity, and electrical conductivity, preventing gas exchange with the
creativecommons.org/licenses/by/ atmosphere or modifying the autochthonous microbiota. In addition, they have toxic
4.0/). properties causing serious effects on human and environmental health [4].

Appl. Sci. 2022, 12, 7720. https://doi.org/10.3390/app12157720 https://www.mdpi.com/journal/applsci

Appl. Sci. 2022, 12, 7720 2 of 15

The most frequent techniques for the recovery of contaminated soils with hydrocar-
bons are the extraction with solvents, washing with surfactants and thermal treatments.
The first method consists of the application of an organic solvent for removing the con-
taminants. The washing with surfactants uses a washing solution of water and chemical
additives. The contaminants are retained in this solution, whether dissolved or suspended.
Finally, thermal treatments consist in applying different temperatures ranging from 90 ◦ C
to 600 ◦ C, with the aim of volatilizing or decomposing organic contaminants [5]. All of
them are ex-situ techniques that are expensive and consume a lot of energy. Although the
resulting soil is clean, it remains biologically and environmentally damaged [6].
Bioremediation represents a less expensive and environmentally friendly alternative
to the physico-chemical techniques previously mentioned. Bioremediation consists of the
recovery of contaminated soil by the action of living organisms [7]. Enhanced contaminants
biodegradation might be achieved by managing soil temperature, and moisture, and
the abundance of nutrients (carbon, nitrogen, and phosphorus) among others factors [8].
There are two main strategies to bioremediate contaminants from soils, biostimulation and
bioaugmentation [9]. The purpose of biostimulation is to favor the metabolism of the native
microbiota of the soil to degrade contaminants by the addition of nutrients, increasing the
level of oxygen and regulating the temperature and soil moisture. In bioaugmentation,
specialized exogenous microorganisms, such as fungi or bacteria, are inoculated into the
soil for increasing contaminants biodegradation [8].
Bioremediation is a promising technique for the amelioration of soils contaminated
with TPHs. However, for the heavier hydrocarbons, the efficiency of this technique is
limited due to their low bioavailability, which makes bacterial degradation difficult [10],
since these organisms have to absorb the contaminant to degrade it intracellularly. To solve
this problem, a new procedure known as mycoremediation has been proposed. In this
approach, fungi are applied to soil for enhancing contaminants removal [5]. Fungi are very
interesting organisms to decontaminate soil because their multicellular fungal mycelium
can reach vast volumes of soil. Compared to bacteria, fungi are also more resistant to
high concentrations of toxins and can produce many useful bioactive compounds, such as
extracellular enzymes, among other advantages [11]. Fungi can degrade a wide variety of
organic contaminants thanks to their non-specific metabolism [11,12]. This degradation
occurs extracellularly through enzymes such as laccase, versatile peroxidase and manganese
peroxidase (MnP) for ligninolytic fungi and intracellularly [13] or by the cytochrome
P450 intracellular enzyme system [14]. In both cases, more polar substances are obtained
because of the degradation of these toxic compounds. The use of filamentous fungi in this
procedure is essential due to the fact that the networks formed by hyphae and mycelium
favor soil colonization and the establishment of a synergic relation between fungi and
bacteria [15,16]. The great disadvantage of this technique is the time it takes for the
degradation of the contaminants, which is greater when compared to the physico-chemical
techniques mentioned above. In addition, it must be considered that the colonization of the
fungus could be limited or impaired due to the interaction with the microbial population
of the soil or even by high concentrations of contaminants [17].
Currently, mushroom cultivation is carried out worldwide. After mushroom produc-
tion, the substrate plus the cultivated mycelium generates the spent mushroom substrate
(SMS) waste. Every 1 kg of fresh mushroom generates between 2.5 and 5 kg of fresh
SMS [18]. The disposal of these residues is an environmental problem, being the most
frequent those from the crop of Agaricus bisporus and Pleurotus spp. [19]. Some alternatives
to disposal are described or implemented, for example: [20] amendment and fertilizer for
agricultural soils, plant biostimulant, as animal feed or in aquaculture, in pest and disease
management, as energy feedstock (alternative fuel, production of biogas), in vermiculture
or as a component of casing material or ingredient of new growth substrates for further
mushroom growing cycles [18,20–22]. All these possibilities promote the circular economy
concept and are in line with the Green Deal of the European Union in contrast to the
traditional disposal of SMS [19]. SMS wastes are based on lignocellulosic materials [23]
Appl. Sci. 2022, 12, 7720 3 of 15

and contain viable mycelium and specific microbiota able to colonize polluted soils and
degrade a wide variety of soil contaminants [24–26]. The re-use of SMSs in environmental
remediation is a promising way to covert this agricultural waste into a sustainable biore-
source promoting the green development of the global mushroom industry [20]. Different
SMSs were used to adsorb organic and inorganic contaminants or to degrade organic con-
taminants from water [21,23,27]. The composted SMS is an effective adsorbent to minimize
the availability of metals in soil and enhanced the phytoremediation of mining soil [28],
reduced the leaching of pesticides from soil [29] and minimized the impact of herbicides
in soil microbial community [30]. The ability of SMS to remove a wide variety of organic
contaminants from soils was reported in different publications [20,25,31]. Hence, we hy-
pothesize that the use of SMS to remediate polluted soils with petroleum hydrocarbons is a
valuable strategy to re-use this agricultural waste.
The main objective of this work is to assess the capability of four SMS, Pleurotus eryngii
(king mushroom), Lentinula edodes (shiitake), Pleurotus ostreatus (oyster mushroom) and
Agaricus bisporus (champignon) to enhance biodegradation of petroleum hydrocarbons
from a soil highly contaminated with crude oil.

2. Materials and Methods

2.1. Spent Mushroom Substrates
SMSs were collected from the Technological Center for Mushroom Research in La
Rioja (CTICH, La Rioja, Spain). In its composition there are remains of the mycelia of the
cultivated fungi and of the constituent components of their growth substrates; for oyster
mushroom (P. eryngii), hardwood sawdust, seed hulls and wheat bran; for shiitake (L. edodes)
steam pasteurized straw, sawdust; in the case of the oyster mushroom (P. ostreatus) wheat
straw and finally for the mushroom (A. bisporus), the substrate was based on composted
wheat straw and chicken manure.

2.2. Contaminated Soil

The soil was obtained from a collection of contaminated soils from accidental oil spills
in a refinery located in Huelva, where they are stored until their final management by
authorized companies. According to an analysis carried out in a certified laboratory, the
soil had a concentration of 12,000 ± 1414 mg/kg of total petroleum hydrocarbons (TPH).
These values exceed the reference values by more than 100 times according to Spanish
legislation [32]. This soil can be considered directly as contaminated soil without the need
to carry out a risk assessment.
The soil basic parameters determined were: %sand, %loam, % clay, maximum water
retention capacity (MWHC), pH (water and 1 M KCl), electrical conductivity, oxidizable
organic matter, total carbonates, available phosphorus, and trace elements [33].
The percentage of sand, silt and clay contained in the soil sample was determined by
the Bouyoucos method. The pH of the soil was determined in water and KCl 1M extract
1:2.5 (w:v) by pHmeter. Electrical conductivity was determined in extract 1:5 (p:v) using
a conductivity meter. Oxidizable organic matter was determined by the Walkley–Black
method using K2 Cr2 O7 . CaCO3 content was analyzed by calcimetry. Available phospho-
rous was extracted with NaCO3 (0.25 M, pH 8.5) and analyzed spectrophotometrically at
660 nm using the Duval reagent. Trace elements were extracted by pseudo-total digestion
assisted by microwaves oven with HCl:HNO3 and analyzed by ICP-MS [33].

