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\DOI{10.5802/crchim.234}
\datereceived{2023-03-06}
\daterevised{2023-05-03}
\dateaccepted{2023-05-15}
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\dateposted{2023-08-21}
\begin{document}

\begin{noXML}

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{Materials and Clean Processes for Sustainable Energy and Environmental Applications}
%\def\cdr@specialtitle@french{Mat\'eriaux et proc\'ed\'es propres pour des applications \'energ\'etiques et environnementales}
%\makeatother

\title{Short-term effects of olive-mill-wastes-derived biochars
amendment and arbuscular mycorrhizal fungi inoculation on growth of
maize (\protect\textit{Zea mays}) and mycorrhizal colonization}

\alttitle{Effets \`a court terme de l'amendement de biochars
d\'eriv\'es de d\'echets d'olives et de l'inoculation de champignons
mycorhiziens \`a arbuscules sur la croissance du ma\"{\i}s et la
colonisation mycorhizienne}

\shortrunauthors

\author{\firstname{Christiane} \lastname{Minkosse}}
\address{Rittmo Agroenvironnement, ZA Biop\^{o}le, 37 rue de
Herrlisheim, CS 80023, F-68025 Colmar Cedex, France}
\email[C. Minkosse]{christiane.minkosse@rittmo.com}

\author{\firstname{Aude} \lastname{Langenfeld}}
\addressSameAs{1}{Rittmo Agroenvironnement, ZA Biop\^{o}le, 37 rue de
Herrlisheim, CS 80023, F-68025 Colmar Cedex, France}
\email[A. Langenfeld]{aude.Langenfeld@rittmo.com}

\author{\firstname{Ahmed Amine} \lastname{Azzaz}\CDRorcid{0000-0003-3516-9722}}
\address{Universit\'{e} de Haute-Alsace, CNRS, Institut de Science des
Mat\'{e}riaux de Mulhouse (IS2M) UMR 7361, F-68100 Mulhouse, France}
\address{Universit\'{e} de Strasbourg, F-67081 Strasbourg, France}
\email[A. A. Azzaz]{azzaz.ahmedamine@gmail.com}

\author{\firstname{Mejdi} \lastname{Jeguirim}\CDRorcid{0000-0003-2401-5824}\IsCorresp}
\addressSameAs{2}{Universit\'{e} de Haute-Alsace, CNRS, Institut de
Science des Mat\'{e}riaux de Mulhouse (IS2M) UMR 7361, F-68100
Mulhouse, France}
\addressSameAs{3}{Universit\'{e} de Strasbourg, F-67081 Strasbourg, France}
\email[M. Jeguirim]{mejdi.jeguirim@uha.fr}

\author{\firstname{Leila} \lastname{El-Bassi}\CDRorcid{0000-0002-3144-4435}}
\address{Laboratory of Wastewater and Environment, Center of Water
Research and Technologies (CERTE), Borj Cedria Ecopark, P.B. 273 - 8020
Soliman, Tunisia}
\email[L. El-Bassi]{l.elbassi@gmail.com}

\author{\firstname{Hanene} \lastname{Akrout}\CDRorcid{0000-0002-4878-6108}}
\addressSameAs{4}{Laboratory of Wastewater and Environment, Center of
Water Research and Technologies (CERTE). Borj Cedria Ecopark, P.B. 273
- 8020 Soliman, Tunisia}
\email[H. Akrout]{hanene.akrout@gmail.com}

\author{\firstname{Salah} \lastname{Jellali}\CDRorcid{0000-0002-4095-4154}\IsCorresp}
\address{Centre for Environmental Studies and Research (CESAR), Sultan
Qaboos University, Al-Khoud 123, Muscat, Oman}
\email[S. Jellali]{s.jelali@squ.edu.om}

\author{\firstname{Cam\'{e}lia Matei} \lastname{Ghimbeu}\CDRorcid{0000-0003-3600-5877}}
\addressSameAs{2}{Universit\'{e} de Haute-Alsace, CNRS, Institut de
Science des Mat\'{e}riaux de Mulhouse (IS2M) UMR 7361, F-68100
Mulhouse, France}
\addressSameAs{3}{Universit\'{e} de Strasbourg, F-67081 Strasbourg, France}
\email[C. M. Ghimbeu]{camelia.ghimbeu@uha.fr}