2.3. Mycoremediation Assay

Untreated soil (control) was compared against four bioaugmentation treatments by
adding SMS of P. eryngii, L. edodes, P. ostreatus and A. bisporus. For this, 500 g of polluted
soil were mixed with 50 g of each SMS in closed glass containers of 1 L. The control and the
treatments were performed in quadruplicate and were kept in the dark for 40 days at 20 ◦ C
and 70% MWHC. During this period, the glass containers were opened once per week to
oxygenate them and to add water, when needed to keep the soil moisture. After incubation,
Appl. Sci. 2022, 12, 7720 4 of 15

sampling, processing and analysis of hydrolase and dehydrogenase activities, ergosterol

content, ligninolytic enzymatic activities total TPHs and its aliphatic and aromatic fractions
were carried out.
To determine the soil microbial activity, two soil enzyme activities were determined,
dehydrogenase and total hydrolase activity. Soil dehydrogenase activity determination
was performed by reducing the triphenyltetrazolium chloride to 1,3,5-triphenylformazan.
To this, 1000 g of soil was incubated with 1 mL of 0.1 mM Tris-HCl buffer at pH 7.6
with 1.5% triphenyltetrazolium chloride (TCC) for 24 h at 30 ◦ C. After this time, the
reaction was stopped and the 1,3,5-triphenylformazan produced was extracted with acetone,
centrifuged and measured in the spectrophotometer at 546 nm (ε = 15.4 mM−1 cm−1 ) [34].
To determine the total hydrolase activity, the fluorescein produced from the hydrolysis
of fluorescein diacetate was measured [35]. The soil sample (1 g) was incubated with
60 mM KH2 PO4 /K2 HPO4 buffer at pH 7.6 and fluorescein diacetate for 20 min at 30 ◦ C.
After that time, acetone was added to stop the reaction and then samples were centrifuged
at 5000 rpm for 5 min after a manual stirring. The absorbance of the supernatant was
measured at 490 nm in the spectrophotometer (ε = 80.3 M−1 cm−1 ).
Ergosterol is a membrane lipid found in fungi that allows for determining the fungal
biomass present in the soil [36]. A suspension of 0.5 g of milled soil sample was extracted
with 3 mL of KOH 10% w/v in methanol and 1 mL of hexane. This is sonicated for 90 min
in a hot water bath at 70 ◦ C. Subsequently, the extract was filtered, and the soil was washed
with hexane. The purification of the extract was carried out by liquid-liquid extraction in a
separating funnel. A total of 1 mL of H2 O was added to the methanol:hexane extract and
extracted three times with 2 mL of hexane. The hexane phases were combined and dried
over a stream of nitrogen. Subsequently, the solid residue was redissolved in methanol [37].
The extracts were analyzed by high-performance liquid chromatography coupled to a
photodiode array detector (HPLC-PDA). Chromatographic separation of ergosterol was
achieved with a Phenomenex Luna C18 (2) (150 × 4.6 mm; size of 5 µm particle; 100 Å pore
size) column using an isocratic elution program with methanol:water (95:5) at a flow rate of
1 mL min−1 . The column temperature was set at 30 ◦ C. The elution profile was monitored
at 282 nm. Peak identification of each sample was based on retention time (15.99 min) and
UV spectrum (200–400 nm) of commercially available standard (Thermo Fisher Scientific,
Waltham, MA, USA).
Before the ligninolytic enzyme determination, an extraction of 3000 g of the soil
sample was carried out with 30 mL of extracting solution composed of 2.94 g/L CaCl2 H2 O,
4.10 g/L of sodium acetate, 2.86 mL/L of glacial acetic acid and 0.50 mL/L of Tween 80.
The mixture was stirred at 4 ◦ C for 60 min, centrifuged for 15 min at 5000 rpm and finally,
the supernatant was collected [7]. To analyze the laccase enzymatic activity, the oxidation of
2,6-dimethoxiphenol (DMP) was monitored at 477 nm in the spectrophotometer in kinetic
mode for 2 min [38]. Versatile Peroxidase was determined by oxidation of ABTS at 420 nm
for 10 min in presence of H2 O2 [39]. To analyze the manganese peroxidase (MnP) activity,
the production of the malonate-Mn3+ complex was measured at 270 nm [7].
The analysis of TPHs was performed by GC-FID after microwave extraction. The
contaminated soil samples were firstly air dried and then sieved at 2 mm to eliminate the
SMS, and secondly sieved again after its grinding at 0.02 mm to homogenize them. The
extraction solvent, 20 mL of acetone and n-hexane 1:1 (v/v) was added to 1 g of dried soil
sample. The mixture was subjected to microwave extraction (Ethos Sel, Milestone, Sorisole,
Italy). The extraction program consisted of 20 min at 150 ◦ C and 1000 W. The extract was
filtered through a polytetraflourethylene membrane with a pore size of 20 µm. The filtered
extracts were then evaporated with nitrogen to a volume of 1 mL. The reduced extracts were
fractionated following a clean-up procedure involving Isolute EPH cartridges (25 mL/5 g).
The cartridges were conditioned prior to sample loading with 30 mL of hexane. After
loading the sample, the elution was performed using 12 mL of hexane, and then 20 mL of
dichloromethane, both at a flow rate of 2–3 mL min−1 . The cleaned extract was dried by
N2 flow to 1 mL and injected into gas chromatography (Agilent 7820A, Santa Clara, CA,
Appl. Sci. 2022, 12, 7720 5 of 15

USA) coupled to a flame ionization detector (FID Agilent G4513A, Santa Clara, CA, USA)
equipped with an autosampler with an HP5-MS and capillary column 30 m × 0.32 mm
i.d.; nominal film thickness 0.25 mm. Split less injection was used with a deactivated
inlet liner (4 mm i.d.) with glass wool and a single taper. The injection temperature was
250 ◦ C, and the injection volume was 3 µL. Helium was the carrier gas (74 kPa). The oven
operating conditions were: initial temperature of 80 ◦ C, increasing to 200 ◦ C at a rate of
7 ◦ C min−1 , then increasing to 300 ◦ C for 17 min at a rate of 11 ◦ C min−1 . FID was operated
at 325 ◦ C and 20 Hz. The aliphatic and aromatic fractions detected are defined based on
their equivalent carbon [40].

2.4. Statistical Analysis

The statistical test was carried out using the IBM SPSS statistical software package
v26 (Armonk, NY, USA). The analysis of variance was performed after a Levene variance
homogeneity test. To compare the differences between treatments, the Duncan test was
performed for samples that present homogeneity of variances and the Games–Howell test
for those that present heterogeneity with p < 0.05 for both cases. The principal component
analysis (PCA) was performed to determine the relationship between the degradation of
aliphatic and aromatic fractions of TPHs and the bioaugmentation treatments. This test
was performed using the PAST V. 4.02 software (Natural History Museum, University of
Oslo, Oslo, Norway).

3. Results
3.1. Soil Characterization
The studied soil had a sandy texture, a basic pH, high concentration of organic matter
due to the nature of the contamination, low electrical conductivity and low concentration
of nutrients (Table 1). The nutrients are unbalanced with C:N:P of 100:1.5:0.08 far from
the optimal 100:10:1 [4]. The results obtained for trace elements were below the limits
established by the Spanish Legislation [32].

Table 1. Soil basic characterization (mean ± standard deviation) n = 3.

MHWC (%) 13 ± 4
pH (H2 O) 8.66 ± 0.05
pH (KCl) 7.42 ± 0.04
Electric Conductivity (dS/cm) 0.7 ± 0.2
Total Limestone n.d.
Sand (%) 91.20 ± 0.01
Silt (%) 2.5 ± 0.3
Clay (%) 6.3 ± 0.3
Texture Sandy
Organic Matter (g/kg) 23 ± 4
Assimilable Phosphorus (g/kg) 0.011 ± 0.001
Nitrogen (g/kg) 0.2 ± 0.0
Trace elements (mg/kg) Legislated values (mg/kg) *
Sb 4±2 80
As 16 ± 4 40
Cd 0.3 ± 0.0 300
Cu 315 ± 92 8000
Co 4.0 ± 0.2 1500
Cr n.d. 2300
Sn 6.75 ± 0.07 100,000
Hg 0.285 ± 0.007 15
Mo 8±1 1500
Ni 8.0 ± 0.9 15,600
Pb 80.5 ± 22 2700
Zn 290 ± 42 100,000
n.d.: not detected. MWHC: maximum water holding capacity * According to Spanish Legislation for industrial soils.
 Hg 0.285 ± 0.007 15
Mo 8±1 1500
Ni 8.0 ± 0.9 15,600
Pb 80.5 ± 22 2700
Appl. Sci. 2022, 12, 7720
Zn 290 ± 42 100,000 6 of 15
n.d.: not detected. MWHC: maximum water holding capacity * According to Spanish Legislation for
industrial soils.

3.2.
3.2.Mycoremediation
MycoremediationAssay
Assay
Figure
Figure1 1shows
showsthethevisual
visualaspect
aspectofofthe
thefour
fourmicrocosms
microcosmsinoculated
inoculatedwith
withthethedifferent
different
SMSs after 40 days of incubation. The microcosms inoculated with SMS of
SMSs after 40 days of incubation. The microcosms inoculated with SMS of P. eryngii P. eryngii A.
andand
bisporus showed extensive colonization of the contaminated soil. The fungi P. ostreatus
A. bisporus showed extensive colonization of the contaminated soil. The fungi P. ostreatus and
L.and
edodes showed
L. edodes lowerlower
showed colonization than the
colonization thanprevious ones. ones.
the previous In theIncase
the of P. ostreatus
case the
of P. ostreatus
colonization was not clearly observed until the 30th day.
the colonization was not clearly observed until the 30th day.

SMS P. Eryngii SMS L. Edodes SMS P. Ostreatus SMS A. Bisporus

Figure1.1.Visual
Figure Visualstate
stateofofthe
thebioaugmented
bioaugmentedmicrocosms
microcosmswith
withfour
fourdifferent
differentSMS
SMS(P.
(P.eryngii,
eryngii,L.L.edodes,
edodes,
P.P. ostreatus
ostreatus and
and A.A. bisporus)
bisporus) after
after 4040 days
days of of incubation.
incubation.

Meanvalues
Mean values ofof hydrolase
hydrolase and
and dehydrogenase
dehydrogenase activities
activities werewere higher
higher for for
the the A. bispo-
A. bisporus
rus microcosm
microcosm than than forcontrol.
for the the control. The other
The other treatments
treatments showed showed
higherhigher enzymatic
enzymatic activ-
activities
itiesthe
than than the control
control but werebutnot
were not statistically
statistically different
different due to due to thedeviations
the high high deviations
between between
repli-
replicates
cates (Table(Table 2). Hydrolase
2). Hydrolase and dehydrogenase
and dehydrogenase activities
activities were 17 were
and17 and 4 times,
4 times, respec-
respectively,
higher
tively,for A. bisporus
higher for A.than for the
bisporus control,
than denoting
for the control,the high metabolic
denoting the highactivity in A.activity
metabolic bisporusin
treatment
A. bisporuswith respect to
treatment control.
with respectThetomean values
control. Theofmean
ergosterol
valuesdidof not presentdid
ergosterol significant
not pre-
differences between
sent significant bioaugmented
differences between treatments (Tabletreatments
bioaugmented 2). However, the increment
(Table 2). However, of ergos-
the in-
terol content
crement in those treatments
of ergosterol content inwith
thoserespect to thewith
treatments control indicates
respect to thethe soil colonization
control indicates the
of the four fungi from their respective SMS. The ligninolytic activity of the five microcosms
was determined by three different activities: laccase, versatile peroxidase and MnP. Laccase
was proposed as a bioindicator of the mycelial growth of A. bisporus [41]. This enzyme only
was detected in A. bisporus and P. eryngii treatments, denoting the extensive colonization
of the soil and their metabolic activity. The laccase activity of A. bisporus microcosms was
significantly higher than the laccase activity of P. eringii microcosms. Peroxidase activities
were detected in control and all the bioaugmented microcosms irrespective of the SMS
inoculated. However, no significant differences were detected for versatile peroxidase
and MnP activities between treatments. The detection of peroxidase activity suggests the
metabolic activity of the inoculated fungi and the inherent microbiota of soil.
Appl. Sci. 2022, 12, 7720 7 of 15

Table 2. Enzymatic activities (total hydrolase, dehydrogenase, laccase, versatile peroxidase, Mn-
peroxidase) and ergosterol content of the control microcosms (C), and microcosms inoculated with
SMS of P. eryngii (Pe), L. edodes (Le), P. ostreatus (Po) and A. bisporus (Ab) at the end of the incubation.
Data are presented as mean values ± standard deviation (n = 3). Same letters indicate lack of
statistically significant difference (p < 0.05) between treatments. n.d.: parameter not detected.

Versatile
Hydrolase Dehydrogenase Ergosterol Mn-Peroxidase
Lacasse (U/kg) Peroxidase
(µmol·h−1 ·g−1 ) (nmol·h−1 ·g−1 ) (mg/kg) (U/kg)
(U/kg)
C 22.5 ± 0.7 b 0.80 ± 0.08 b n.d. n.d. 70 ± 14 a 4±4a
Pe 71 ± 44 b 2 ± 1 ab 1.0 ± 0.5 a 10 ± 7 b 99 ± 30 a 17 ± 12 a
Le 76 ± 56 b 1.0 ± 0.4 b 0.7 ± 0.5 a n.d. 63 ± 50 a 9±8a
Po 26 ± 7 b 1.3 ± 0.5 ab 0.6 ± 0.7 a n.d. 74 ± 57 a 10 ± 10 a
Ab 378 ± 243 a 3±2a 0.7 ± 0.4 a 54 ± 38 a 48 ± 39 a 10 ± 6 a
n.d. not detected.

All the bioaugmented treatments reached a final concentration of TPHs significantly

lower than the control. The A. bisporus microcosms were the most effective treatment for the
TPHs removal, reaching the lowest concentration of these contaminants at the end of the
assay. This treatment showed a degradation rate of 48% higher than the control, followed
by L. edodes (34%), P. ostreatus (29%) and P. eryngii (12%). The assessment of the petroleum
hydrocarbons removal was analyzed considering the aliphatic and aromatic nature of the
contaminants. Moreover, the aliphatic and aromatic compounds were analyzed according
to the length of their carbon chains (Table 3).

Table 3. Concentration of TPHs and aliphatic and aromatic fractions by chains (mean ± standard
deviation) of the control microcosms (C), and microcosms inoculated with SMS of P. eryngii (Pe), L.
edodes (Le), P. ostreatus (Po) and A. bisporus (Ab) at the end of the incubation. Data is presented as
mean values ± standard deviation (n = 3). The same letters indicate a lack of statistically significant
difference (p < 0.05) between treatments.

mg/kg C Pe Le Po Ab
Aliphatic
>C10 −C12 359 ± 44 a 141 ± 51 b 190 ± 31 b 166 ± 12 b 73 ± 35 c
>C12 −C16 4629 ± 269 a 3599 ± 283 b 2902 ± 206 c 2960 ± 114 c 2071 ± 343 d
>C16 −C21 5854 ± 245 a 5180 ± 250 b 3951 ± 253 c 4100 ± 248 c 3144 ± 503 d
>C21 −C35 3204 ± 158 a 2916 ± 212 ab 2334 ± 130 cd 2611 ± 392 bc 2093 ± 278 d
>C35 30 ± 9 ab 22 ± 13 ab 15 ± 5 b 39 ± 16 a 27 ± 10 ab
Σ Aliphatic 14,077 ± 656 a 11,857 ± 617 b 9392 ± 599 c 9875 ± 728 c 7407 ± 1151 d
Aromatic
>EC10 −EC12 18 ± 7 a 11 ± 2 b 12 ± 3 ab 8 ± 2 bc 4±1c
>EC12 −EC16 638 ± 104 a 451 ± 65 b 294 ± 98 c 345 ± 36 bc 127 ± 26 d
>EC16 −EC21 2667 ± 264 a 2765 ± 298 a 1811 ± 187 b 1990 ± 98 b 1255 ± 111 c
>EC21 −EC35 516 ± 44 ab 614 ± 180 a 412 ± 49 b 522 ± 118 ab 413 ± 31 b
>EC35 56 ± 11 a 85 ± 42 a 72 ± 39 a 71 ± 25 a 58 ± 10 a
Σ Aromatic 3895 ± 410 a 3926 ± 540 a 2601 ± 249 b 2935 ± 223 b 1857 ± 155 c
ΣTPHs 17,971 ± 1061 a 15,784 ± 1061 b 11,993 ± 836 c 12,811 ± 948 c 9264 ± 1230 d