\author{\firstname{Najat} \lastname{Nassr}}
\addressSameAs{1}{Rittmo Agroenvironnement, ZA Biop\^{o}le, 37 rue de
Herrlisheim, CS 80023, F-68025 Colmar Cedex, France}
\email[N. Nassr]{najat.nassr@rittmo.com}

\begin{abstract}
The viability of arbuscular mycorrhizal fungi (AMF) spores formulated
with biochars derived from the pyrolysis of raw (ROP) or impregnated
olive pomace with olive mill wastewaters (IROP) was investigated under
laboratory-controlled conditions. A greenhouse experiment was also
conducted to study the effect of AMF spores and biochars adding on the
growth of maize plants. Nine treatments, combining one biochar (ROP or
IROP) at a level of 2.5 g/kg of soil and inoculation or not with
\textit{Funneliformis mosseae} at two concentrations (30 and 125
spores/kg of soil), were arranged in an entirely randomized block
design. The results of the formulation trial showed that arbuscular
mycorrhizal fungi (AMF) spores had short-term viability and are
sensitive to the presence of biochar. This effect is more pronounced
when the biochar is made from IROP. Both the AMF and biochars
\mbox{treatments} did not significantly enhance the growth of maize plants,
biomass production,\pagebreak
and leaf chlorophyll \mbox{contents.} However, the most
significant effect on nutrients uptake was observed in the modalities
treated with AMF alone (30 spores/kg of soil) and AMF combined with the
biochar made from IROP (IROP~$+$~30~spores/kg of soil). In those
modalities, the N uptake was improved by 45\% and 33\%, respectively.
Moreover, biochar addition to the soil did not particularly enhance
root colonization by AMF and no negative effect of its application was
observed either. However, the inoculation of spores enhanced propagules
production and maize root colonization.
\end{abstract}

\begin{altabstract}
La viabilit\'e des spores de champignons mycorhiziens \`a arbuscules
(AMF) en formulation avec des biochars d\'eriv\'es de la pyrolyse de
grignons d'olive bruts (ROP) ou impr\'egn\'es d'eaux us\'ees de moulins
\`a olives (IROP) a \'et\'e \'etudi\'ee dans des conditions
contr\^ol\'ees en laboratoire. Les r\'esultats ont montr\'e que les
spores des champignons mycorhiziens \`a arbuscules (AMF) avaient une
viabilit\'e \`a court terme et \'etaient sensibles \`a la pr\'esence de
biochar. Cet effet est plus prononc\'e lorsque le biochar est
fabriqu\'e \`a partir d'IROP. Une exp\'erience en serre a \'egalement
\'et\'e men\'ee pour \'etudier l'effet de l'addition de spores d'AMF et
de biochars sur la croissance des plants de ma\"{\i}s. Neuf
traitements, combinant un biochar (ROP ou IROP) \`a un niveau de
2,5~g/kg de sol et l'inoculation ou non de \textit{Funneliformis mosseae} \`a
deux concentrations (30 et 125 spores/kg de sol), ont \'et\'e
dispos\'es dans un plan en damier enti\`erement al\'eatoire. Les
r\'esultats ont montr\'e que les traitements par AMF et par biochars
n'ont pas am\'elior\'e de mani\`ere significative la croissance des
plants de ma\"{\i}s, la production de biomasse et la teneur en
chlorophylle des feuilles. Cependant, l'effet le plus significatif sur
l'absorption des nutriments a \'et\'e observ\'e dans les modalit\'es
trait\'ees avec l'AMF seul (30 spores/kg de sol) et l'AMF combin\'e
avec le biochar fabriqu\'e \`a partir de grignons d'olive impr\'egn\'es
(IROP $+$ 30 spores/kg de sol). Dans ces modalit\'es, l'absorption de
l'azote a \'et\'e am\'elior\'ee de 45~\% et 33~\%, respectivement. De
plus, l'ajout de biochar au sol n'a pas particuli\`erement am\'elior\'e
la colonisation des racines par les AMF et aucun effet n\'egatif de son
application n'a \'et\'e observ\'e non plus. Cependant, l'inoculation de
spores a am\'elior\'e la production de propagules et la colonisation
des racines du ma\"{\i}s.
\end{altabstract}

\keywords{\kwd{Arbuscular mycorrhizae}
\kwd{Biochar}
\kwd{Root colonization}
\kwd{Nitrogen}
\kwd{Viability assay}}

\altkeywords{\kwd{Mycorhizes \`a arbuscules}
\kwd{Biochar}
\kwd{Colonisation des racines}
\kwd{Azote}
\kwd{Essai de viabilit\'e}}