Regarding the total aliphatic hydrocarbon removal, once again, all the bioaugmented
microcosms showed significantly lower concentrations than the control microcosms at the
end of the incubation. The SMS of A. bisporus achieved the lowest concentration of total
aliphatic hydrocarbons. In contrast, among the bioaugmented treatments, P. eryngii was
less effective for the removal of aliphatic hydrocarbons.
The most abundant aliphatic hydrocarbons are compounds with chains of C12 –C16 ,
C16 –C21 and C21 –C35 . The concentration of lighter hydrocarbons (C10 –C12 ) was one-fold
Appl. Sci. 2022, 12, 7720 8 of 15

lower than the previous fractions. Finally, the heaviest fraction (>C35 ) was the less abun-
dant, with a concentration two-folds lower than the most abundant aliphatic hydrocarbons.
The control microcosms achieved the highest concentration for most of the aliphatic groups.
In contrast, the bioaugmented microcosms improved the degradation of aliphatic hydrocar-
bons with all having significantly lower concentrations than control microcosms. Therefore,
the four SMSs were useful to improve the aliphatic hydrocarbon’s removal, excepting the
heaviest fraction (>C35 ), for which no significant differences were found between the control
and any of the bioaugumented microcosms. For fractions lower to C35 significant differ-
ences were observed between the four bioaugmented treatments. A. bisporus microcosms
reached the lowest concentration of all the aliphatic groups except for the heaviest group
(>C35 ). L. edodes and P. ostreatus treatments also achieved significantly higher degradation
than control. P. eryngii microcosms achieved significantly higher concentrations of C10 –C12 ,
C12 –C16 , C16 –C21 and C21 –C35 hydrocarbons than A. bisporus microcosms.
The most abundant aromatic hydrocarbons were EC16 –EC21 . The aromatic hydrocar-
bons with chains EC12 –EC16 and EC21 –EC35 were one-fold lower that EC16 –EC21 . Finally,
the lightest (EC10 –EC12 ) and heaviest (>EC35 ) aromatic hydrocarbons were the less abun-
dant with concentrations 2-fold lower than the most abundant aromatic fraction. Three
of the four bioaugmented treatments, L. edodes, P. ostreatus and A. bisporus, got significant
lower concentrations of total aromatic hydrocarbons than the control treatment. Between
the effective treatments, A. bisporus microcosms achieved a significantly lower concen-
tration of total aromatic hydrocarbons. However, the improvement in the degradation
effectiveness of SMS treatments was only detected for the light aromatic hydrocarbons EC10 –
EC12 , EC12 –EC16 and EC16 –EC21 . Therefore, these treatments were not effective to improve
the degradation rate of heavy aromatic hydrocarbons with respect to the autochthonous
microbiota. For the light fractions, A. bisporus SMS got a significantly lower concentration
of these contaminants than L. edodes, P. ostreatus and P. eryngii microcosms. Despite P. eryngii
SMS did not show significant differences with respect to the control treatment for the total
aromatic hydrocarbons, this treatment reached a significantly lower concentration for the
two most light fractions (EC10 –EC12 and EC12 –EC16 ).
The PCA analysis of the remaining concentration of TPHs and aliphatic and aromatic
chains for the control treatment and the four SMSs microcosms are shown in Figure 2. The
percent variability explained by PC1 was 66.4% and for the PC2 was 14.9%. The PC1 was
positively related to the remaining concentration of TPHs and all the hydrocarbon fractions
except to >C35 and the heaviest aromatic fractions (EC21 –EC35 and >EC35 ) which were
positively related to the PC2. The control microcosm was located at the right of the PCA
diagram denoting the high concentration of hydrocarbons at the end of the incubation time.
The bioaugmented microcosms were progressively displaced to the left of the PCA diagram
according to the effectivity of hydrocarbons degradation. P. eryngii microcosms were closer
to the control, both in the positive section of PC1. The other bioaugmented microcosms
were in the negative section of the PC1 denoting their ability to remove TPHs and a high
variety of aliphatic and aromatic chains. L. edodes and P. ostreatus were overlapped denoting
similar effectivity to remove petroleum hydrocarbons. Finally, A. bisporus microcosms were
clearly away from the rest of the microcosms with negative values of PC1. This location
was negatively related to a high concentration of hydrocarbons demonstrating once again
the effectivity of this SMS to remove petroleum hydrocarbons. The low score of PC2 of
A. bisporus and the other bioaugmented treatments or even the positive score of P. eryngii
showed the low effectiveness of these SMSs to remove heavy aromatic hydrocarbons.
 of PC1. This location was negatively related to a high concentration of hydrocarbons
demonstrating once again the effectivity of this SMS to remove petroleum hydrocarbons.
The low score of PC2 of A. bisporus and the other bioaugmented treatments or even the
positive score of P. eryngii showed the low effectiveness of these SMSs to remove heavy
aromatic hydrocarbons.
Appl. Sci. 2022, 12, 7720 9 of 15

Figure 2. Principal component analysis of the control microcosms (C) and the bioaugmented micro-
Figure 2. Principal component analysis of the control microcosms (C) and the bioaugmented micro-
cosms with SMS of P. eryngii (P.e.), L. edodes (L.e.), P. ostreatus (P.o.), and A. bisporus (A.b.) showing
cosms with SMS of P. eryngii (P.e.), L. edodes (L.e.), P. ostreatus (P.o.), and A. bisporus (A.b.) showing
loading
loading scores of of
scores thetheresidual concentration
residual concentration of TPHs
of TPHsandand theirtheir
aliphatic (C10 –C
aliphatic , C1212, –C
(C1012–C C1216
–C ,C –C21 ,21,
16,16C16–C
C21
C–C 3535and
21–C and>C>C3535) )and
andaromatic
aromatic(EC
(EC –EC
1010 1212, ,EC
–EC EC1212–EC
–EC1616,, EC
EC16 –EC21,
16–EC EC2121–EC
21, EC –EC3535and
and>EC >EC3535 ) fractions.
) fractions.

4. Discussion
4. Discussion
The colonization of this polluted soil confirmed the ability of P. eryngii, L. edodes,
The colonization of this polluted soil confirmed the ability of P. eryngii, L. edodes, P.
P. ostreatus and A. bisporus to adapt to highly contaminated environments. This fact is also
ostreatus and A. bisporus to adapt to highly contaminated environments. This fact is also
more relevant because the fungi were inoculated from SMSs. This agricultural waste is
more relevant because the fungi were inoculated from SMSs. This agricultural waste is
generated after the industrial culture of edible fungi when the culture of more mushrooms
generated after the industrial culture of edible fungi when the culture of more mushrooms
is not economically viable. SMSs are characterized by their low level of nutrients for
is notsuch
fungi economically
as cellulose viable.
and SMSs are characterized
hemicellulose which wereby their low level
consumed of nutrients
during for fungi
the culture of
such as cellulose and hemicellulose which were consumed during
mushrooms [41]. However, they still contained the living organism, including myceliumthe culture of mush-
rooms
that [41].
can be However,
used they still contained
for soil bioremediation the living
between organism, including
other possibilities [18,21,26].mycelium that
canHence,
be used for soil
SMSs bioremediation
are organic between
wastes with a wideother possibilities
variety [18,21,26].[26,41]. The use
of microorganisms
Hence, SMSs are organic wastes with a wide variety of microorganisms
of these wastes in soil bioremediation provides multiple benefits such as their recycling, [26,41].the
The
use of these wastes in soil bioremediation provides multiple benefits such
alleviation of the disposal problem and the cost decrease in waste treatment [42], possible as their recy-
cling, the alleviation
bioaugmentation of the disposal problem
of hydrocarbonoclastic and
bacteria in the cost decrease
contaminated in[26]
soils waste
and treatment
inoculation[42],
possible bioaugmentation of hydrocarbonoclastic bacteria in contaminated
of lignin-degrading fungi [24,25,43], biostimulation of autochthonous soil microbiota by soils [26] and
inoculation
adding of lignin-degrading
nutrients and organic matter fungiand
[24,25,43],
servingbiostimulation of autochthonous
as a bulking agent increasing oxygensoil mi-
crobiota[26,42].
diffusion by adding nutrients and organic matter and serving as a bulking agent increasing
oxygen
One diffusion
of the key[26,42].
factors for bioremediation processes is microbial activity since it is
an indicatorofof
One thethe
key factors
ability offor
thebioremediation processes
microbiota to grow and is microbial
degrade theactivity sinceunder
pollutants it is an
indicator of the ability of the microbiota to grow and degrade the pollutants
certain environmental conditions [44]. The soil biological activity of the microcosms was under certain
determined by dehydrogenase and total hydrolase activities (Table 2). Dehydrogenase is
an oxidoreductase that is related to the abundance and metabolic activity of soil microor-
ganisms and catalyzed the biological oxidation of organic compounds [45]. It is the most
representative soil enzyme among soil oxidoreductases, and its activity is one of the most
used parameters for evaluating the microbiological status of the soil [46]. This enzymatic
activity is clearly related to the degradation of TPHs in soil and was purposed as an in-
dicator of the potential of soil microbiota to degrade TPHs [44]. Hydrolases are enzymes
capable of catalyzing the hydrolysis of a chemical bond using water. In the soil, these
enzymes are used for the degradation of complex organic matter, generating molecules that
are more easily accessible to soil organisms [47]. It is clear in Table 2 the positive impact of
A. bisporus SMS on soil microbial activity. This SMS was the only one that include chicken
Appl. Sci. 2022, 12, 7720 10 of 15

manure in its composition because A. bisporus is a secondary decomposer that requires

materials with a lower C/N ratio and higher cellulose, hemicellulose and nitrogen contents
(manure and compost) than primary decomposers such as the rest of fungi tested in this
work who required high C/N ratio and lignin content [18]. Hence, A. bisporus SMS had the
highest nutritional content of the four SMSs tested [20], and therefore, the SMS with the
highest biostimulation capacity of the soil microbiota. Simultaneously to soil biostimula-
tion, the inherent microbiota of SMS can colonize the soil and produce bioaugmentation
of the soil microbiota with microorganisms able to degrade organic pollutants [24]. The
other important factor that could enhance the hydrolase and dehydrogenase activity at
the end of the incubation period was the lower TPHs concentration of this microcosm.
Low activities of these enzymes are indicators of soil toxicity [1,34]. Therefore, the higher
soil enzymatic activity of A. bisporus treatment with respect to the other treatments could
be a combination of the positive effects of the SMS in soil microbiota and the reduction
in soil toxicity because of the high effectivity of this treatment to biodegrade TPHs. The
hydrolase and dehydrogenase activities for P. eryngii, L. edodes and P. ostreatus treatments
were not significantly higher than the control, probably because a combination of multiple
factors such as the low nutrient status of these SMSs and their lignocellulosic base does not
promote the biostimulation of soil microbiota at long lasting. or antagonistic interactions
between autochthonous soil microbiota and SMS microbiota and finally, the lower effectiv-
ity to reclaim and detoxify the soil than A. bisporus SMS. In this regard, Becarelli et al. [48]
described very low TPHs degradation by P. ostreatus inoculated from SMS. In addition,
these authors found that despite the bacterial community of the SMS was competent to
degrade TPHs, the bacteria prioritized the transformation of the more biodegradable SMS
organic matter.
Despite the different visual colonization of the four fungi (Figure 1), the ergosterol
content was not different between treatments (Table 2). P. eryngii and A. bisporus were the
ones with the highest colonization. While P. ostreatus and L. edodes only showed colonization
at the end of the experiment, but not as vigorous as the other two species. Ergosterol variesi
depending on some factors such as the growth medium, the culture conditions and the
growth phase in which it is found [36]. For this mycoremediation test, each microcosm
presented a different culture phase of each fungus after 40 days of incubation and the
growth medium was also different since each one had a different SMS. The low ability of
P. ostreatus to colonize this soil was not expected. There are multiple works that reported
good performance of this fungus for the mycoremediation of soil [4,49,50]. There are
also abundant works about the potential of P. eryngii, L. edodes and A. bisporus to colonize
contaminated soils by PAHs, PCBs or pesticides; however, there is limited or even zero
information on the fungal colonization of TPHs contaminated soil by these fungi [20].
The inoculation of lignin-degrading fungi by SMS is a valuable source of ligninolytic
enzymes such as laccase and different peroxidases [21]. Many transforming interactions
between fungi and different pollutants depend on a variety of extracellular excreted sub-
stances and metabolites. Fungi are capable of degrading petroleum hydrocarbons by
secreting enzymes [51]. For example, Zhou et al. [26] reported the increment of Laccase,
MnP and Lignin peroxidase of PAH-polluted soils incubated with SMS of P. ostreatus,
P. eryngii and Auricularia auricular and García-Delgado et al. [24,52] reported laccase and
MnP activity in PAH and PAH-Pb contaminated soils using A. bisporus SMS. In this work,
the species P. eryngii and A. bisporus were the only ones with laccase activity at the end
of the assay. Laccase is a nonspecific enzyme with a high redox potential that can oxi-
dize different compounds such as phenols and polyphenols [53]. This enzyme uses O2
as an electron acceptor, while for peroxidase it uses H2 O2 . Despite both fungi, P. eryngii
and A. bisporus, showing laccase activity, the highest laccase activity was measured for
A. bisporus treatment. This agrees with the visual colonization of the microcosms that
was more vigorous for A. bisporus and P. eryngii than for the other fungi. MnP has a high
redox potential and is characterized by oxidizing phenolic structures [54]. It was observed
that all the bioaugmented treatments showed a relatively higher value than the control,
Appl. Sci. 2022, 12, 7720 11 of 15

highlighting again P. eryngii and A. bisporus. A possible explanation for these better results
is, once again, that they were the fungi that better colonized the soil and these enzymes
were indicators of fungal activity [41]. Both for P. ostreatus and for L. edodes, the fungi that
colonized the soil the least, possibly the enzymatic activity that they presented came from
the mycelium that remained alive in the substrates. Finally, versatile peroxidase catalyzes
oxidation for both organic and inorganic substrates, phenols or aromatic amines, and is
characterized by oxidizing substrate compounds with high and low redox potential [55].
Because of these results, it is clear that A. bisporus treatment reached the highest
biodegradation of TPHs (Table 3). These results agreed with the visual aspect of the
microcosms, total hydrolase, dehydrogenase and ligninolytic activities. Mohammadi-
Sichani et al. [50] reported that A. bisporus was more effective than P. ostreatus SMS in a
TPHs contaminated soil after biostimulation with NPK. I [48]. However, L. edodes and P.
ostreatus SMSs showed the second highest removal of TPHs. This result was unexpected
since vigorous fungal colonization was not observed (Figure 1). Furthermore, the enzymatic
activities were not significantly higher than the control treatment.
Therefore, it can be deduced that, for both fungal species, a greater degradation oc-
curred because their respective SMSs favored the soil microbiota that is already resistant to
high contamination levels. Jabbar et al. [4] highlighted that the microbial variety is reduced
in the contaminated soils due to the influences of the toxic petroleum hydrocarbons upon
the organisms. Therefore, the population of degrading microbes ranges from 1% to 10%
of the population in a contaminated environment, whereas those in an uncontaminated
environment are less than 1% of the population. Another explanation could be that L. edodes
and P. ostreatus SMSs contain inherent microbiota highly specific to degrade hydrocarbons
such as Proteobacteria, Microbacterium, Brevundimonas, and Devosia as was previously re-
ported for P. ostreatus SMS [26]. Covino et al. [49] highlighted the key role of the synergistic
effects of soil microbiota and lignin-modified fungi to promote the degradation of aliphatic
hydrocarbons, specifically C28 –C34 .
The assessment of TPH degradation by chains is not a common issue. In this work,
the TPHs biodegradation was assessed by degradation of TPHs, aliphatic and aromatic
chains and by the number of carbons of these chains. The degradation effectiveness of each
treatment by aliphatic and aromatic chains followed the same pattern than TPHs degrada-
tion, A. bisporus SMS > P. ostreatus SMS and L. edodes SMS > P. eryngii SMS (Table 3) and it
was maintained in many of the specific aliphatic and aromatic chains. The interpretation of
the degradation effectiveness of each SMS by the carbon chain length demonstrated the
ability of all the tested SMSs to enhance the biodegradation of light aliphatic and aromatic
hydrocarbons. However, P. eryngii SMS was the only which was not able to improve the
degradation of the heavy aliphatic fraction C21 –C35 with respect to control. For the heavy
aromatic hydrocarbons (EC21 –EC35 ), none of the Pleurotus SMSs improved the degradation
of the control treatment but L. edodes and A. bisporus SMSs were able to stimulate the degra-
dation of EC21 –EC35 . Therefore, despite all the SMSs were able to improve the hydrocarbon
degradation, their potential to remove heavy fractions was different or even seem impaired
for the heaviest fractions (>C35 and >EC35 ). For example, aromatic hydrocarbons such
as PAHs with high molecular weight having more than three benzene rings are difficult
to degrade by soil microbiota because of their low availability [56] as was seen in this
work (Table 3). Because the bioremediation process depends on the bioavailability of
contaminants and the biodegradation performed by the microbial communities [57], an
additional strategy could be the use of surfactants such as saponin or Tween 80, between
other possibilities, that enhance TPH removal through increasing its solubility [58]. These
surfactants also did not negatively affect the fungi growth stimulating also the ligninolytic
peroxidase activities [59]. Lignin-degrading fungi produce biosurfactants [50]. There-
fore, the promotion of this ability could be a good alternative to exogenous surfactants
to improve the bioavailability of heavy hydrocarbons and consequently, enhance their
biodegradation. Some authors have published the usefulness of lignin-modified fungi to
degrade low-available aliphatic and aromatic hydrocarbons [24,49].
Appl. Sci. 2022, 12, 7720 12 of 15