\maketitle

\vspace*{1pc plus 1pt minus 1pt}

\twocolumngrid

\end{noXML}

\section{Introduction}\label{sec1}

Since the middle of the 20th century, the global agri-food industry is
highly dependent on a massive input of synthetic fertilizers to
increase crop yields to satisfy the rising demand for food, animal
feed, and biofuels~\cite{1}. Fertilizers are defined as compounds or
mixtures delivered as solids, liquids, or gases, that supply essential
nutrients like nitrogen (N), phosphorous (P), and potassium (K) to
crops in soluble forms. Their origin can be natural or
synthetic~\cite{2}. Synthetic N fertilizers use is expected to
increase by 50\% from 2012 to 2050, which will lead to an even larger
amount of nitrous oxide (N$_{2}$O) emissions from
agricultural soils Only part of the nitrogen fertilizer applied to the
soil is absorbed by plants, while the other part is used by
microorganisms to produce N$_{2}$O, one of the principal
greenhouse gases responsible for the deterioration of the ozone layer.
Moreover, loss of nitrogen through leaching can also cause soil
acidification and eutrophication of ground and surface waters, and
consequently affect biodiversity, fish mortality, algal blooms, and
aquatic ecosystems\break \cite{7,8,9}.

Hence, it is important to develop alternatives to synthetic fertilizers
for the protection of the environment and a more sustainable
agricultural system. For this purpose, this investigation is focused on
the by-products (solids and liquids wastes) of the extraction of olive
oil in the Mediterranean Basin Countries. Olive oil production is
mainly concentrated in the Mediterranean region and it constitutes
around 98\% of the overall produced amount. According to the
International Olive Council, worldwide olive oil production was as high
as 3.01 million tons for the crop year 2020/2021. The five largest
olive-oil-producing countries in that year were Spain (1389,000 tons),
Greece (275,000 tons), Italy (273,500 tons), Turkey (210,000 tons), and
Morocco (160,000 tons). Meanwhile, Tunisia produced 140,000 tons of
olive oil\break during the 2020/2021 harvest season. This represents a
decrease of 31\% compared to the previous season, due to unfavorable
weather conditions such as drought~\cite{10}.

Despite its important economic value in the producing countries, the
extraction processes generate huge quantities of waste: olive mill
solid waste (OMSW) and olive mill wastewater (OMWW), which represent a
real concern for the environment~\cite{11,12} if no reuse
approaches are considered (European Directive 2008/98/EC). These wastes
are generally hard to manage and usually burnt or disposed of in
landfills or discharged near lakes, rivers, or seas~\cite{13,14}.
In Mediterranean basin countries, production and irrational disposal of
blunt amounts of olive mill wastes in short periods create severe
environmental problems~\cite{83}. With the absence of treatments plants
at the mills, wastewaters are left out in outdoor storage or
evaporation lagoons and can reach water bodies during periods of high
precipitations causing the deterioration of the environment such as
coloring, and pollution of surface and ground waters, soil surface, and
foul odors problems~\cite{18,84,85}. The waste waters are usually very
smelly, have a high organic load and high content of phytotoxic and
antibacterial phenolic substances, and therefore resist biological
degradation~\cite{18,86,87,88,89,90}.

The valorization of some of these residues can contribute to reducing
the quantities of wastes dumped in the
environment~\cite{15,16,17}. In that perspective, OMWW and ROP
were converted into biochars to be valorized as eco-friendly materials
for agricultural purposes. Throughout the years, several
physicochemical methods have been elaborated for treating olive mill
wastes as a pollutant or for alleviating their
toxicity~\cite{17,18,19,20}. However, only a small focus on
developing strategies to convert these wastes into biochars through
pyrolysis has been reported and then to be used as alternatives or
complementary amendments to synthetic fertilizers. One of those
strategies is the utilization of biobed for the treatment of olive
mills wastewaters. This technique is usually used for the reduction of
pesticides by adsorption and degradation~\cite{82}. In 2021, a study
was done on the efficiency of the treatment, purification, and
detoxification of a highly loaded olive oil wastewater effluent
concluded that the use of a biobed layer was efficient in pollutant
removal~\cite{88}. A decrease in biochemical oxygen demand, chemical
oxygen demand, total phenols, total Kjedahl nitrogen, and
NH$_{4}^+$ respectively decrease by 96\%,
92\%, 88\%, 85\%, and 100\%~\cite{91}.