To use fungi successfully for bioremediation, knowledge must be taken from the fields
with the four-phase strategy: bench-scale treatability, on-site pilot testing, production of
inoculum, and finally full-scale application [60]. The challenge in the development of
mycoremediation to large-scale field applications on petroleum-contaminated soils lies in
incorporating ideal environmental, edaphic and climatic factors of a typical contaminated
site into the process [61] and combining with the selected fungi and autochthonous hy-
drocarbonoclastic bacteria. Mixed (multi-domain) microbial communities exhibit unique
associations and interactions that could result in more efficient systems for the biodegra-
dation of organic pollutants as a result of sequential breakdown by fungi performing an
initial oxidation step producing metabolites that are available for bacterial degradation [62].
Myco-augmentation of contaminated matrices is operational via substrate-unspecific extra-
cellular and intracellular oxide reductases, laccases, and Mn-dependent and independent
peroxidases, enabling them to transform pollutants [63]. However, the fungal degradation
system is poorly studied and understanding of the genetic basis for biochemical activ-
ity is still incomplete compared to known bacterial degradation pathways of aromatic
pollutants [64].
Accelerating the global implementation of recycling and utilization of SMS is an
important way to realize the transformation and upgrading of the green development of
the edible mushroom industry [20]. The results of this work seem a good perspective for
the scaling up of mycoremediation as a real alternative to reclaim contaminated soils with
TPHs. The fact that SMS is useful as fungal and hydrocarbonoclastic bacteria inoculum to
reclaim contaminated soils, involves one more option to recycle this abundant and available
all over the world agricultural waste.

5. Conclusions
All of the four different spent mushroom substrates (P. eryngii, L. edodes, P. ostreatus,
and A. bisporus) evaluated in this work showed significantly higher removal of TPHs than
the non-inoculated soil demonstrating the usefulness of these wastes in the remediation
of contaminated soils with petroleum hydrocarbon. A. bisporus and P. eryngii clearly
colonize the contaminated soil. However, the mere colonization of the soil did not assure
high removal of TPHs since the inherent microbiota of the SMS and its interaction with
the autochthonous soil microbiota are also important factors. The SMS of A. bisporus
was the most effective SMS for the bioremediation of contaminated soils with petroleum
hydrocarbon. L. edodes SMS reached high degradation of the heaviest aliphatic chains (>C35 )
and the aromatic chains >EC12 –EC16 . SMS of P. ostreatus was the most effective SMS for the
degradation of the aliphatic chains >C10–C12 and aromatic chains EC10 –EC12 . Finally, P.
eryngii SMS was only useful for the biodegradation of the light aliphatic chains >C10 –C12 .
This work demonstrates the feasibility of the bioremediation of contaminated soils
with petroleum hydrocarbons using abundant agricultural waste such as SMS of different
fungi. The re-use of SMS for this purpose promotes the circular economy and the Green
Deal of the European Union reducing the environmental and climate footprint of the
food system.

Author Contributions: Conceptualization, R.A.-H., C.G.-D. and E.E.; methodology, B.M. and N.B.;
validation and formal analysis, R.A.-H., B.M. and N.B.; investigation, R.A.-H., N.B., C.G.-D. and
E.E.; data curation, R.A.-H. and N.B.; writing—original draft preparation, R.A.-H. and C.G.-D.;
writing—review and editing, R.A.-H., C.G.-D., B.M., L.D.-M. and E.E.; supervision, C.G.-D. and E.E.;
project administration, E.E.; funding acquisition, E.E. and C.G.-D. All authors have read and agreed
to the published version of the manuscript.
Funding: This article is result of the European Union’s LIFE Program under the grant contract
number LIFE20 ENV/ES/000416.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Appl. Sci. 2022, 12, 7720 13 of 15

Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: We thank the Technological Center for Mushroom Research in La Rioja (CTICH,
La Rioja, Spain) to provide the spent mushroom substrates and the Centro de Investigaciones
Energéticas, Medioambientales y Tecnológicas (CIEMAT, Madrid, Spain) for the analysis of the TPHs.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data and in the writing of the manuscript.

References
1. García-Delgado, C.; Alfaro-Barta, I.; Eymar, E. Combination of biochar amendment and mycoremediation for polycyclic aromatic
hydrocarbons immobilization and biodegradation in creosote-contaminated soil. J. Hazard. Mater. 2015, 285, 259–266. [CrossRef]
[PubMed]
2. Aioub, A.A.A.; Li, Y.; Qie, X.; Zhang, X.; Hu, Z. Reduction of soil contamination by cypermethrin residues using phytoremediation
with Plantago major and some surfactants. Environ. Sci. Eur. 2019, 31, 26. [CrossRef]
3. Aioub, A.A.A.; Zuo, Y.; Aioub, A.A.A.; Hu, Z. Biochemical and phytoremediation of Plantago major L. to protect tomato plants
from the contamination of cypermethrin pesticide. Environ. Sci. Pollut. Res. 2021, 28, 43992–44001. [CrossRef] [PubMed]
4. Jabbar, N.M.; Alardhi, S.M.; Mohammed, A.K.; Salih, I.K.; Albayati, T.M. Challenges in the implementation of bioremediation
processes in petroleum-contaminated soils: A review. Environ. Nanotechnol. Monit. Manag. 2022, 18, 100694. [CrossRef]
5. Chukwunonso, I.; Ahmed, A.; Hassan, A.; Shahul, F. Environmental Technology & Innovation Remediation of soil and water
contaminated with petroleum hydrocarbon: A review. Environ. Technol. Innov. 2020, 17, 100526. [CrossRef]
6. Rathankumar, A.K.; Saikia, K.; Kumar, P.S.; Varjani, S.; Kalita, S.; Bharadwaj, N.; George, J.; Vaidyanathan, V.K. Surfactant-aided
mycoremediation of soil contaminated with polycyclic aromatic hydrocarbon (PAHs): Progress, limitation, and countermeasures.
J. Chem. Technol. Biotechnol. 2022, 97, 391–408. [CrossRef]
7. D’Annibale, A.; Rosetto, F.; Leonardi, V.; Federici, F.; Petruccioli, M. Role of autochthonous filamentous fungi in bioremediation
of a soil historically contaminated with aromatic hydrocarbons. Appl. Environ. Microbiol. 2006, 72, 28–36. [CrossRef] [PubMed]
8. Das, N.; Chandran, P. Microbial Degradation of Petroleum Hydrocarbon Contaminants: An Overview. Biotechnol. Res. Int. 2011,
2011, 1–13. [CrossRef]
9. Lladó, S.; Solanas, A.M.; de Lapuente, J.; Borràs, M.; Viñas, M. A diversified approach to evaluate biostimulation and bioaugmen-
tation strategies for heavy-oil-contaminated soil. Sci. Total Environ. 2012, 435–436, 262–269. [CrossRef]
10. Maletić, S.P.; Dalmacija, B.D.; Rončević, S.D.; Agbaba, J.R.; Perović, S.D.U. Impact of hydrocarbon type, concentration and
weathering on its biodegradability in soil. J. Environ. Sci. Heal. Part A Toxic/Hazard. Subst. Environ. Eng. 2011, 46, 1042–1049.
[CrossRef]
11. Treu, R.; Falandysz, J. Mycoremediation of hydrocarbons with basidiomycetes—A review. J. Environ. Sci. Health Part B Pestic.
Food Contam. Agric. Wastes 2017, 52, 148–155. [CrossRef] [PubMed]
12. Akhtar, N.; Mannan, M.A.-u. Mycoremediation: Expunging environmental pollutants. Biotechnol. Rep. 2020, 26, e00452.
[CrossRef]
13. Levasseur, A.; Drula, E.; Lombard, V.; Coutinho, P.M.; Henrissat, B. Expansion of the enzymatic repertoire of the CAZy database
to integrate auxiliary redox enzymes. Biotechnol. Biofuels 2013, 6, 1–14. [CrossRef] [PubMed]
14. Syed, K.; Porollo, A.; Lam, Y.W.; Yadav, J.S. A fungal P450 (CYP5136A3) capable of oxidizing polycyclic aromatic hydrocarbons
and endocrine disrupting alkylphenols: Role of Trp129 and Leu324. PLoS ONE 2011, 6, e28286. [CrossRef]
15. Harms, H.; Schlosser, D.; Wick, L.Y. Untapped potential: Exploiting fungi in bioremediation of hazardous chemicals. Nat. Rev.
Microbiol. 2011, 9, 177–192. [CrossRef] [PubMed]
16. Cabral, L.; Giovanella, P.; Pellizzer, E.P.; Teramoto, E.H.; Kiang, C.H.; Sette, L.D. Microbial communities in petroleum-
contaminated sites: Structure and metabolisms. Chemosphere 2022, 286. [CrossRef] [PubMed]
17. Gramss, G.; Voigt, K.D.; Kirsche, B. Degradation of polycyclic aromatic hydrocarbons with three to seven aromatic rings by higher
fungi in sterile and unsterile soils. Biodegradation 1999, 10, 51–62. [CrossRef]
18. Zied, D.C.; Sánchez, J.E.; Noble, R.; Pardo-Giménez, A. Use of spent mushroom substrate in new mushroom crops to promote the
transition towards a circular economy. Agronomy 2020, 10, 1239. [CrossRef]
19. Royse, D.J.; Baars, J.; Tan, Q. Current Overview of Mushroom Production in the World. Edible Med. Mushrooms 2017, 2010, 5–13.
[CrossRef]
20. Leong, Y.K.; Ma, T.W.; Chang, J.S.; Yang, F.C. Recent advances and future directions on the valorization of spent mushroom
substrate (SMS): A review. Bioresour. Technol. 2022, 344, 126157. [CrossRef]
21. Ghose, A.; Mitra, S. Spent waste from edible mushrooms offers innovative strategies for the remediation of persistent organic
micropollutants: A review. Environ. Pollut. 2022, 305, 119285. [CrossRef] [PubMed]
22. Upadhyay, S.K.; Chauhan, P.K. Optimization of eco-friendly amendments as sustainable asset for salt-tolerant plant growth-
promoting bacteria mediated maize (Zea mays L.) plant growth, Na uptake reduction and saline soil restoration. Environ. Res.
2022, 211, 113081. [CrossRef]
Appl. Sci. 2022, 12, 7720 14 of 15

23. Frutos, I.; García-Delgado, C.; Gárate, A.; Eymar, E. Biosorption of heavy metals by organic carbon from spent mushroom
substrates and their raw materials. Int. J. Environ. Sci. Technol. 2016, 13, 2713–2720. [CrossRef]
24. García-Delgado, C.; D’Annibale, A.; Pesciaroli, L.; Yunta, F.; Crognale, S.; Petruccioli, M.; Eymar, E. Implications of polluted
soil biostimulation and bioaugmentation with spent mushroom substrate (Agaricus bisporus) on the microbial community and
polycyclic aromatic hydrocarbons biodegradation. Sci. Total Environ. 2015, 508, 20–28. [CrossRef] [PubMed]
25. Siracusa, G.; Becarelli, S.; Lorenzi, R.; Gentini, A.; Di Gregorio, S. PCB in the environment: Bio-based processes for soil
decontamination and management of waste from the industrial production of Pleurotus ostreatus. N. Biotechnol. 2017, 39, 232–239.
[CrossRef]
26. Zhou, J.; Ge, W.; Zhang, X.; Wu, J.; Chen, Q.; Ma, D.; Chai, C. Effects of spent mushroom substrate on the dissipation of polycyclic
aromatic hydrocarbons in agricultural soil. Chemosphere 2020, 259, 127462. [CrossRef] [PubMed]
27. García-Delgado, C.; Alonso-Izquierdo, M.; González-Izquierdo, M.; Yunta, F.; Eymar, E. Purification of polluted water with spent
mushroom (Agaricus bisporus) substrate: From agricultural waste to biosorbent of phenanthrene, Cd and Pb. Environ. Technol.
2017, 38, 1792–1799. [CrossRef]
28. Frutos, I.; García-Delgado, C.; Cala, V.; Gárate, A.; Eymar, E. The use of spent mushroom compost to enhance the ability of
Atriplex halimus to phytoremediate contaminated mine soils. Environ. Technol. 2017, 38, 1075–1084. [CrossRef]
29. Carpio, M.J.; Rodríguez-Cruz, M.S.; García-Delgado, C.; Sánchez-Martín, M.J.; Marín-Benito, J.M. Mobility monitoring of two
herbicides in amended soils: A field study for modeling applications. J. Environ. Manag. 2020, 260, 110161. [CrossRef]
30. Carpio, M.J.; Marín-Benito, J.M.; García-Delgado, C.; Sánchez-Martín, M.J.; Rodríguez-Cruz, M.S. Soil microbial community
changes in a field treatment with chlorotoluron, flufenacet and diflufenican and two organic amendments. Agronomy 2020, 10,
1166. [CrossRef]
31. Mayans, B.; Camacho-Arévalo, R.; García-Delgado, C.; Alcántara, C.; Nägele, N.; Antón-Herrero, R.; Escolástico, C.; Eymar, E.
Mycoremediation of soils polluted with trichloroethylene: First evidence of pleurotus genus effectiveness. Appl. Sci. 2021, 11,
1354. [CrossRef]
32. Agencia Estatal Boletín Oficial del Estado, Ministerio de la Presidencia. Real Decreto 9/2005, de 14 de enero; Agencia Estatal Boletín
Oficial del Estado, Ministerio de la Presidencia: Madrid, Spain, 2005; Volume 2005, pp. 1833–1843.
33. García-Delgado, C.; Cala, V.; Eymar, E. Influence of chemical and mineralogical properties of organic amendments on the selection
of an adequate analytical procedure for trace elements determination. Talanta 2012, 88, 375–384. [CrossRef] [PubMed]
34. Dawson, J.J.C.; Godsiffe, E.J.; Thompson, I.P.; Ralebitso-Senior, T.K.; Killham, K.S.; Paton, G.I. Application of biological indicators
to assess recovery of hydrocarbon impacted soils. Soil Biol. Biochem. 2007, 39, 164–177. [CrossRef]
35. Adam, G.; Duncan, H. Development of a sensitive and rapid method for the measurement of total microbial activity using
fluorescein diacetate (FDA) in a range of soils. Soil Biol. Biochem. 2001, 33, 943–951. [CrossRef]
36. Barajas-Aceves, M.; Hassan, M.; Tinoco, R.; Vazquez-Duhalt, R. Effect of pollutants on the ergosterol content as indicator of fungal
biomass. J. Microbiol. Methods 2002, 50, 227–236. [CrossRef]
37. Covino, S.; Čvančarová, M.; Muzikář, M.; Svobodová, K.; D’annibale, A.; Petruccioli, M.; Federici, F.; Křesinová, Z.; Cajthaml, T.
An efficient PAH-degrading Lentinus (Panus) tigrinus strain: Effect of inoculum formulation and pollutant bioavailability in
solid matrices. J. Hazard. Mater. 2010, 183, 669–676. [CrossRef] [PubMed]
38. García-Delgado, C.; Eymar, E.; Camacho-Arévalo, R.; Petruccioli, M.; Crognale, S.; D’Annibale, A. Degradation of tetracyclines
and sulfonamides by stevensite- and biochar-immobilized laccase systems and impact on residual antibiotic activity. J. Chem.
Technol. Biotechnol. 2018, 93, 3394–3409. [CrossRef]
39. Ruiz-Dueñas, F.J.; Morales, M.; García, E.; Miki, Y.; Martínez, M.J.; Martínez, A.T. Substrate oxidation sites in versatile peroxidase
and other basidiomycete peroxidases. J. Exp. Bot. 2009, 60, 441–452. [CrossRef]
40. Pindado, O.; Pérez-Pastor, R.M.; Escolano, O. An analytical method for quantifying petroleum hydrocarbon fractions in soils, and
its associated uncertainties. Anal. Methods 2014, 6, 5527–5536. [CrossRef]
41. Carrasco, J.; García-Delgado, C.; Lavega, R.; Tello, M.L.; De Toro, M.; Barba-Vicente, V.; Rodríguez-Cruz, M.S.; Sánchez-Martín,
M.J.; Pérez, M.; Preston, G.M. Holistic assessment of the microbiome dynamics in the substrates used for commercial champignon
(Agaricus bisporus) cultivation. Microb. Biotechnol. 2020, 13, 1933–1947. [CrossRef]
42. Chiu, S.W.; Gao, T.; Chan, C.S.S.; Ho, C.K.M. Removal of spilled petroleum in industrial soils by spent compost of mushroom
Pleurotus pulmonarius. Chemosphere 2009, 75, 837–842. [CrossRef] [PubMed]
43. Di Gregorio, S.; Becarelli, S.; Siracusa, G.; Ruffini Castiglione, M.; Petroni, G.; Masini, G.; Gentini, A.; de Lima e Silva, M.R.;
Lorenzi, R. Pleurotus ostreatus spent mushroom substrate for the degradation of polycyclic aromatic hydrocarbons: The case
study of a pilot dynamic biopile for the decontamination of a historically contaminated soil. J. Chem. Technol. Biotechnol. 2016, 91,
1654–1664. [CrossRef]
44. Calvo, C.; Rodríguez-Calvo, A.; Robledo-Mahón, T.; Manzanera, M.; González-López, J.; Aranda, E.; Silva-Castro, G.A. Biostimu-
lation of crude oil-polluted soils: Influence of initial physicochemical and biological characteristics of soil. Int. J. Environ. Sci.
Technol. 2019, 16, 4925–4934. [CrossRef]
45. Silva-Castro, G.A.; Uad, I.; Rodríguez-Calvo, A.; González-López, J.; Calvo, C. Response of autochthonous microbiota of diesel
polluted soils to land-farming treatments. Environ. Res. 2015, 137, 49–58. [CrossRef] [PubMed]
Appl. Sci. 2022, 12, 7720 15 of 15