According to the European Biochar Certificate (EBC), the so-called
``biochar'' is a porous, carbonaceous material produced by the pyrolysis
of biomass in an oxygen-limited environment. Recently, a special
interest was accorded to biochar use in agriculture as an eco-friendly
fertilizer in the context of sustainable
agriculture~\cite{21,22}. The meta-study of 109 independent
studies reported that biochar application to soil significantly
increased crop yield by 13\% and that effect was even more pronounced
in acidic soil (40\% increase for soil with pH $<$ 5). The
co-application of biochar and fertilizers had also an effect on crop
yield increase in temperate climates~\cite{23}. In addition, it was
reported that biochar has significantly impacted the abundance of soil
microorganisms (total phospholipid fatty acid), bacteria, fungi,
actinomycetes, Gram$+$ and Gram$-$ bacteria by 8\%, 20\%, 19\%, 9\%, 11\%,
and 13\% respectively~\cite{24}. Moreover, the role of biochar in
carbon sequestration in soils was also proved since the early
2000s~\cite{25,26,27}. Indeed, it was demonstrated that the
proportions of small labile C-fraction and recalcitrant C pools in
biochar are respectively 3\% and 97\%~\cite{28}. The mean residence
time (MRT) was estimated to be one year and 556 years respectively for
labile C-fraction and recalcitrant C pools~\cite{28}. Biochar also
had a positive role on soil physico-chemical properties such as water
holding capacity~\cite{29,30}, and soil stability by altering the
size of aggregates and regulating soil water~\cite{31,32}. By
improving the physical properties of the soil, it can allow better
development of fungal mycelium of Arbuscular mycorrhizal\break fungi (AMF).

The symbiosis of AMF with plants has been proven to promote nutrient
absorption, enhance soil fertility, stabilize soil structure, and also
improve water absorption efficiency~\cite{33,34,35}. In
water-deficient soil, mycorrhizas can connect through their hyphae and
form an absorption network conferring more efficient water absorption
than roots to plants~\cite{36,37}. AMF also has the potential to
solubilize inorganic phosphate and supply it to plants in an
assimilable form~\cite{38}. Moreover, AMF can release organic acids
and reduce the pH of the medium which increases P availability in soils
where is it deficient~\cite{39}. Thus, the combined effect of
biochar and AMF could be considered an attractive approach to enhance
crop productivity and promote an eco-friendly fertilizer. Previous
research works have demonstrated that biochars and AMF application can
considerably affect the growth of different crops like cacao and
chickpea~\cite{40,41}. This combined effect was attributed to
nitrogen and phosphorous uptake, photosynthesis, and chlorophyll
synthesis improvement~\cite{40}. Moreover, the use of mycorrhizae
and biochars combined treatments showed interesting results regarding
the remediation of crude oil-contaminated soils~\cite{42}. Later, a
study confirmed that the association of AMF, biochar, and N fertilizer
improved chicory (\textit{Cichorium intybus} L.) growth and nutrient uptake
while reducing cadmium (Cd) uptake in a Cd-contaminated
soil~\cite{43}. Thus, the present study aims to verify the following
hypothesis: could olive mills' wastes-based biochars be used as
fertilizer, and do their use in combination with AMF enhance plant
mycorrhization and nutrition? To verify these hypotheses, biochars have
been chemically characterized, their compatibility with AMF has been
tested in a viability assay and the effect of diverse biochar/AMF
combination on nutrient uptake, relative chlorophyll contents, and
fungal colonization by mycorrhizae were assessed under a greenhouse
trial. The crop chosen for the greenhouse trial was maize (\textit{Zea mays}
L.). Maize is one of the four main crops used in studies on the effect
of biochar on crops. The biochars generally used in those studies are
produced from residues of those cereals crops (maize, rice, wheat, and
barley) since they are the most produced and traded commodity in the
world~\cite{4}. Also, maize is an important source of carbohydrates for
human diets in developing countries and the harvest wastes (husks and
straw) have been historically used as animal feed, stable litter, soil
amendment, or cellulosic biomass for ethanol production~\cite{5,6}.
Therefore, this makes maize an interesting plant for this study.