46. Campos, J.A.; Peco, J.D.; De Toro, J.A.; Moreno, C.; Amorós, J.A.; Moreno, M.M.; García-Noguero, E.M.; Higueras, P. Approach to
the potential usage of two wood ashes waste as soil amendments on the basis of the dehydrogenase activity and soil oxygen
consumption. J. Soils Sediments 2018, 18, 2148–2156. [CrossRef]
47. Krishnan, A.; Convey, P.; Gonzalez, M.; Smykla, J.; Alias, S.A. Effects of temperature on extracellular hydrolase enzymes from soil
microfungi. Polar Biol. 2018, 41, 537–551. [CrossRef]
48. Becarelli, S.; Siracusa, G.; Chicca, I.; Bernabei, G.; Di Gregorio, S. Ascomycetes versus spent mushroom substrate in mycoremedia-
tion of dredged sediments contaminated by total petroleum hydrocarbons: The involvement of the bacterial metabolism. Water
2021, 13, 3040. [CrossRef]
49. Covino, S.; Stella, T.; D’Annibale, A.; Lladó, S.; Baldrian, P.; Čvančarová, M.; Cajthaml, T.; Petruccioli, M. Comparative assessment
of fungal augmentation treatments of a fine-textured and historically oil-contaminated soil. Sci. Total Environ. 2016, 566, 250–259.
[CrossRef]
50. Mohammadi-Sichani, M.; Mazaheri Assadi, M.; Farazmand, A.; Kianirad, M.; Ahadi, A.M.; Hadian-Ghahderijani, H. Ability of
Agaricus bisporus, Pleurotus ostreatus and Ganoderma lucidum compost in biodegradation of petroleum hydrocarbon-contaminated
soil. Int. J. Environ. Sci. Technol. 2019, 16, 2313–2320. [CrossRef]
51. Li, Q.; Liu, J.; Gadd, G.M. Fungal bioremediation of soil co-contaminated with petroleum hydrocarbons and toxic metals. Appl.
Microbiol. Biotechnol. 2020, 104, 8999–9008. [CrossRef]
52. García-Delgado, C.; Yunta, F.; Eymar, E. Bioremediation of multi-polluted soil by spent mushroom (Agaricus bisporus) substrate:
Polycyclic aromatic hydrocarbons degradation and Pb availability. J. Hazard. Mater. 2015, 300, 281–288. [CrossRef] [PubMed]
53. Fernández-Fernández, M.; Sanromán, M.Á.; Moldes, D. Recent developments and applications of immobilized laccase. Biotechnol.
Adv. 2013, 31, 1808–1825. [CrossRef] [PubMed]
54. Martínez, A.T. Molecular biology and structure-function of lignin-degrading heme peroxidases. Enzyme Microb. Technol. 2002, 30,
425–444. [CrossRef]
55. Knop, D.; Levinson, D.; Makovitzki, A.; Agami, A.; Lerer, E.; Mimran, A.; Yarden, O.; Hadar, Y. Limits of versatility of versatile
peroxidase. Appl. Environ. Microbiol. 2016, 82, 4070–4080. [CrossRef]
56. Mandal, S.K.; Das, N. Phyto-mycoremediation of benzo[a]pyrene in soil by combining the role of yeast consortium and sunflower
plant. J. Environ. Biol. 2018, 39, 261–268. [CrossRef]
57. Guirado, M.; Garrido-Sanz, D.; Pindado, O.; Rodríguez-Rastrero, M.; Merino-Martín, L.; Sierra, M.J.; Escolano, O.; Rivilla,
R.; Millán, R. Effectiveness of biochar application and bioaugmentation techniques for the remediation of freshly and aged
diesel-polluted soils. Int. Biodeterior. Biodegrad. 2021, 163. [CrossRef]
58. Wu, M.; Xu, Y.; Ding, W.; Li, Y.; Xu, H. Mycoremediation of manganese and phenanthrene by Pleurotus eryngii mycelium
enhanced by Tween 80 and saponin. Appl. Microbiol. Biotechnol. 2016, 100, 7249–7261. [CrossRef] [PubMed]
59. Giubilei, M.A.; Leonardi, V.; Federici, E.; Covino, S.; Šašek, V.; Novotny, C.; Federici, F.; D’Annibale, A.; Petruccioli, M. Effect of
mobilizing agents on mycoremediation and impact on the indigenous microbiota. J. Chem. Technol. Biotechnol. 2009, 84, 836–844.
[CrossRef]
60. Rhodes, C.J. Mycoremediation (bioremediation with fungi)-growing mushrooms to clean the earth. Chem. Speciat. Bioavailab.
2014, 26, 196–198. [CrossRef]
61. Dickson, U.J.; Coffey, M.; Mortimer, R.J.G.; Di Bonito, M.; Ray, N. Mycoremediation of petroleum contaminated soils: Progress,
prospects and perspectives. Environ. Sci. Process. Impacts 2019, 21, 1446–1458. [CrossRef]
62. Espinosa-Ortiz, E.J.; Rene, E.R.; Gerlach, R. Potential use of fungal-bacterial co-cultures for the removal of organic pollutants. Crit.
Rev. Biotechnol. 2021, 42, 361–383. [CrossRef] [PubMed]
63. Chun, S.C.; Muthu, M.; Hasan, N.; Tasneem, S.; Gopal, J. Mycoremediation of PCBs by pleurotus ostreatus: Possibilities and
prospects. Appl. Sci. 2019, 9, 4185. [CrossRef]
64. Park, H.; Choi, I.G. Genomic and transcriptomic perspectives on mycoremediation of polycyclic aromatic hydrocarbons. Appl.
Microbiol. Biotechnol. 2020, 104, 6919–6928. [CrossRef] [PubMed]