\section{Materials and methods}\label{sec2}

\subsection{Biochar synthesis and characterization}

Raw olive pomace (ROP) and olive mill wastewater (OMWW) were collected
from a three-phase olive mill press located in Touta city, Nabeul,
Northeast of Tunisia (36\textdegree 38$'$19.1$''$N 10\textdegree 28$'$10.1$''$E).
Before use, ROP was sieved and air-dried. The OMWW was first
homogenized and filtered to discard the large particles, as described
on detail in Jeguirim \etal~\cite{16} and
El-Bassi \etal~\cite{32}. Two biomasses
have been prepared: (i) ROP and (ii) raw olive pomace treated with
olive mill wastewater (200~g of ROP per liter of OMWW) (used to produce
a biochar named IROP) as described by Jeguirim \etal~\cite{16}.
Obtained biomasses (ROP and IROP) were converted into biochars through
a slow pyrolysis process at 500~\textdegree C for 2~h in a tubular furnace
reactor (Thermolyne F 21100). Then, mineral composition and
physical-chemical characterization of the two biochars were carried out
according to the method described in El-Bassi \etal~\cite{32}. The
mineral composition of the generated biochars was performed using an
X-ray fluorescence spectrophotometer (Philips, Eindhoven,
Netherlands-PW2540) for analysis. Proximate analysis was assessed to
determine the materials content in ash, volatile matter, and fixed
carbon. Briefly, 0.5 g of samples were placed in a silicate alumina
crucible and then deposed inside a TGA/DSC3+ apparatus (Mettler-Toledo,
Greifense, Switzerland). Biochars were exposed to heat variation under
argon gas flow as follows: A heating at 30~\textdegree C for 20 min, then
an increase in temperature up to 800~\textdegree C at a 10~\textdegree C/min
rate, followed by an isotherm of two hours at 800~\textdegree C, and
finally an introduction of ambient air at the same temperature to
reduce the sample into ash~\cite{17}. Specific surface area and
porosity of the produced biochars were assessed through physical
CO$_{2}$ adsorption/desorption isotherms. Analysis was
performed using an ASAP 2020 gas adsorption apparatus (Micromeritics,
Georgia, USA). Before analysis, 0.2 g of each biochar was degassed
under vacuum (${<}$10~$\upmu$mHg) for 24 h at
200~\textdegree C. Investigations were afterward performed at
273~K with progressive adsorption of CO$_{2}$ gas
doses. Porosity profile was determined using the CO$_{2}$
NLDFT (Non-localized density functional theory) slit pores
model~\cite{44}. Mineral composition of the biochars \pagebreak was performed
through X-ray fluorescence (XRF) analysis. Generally, 200 mg of ROP and
IROP biochar powders were mixed with 100 mg of boric acid (used as a
binder), transformed into pellets then analyzed using a PHILIPS PW2540
(Amsterdam, Netherlands)~\cite{45}.

\subsection{Viability assay: Compatibility of biochars and AMF}

\textit{Funneliformis mosseae} spores used in this study were extracted
from sporocarps and maintained in distilled water at 4~\textdegree C.
To assess the viability of \textit{Funneliformis mosseae} spores after
formulation with biochars (and thereafter their germination potential),
an assay using 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyl-
2H-tetrazolium bromide (MTT) as an indicator was
performed~\cite{46,47}. The viability rate was evaluated using a
colorimetric assay, based on the reduction of a yellow tetrazolium salt
to purple formazan crystals by metabolically active cells. 

Fifty spores of \textit{Funneliformis mosseae} were inoculated in 1 mL
of demineralized water containing 1~g of synthesized biochars (ROP or
IROP). Two incubation periods (3 and 7 days) were considered. The test
was limited to 7 days due to the fragile character of the glomus that
over time tends to wither in water and at ambient temperature. Spore
recovery was carried out by wet sieving using a stainless-steel sieve
(SAULAS, N\textdegree 0226069) with a mesh sieve of 200~$\upmu$m. 
Recovered spores were stained with a 0.5 g${\cdot}$mL$^{-1}$
solution of MTT and incubated at room temperature in the dark for 48 h,
in contact with air. For control treatments, the corresponding assays
were performed without biochars. All these treatments were done in
three replicates.

At the end of the staining time, spores were observed using a
stereoscopic microscope (Nikon.SMZ-1B). The recovered viable spores
turned pink or purple while non-viable ones remained colorless, black
or blue (Figure~\ref{fig1}). 

\begin{figure*}
\includegraphics{fig01}
\caption{\label{fig1} Spores formulated with biochar (a) and Spores (b)
before and after staining with MTT solution (0.5~g${\cdot}$L$^{-1}$). The viable
spore (b) is clearly stained as defined purples spheres while the
non-viable spore is not stained and remain transparent brown. Images
were observed using a Nikon SMZ-1B stereoscopic microscope under a
total magnification of 100${\times}$.}
\end{figure*}


The viability rate was estimated by the following Equation (\ref{eq1}): 
{\begin{equation}
\label{eq1}
\text{Viability rate } (\%)=\frac{\text{Number of pink or purple spores}}{\text{Number of total recovered spores}}
\end{equation}}\unskip
All data were statistically analyzed by ANOVA (one-way) using XLSTAT
2021.2.2 and the Fisher LSD test was performed at $p = 0.05$.

\subsection{Greenhouse trial on maize crops cultivation}

The experiment was conducted in a greenhouse at temperatures between
22--30~\textdegree C. The soil used was a non sterile agronomic soil
collected from the top layer of soil in Guemar, located in the Grand
Est region of France (48\textdegree 11$'$23.93$''$N 7\textdegree
23$'$25.0$''$E). The sandy silt soil was analyzed in a laboratory and its
characteristics were determined, those are presented in Table~\ref{tab1}.

\begin{table}
\caption{\label{tab1} Physical and chemical characteristics of the used soil}
\fontsize{9.5}{11.5}\selectfont
\tabcolsep=3pt
\begin{tabular}{llcc}  
\thead
Analysis & Method & Results & Unit \\ 
\endthead
\parbox[t]{2.5cm}{\raggedright\hangindent .7pc Organic matter (MO)}\vspace*{2pt} & NF ISO 14235 & 1.6\% & \% \\ 
Soil pH & NF EN 13037 & 7.1 & -- \\ 
Total nitrogen (N) & NF ISO 13878 & 0.1 & \% \\ 
\parbox[t]{2.5cm}{\raggedright\hangindent .7pc Phosphore Olsen (P$_2$O$_5$)}\vspace*{2pt} & NF ISO 11263 & 130 & mg${\cdot}$kg$^{-1}$ \\ 
Potassium (K$_2$O) & NF X31-108 & 412 & mg${\cdot}$kg$^{-1}$ 
\botline
\end{tabular}
\end{table}


After air-drying, the soil was subsequently ground and sieved through a
10-mm sieve, without sterilization. Nine treatments combining one
biochar (ROP or IROP) at a level of 2.5 g/kg of soil and inoculated or
not with \textit{Funneliformis mosseae} at two concentrations (30 and
125 spores/kg of soil) were arranged in an entirely randomized block
design (Table~\ref{tab2}). All treatments were applied locally during the sowing
and replicated 4 times. For each treatment, three maize seeds were
planted into a culture pot containing 1.5 kg of dry soil, which was
reduced to one per pot after seven days. The soil's water content was
maintained during the experiment at 70\% of its field capacity. The
trial last for 47 days or until the 7-leaf stage.

\begin{table}
\caption{\label{tab2} Summary of treatments for greenhouse trials (4
replicates per treatment)}
\fontsize{9.1}{11.5}\selectfont
\tabcolsep=1pt
\begin{tabular}{cccc}
\thead
\parbox[t]{1.5cm}{\centering Treatment name} & 
\parbox[t]{2cm}{\centering Biochar \mbox{quantity (g/kg)}} & 
\parbox[t]{1.5cm}{\centering Biochar \mbox{feedstock}} & 
\parbox[t]{1.7cm}{\centering Mycorrhizal inoculant}\vspace*{2pt} \\ 
\endthead
Control & None & None & None \\ 
Sp30 & None & None & 30 spores/kg soil \\ 
Sp125 & None & None & 125 spores/kg soil \\ 
ROP & 2.5 g/kg & ROP & None \\ 
ROP30 & 2.5 g/kg & ROP & 30 spores/kg soil \\ 
ROP125 & 2.5 g/kg & ROP & 125 spores/kg soil \\ 
IROP & 2.5 g/kg & IROP & None \\ 
IROP30 & 2.5 g/kg & IROP & 30 spores/kg soil \\ 
IROP125 & 2.5 g/kg & IROP & 125 spores/kg soil
\botline
\end{tabular}
\end{table}

The height of the plants was measured using a tape measure. The
investigation of relative chlorophyll content was conducted with a 
SPAD~502 \mbox{(Konica Minolta, Inc.).}
Measurements were taken from the fully
expanded functional leaves at six growth stages; 2nd-leaf, 4th-leaf,
5th-leaf, 5 to 6th-leaf and 7th-leaf stage. Additionnaly, the total
height increment (growth rate) for each treatment was calculated
following this formula: 
{\begin{eqnarray*}
&&\text{Height Increment }(\mathrm{cm}{\cdot}\mathrm{day}^{-1})\\
&&\quad=\frac{\text{Total Height Increment }(\mathrm{cm})}{\text{Total Number of Days}}\\[-7pt]
\end{eqnarray*}}\unskip
Height Increment by Day $=$ Total Height Increment/Total Number of Days.

The total height increment for each treatment was calculated by
subtracting the initial height measurement from the final height
measurement. The total number of days for each treatment was calculated
by subtracting the date of the first measurement from the date of the
last measurement and adding 1 (since we are counting the first and last
days). At the end of the experiment, the dry weight of the aerial
biomass was determined after drying at 40~\textdegree C in an oven for
several days. The dry biomass was then analyzed for C and nutrient
concentration (N, P, and K). The nutrient uptake was then obtained by
the following formula (\ref{eq2}):
{\begin{eqnarray}
\label{eq2}
&&\text{Nutrient uptake }(\mathrm{mg})\nonumber\\
&&\quad=\frac{\text{Nutrient concentration }(\%)\times \text{Dry matter }(\mathrm{mg})}{100}\nonumber\\
\end{eqnarray}}\unskip
Fungal colonization by mycorrhizae was assessed by clearing the roots
in 10\% KOH at 75~\textdegree C for 1.5~h followed by staining
with 0.05\% trypan blue in lactic acid for 15 h. Stained roots were
mounted on a glass microscope slide and assessed for colonization under
a compound microscope at ${\times}$400 magnification~\cite{3}.
Mycorrhizal development such as the frequency of colonization (\%F) and
the intensity of colonization (\%M) was evaluated by the method
described in Trouvelot~\cite{3}. All data were statistically
analyzed by ANOVA (one-way) using XLSTAT 2021.2.2. The Fisher LSD test
was performed at\break $p = 0.05$.

\section{Results and discussions}\label{sec3}

\subsection{Physico-chemical characterization of the biochars}

Biochars produced from the raw and IROP were characterized using
multiple analysis techniques. Firstly, the proximate analysis revealed
a relatively high content of fixed carbon for both materials up to 59\%
and 61\% for ROP and IROP, respectively (Figure~\ref{fig2}). The addition of
OMWW slightly enhanced the organic content in the lignocellulosic
material, which was converted into additional fixed carbon content
after pyrolysis~\cite{12}. A slight increase in the volatile matter
content (Figure~\ref{fig2}) was also noticed, which was set to be 
relatively low
(17\% and 18\%, respectively) compared to the initial feedstocks (67\%
and 69\% for ROP and IROP, respectively~\cite{45,48}).

\begin{figure}
\vspace*{-2pt}
\includegraphics{fig02}
\vspace*{-2pt}
\caption{\label{fig2} Proximate analysis of raw ROP and IROP and its
derivative biochars (results related to the raw materials were imported
from our previous publications~\cite{45,48}).}
\vspace*{-2pt}
\end{figure}

On the other hand, ash content slightly decreased for IROP by about
3\%. Despite the successful retention of some minerals during the
impregnation process, it is possible that the acidic aspect of the OMWW
led to the leaching of some minerals and therefore the decrease in ash
content. These results were further confirmed by mineral analysis
(Table~\ref{tab3}) suggesting a rather specific variation of minerals after
impregnation with an increase in content for potassium, phosphorus,
sodium, and silicon and a decrease in calcium, magnesium, and sulfur
concentrations. The effect of OMWW impregnation was also noticeable
when investigating the porosity of the produced biochars, and the
results were depicted in Figure~\ref{fig3}. The physical adsorption/desorption
isotherm analysis (BET/CO$_{2}$) indicates a decrease in the specific
surface area from about 150 m$^{2}$/g for ROP to 137 m$^{2}$/g for IROP
(Figure~\ref{fig3}). Moreover, mean pores volume decreased from 0.5 to 0.36
cm$^{3}$/g for the same samples, respectively; the
excessive presence of specific minerals such as magnesium may have led
to the blocking of this ultra-micro porosity, thus reducing their
specific surface area and micropores volume~\cite{49}. 

\begin{figure}[t!]
\vspace*{-3pt}
\includegraphics{fig03}
\vspace*{-3pt}
\caption{\label{fig3} (a) CO$_2$ adsorption isotherm and (b) the related
pore size distribution of ROP and IROP (SSA: specific surface area,
m$^{2}$/g; pores width was determined using the slit pores CO$_2$ NLDFT
model).}
\vspace*{-3pt}
\end{figure}

\begin{table}
\caption{\label{tab3} Chemical elemental of ROP and IROP-derived
biochars produced at 500~\textdegree C~\cite{32}}
\begin{tabular}{lcccccc}
\thead
 & Unit & C  & N  & S  & O  & H  \\ 
\endthead
\parbox[t]{1.2cm}{\hangindent .5pc ROP biochar}\vspace*{3pt} & wt\% & 63.83 & 0.98 & 0.07 & 8.06 & 2.54 \\ 
\parbox[t]{1.2cm}{\hangindent .5pc IROP biochar}\vspace*{3pt} & wt\% & 71.08 & 0.71 & 0.04 & 4.91 & 2.44
\botline
\end{tabular}
\end{table}

Furthermore, the impregnation of OMWW onto ROP significantly affected
the final content of carbon (Tables~\ref{tab3} and~\ref{tab4}).
Besides, the XRF analysis highlighted the decrease in mineral
composition after OMWW impregnation where a significant number of
peaks, especially related to CaCO$_{3}$ and KCl presented much lower
intensities when comparing IROP to ROP spectra~\cite{32}. Nevertheless,
a slight increase in potassium content was noted after impregnation
from 4.71 to 5.54\%, attributed to its incorporation and high affinity
with the lignocellulosic structure of olive\break pomace.

\begin{table*}
\caption{\label{tab4} Mineral composition of ROP and IROP-derived
biochars produced at 500~\textdegree C~\cite{32}}
\begin{tabular}{lccccccccc}
\thead
 & Unit & Na  & Mg & Si & P & Cl & K & Ca & Fe \\ 
\endthead
ROP biochar & wt\% & 1.47 & 0.90 & 2.18 & 0.30 & 1.32 & 4.71 & 7.40 & 0.53 \\ 
IROP biochar & wt\% & 0.85 & 0.67 & 1.80 & 0.34 & 1.16 & 5.54 & 5.80 & 0.45 
\botline
\end{tabular}
\end{table*}

{\vspace*{-2pt}}
\subsection{Viability assay of biochar inoculated AMF}
{\vspace*{-2pt}}

The viability of spores inoculated with the ROP and IROP biochars was
assessed using the MTT staining\break assay. In control, a higher proportion
of viable spores was observed after 3-days incubation time (87\% viable
spores) compared to 7-days incubation time (68\% viable spores) 
(Figure~\ref{fig4}).
This is probably due to incubation conditions leading to cells
burst and spores shell fragments. However, although a decrease in spore
viability is observed with biochar after 3 days compared to control,
the viability seems to be constant over time, with slight increase of
viability between 3 and 7 days. The viability of spores was 48 and 55\%
after 3 and 7 days respectively for ROP inoculated biochars. It was
about 25 and 40\% after 3 and 7 days respectively for IROP inoculated
biochars. The inoculation on biochars seems to have an overall negative
impact on spores' viability, but over time biochars appears to limit
the spores' death observed in control between 3 and 7 days (Figure~\ref{fig4}).
Based on these results, AMF and biochars have been applied separately
for greenhouse tests, to limit spores' death.

\begin{figure}
\includegraphics{fig04}
{\vspace*{-2pt}}
\caption{\label{fig4} Viability rate of recovered spores (MTT
treatment) and spores' losses of AMF formulated without or with the two
biochars (ROP and IROP) after an incubation time of 3 days and 7 days
(data are means of three replicates ($n = 3$), letters (a, b and ab)
represents the significant difference at $p  <  0.05$).}
{\vspace*{-2pt}}
\end{figure}

The results of the viability assay showed that AMF spores' viability
(spores' germination) decreased after formulation with biochars.
However, compared to the control treatment (solution of spores in
water), the viability of spores is maintained with biochar formulation.
Biochars seem to have a deleterious effect on spores' germination (more
pronounced with IROP than ROP). The loss of viability of spores might
be due to osmotic pressure causing the collapse of spores' membrane
through cytolysis and/or spores burst caused by the biochars particles.
Numerous studies were carried out regarding the interactions of AMF and
biochar in soil but only few seemed to have focused on the direct
impact of biochar on AMF viability before application to soil. However,
some studies have investigated the colonization of plants by AMF in
adsorptive substrate systems, and their survival and long-term
infectivity in substrate such as peat. In Hu \etal~\cite{52} study,
350 g of a fungal inoculum containing \textit{Rhizophagus irregularis}
(BEG 140, known as \textit{Glomus intraradices)} was added to a layer
of biochar in a PVC column filled with gravel (15~cm depth), biochar
(20 cm depth) and sand (15 cm depth). Hu \etal~\cite{52} 
demonstrated that AMF colonization in biochar systems was lower than in
perlite and vermiculite systems. Thus, AMF development may have been
inhibited by the toxic substances contained in biochar such as dioxins,
ethylene, polycyclic aromatic hydrocarbons, phenolic compounds,
volatile compounds, and heavy metals~\cite{52}. The analysis of the
prepared biochars showed that the volatile matter proportion in ROP and
IROP were respectively 17\% and 18\% which could explain the negative
effect on AMF spore germination. Concerning the evolution of
infectivity of AMF inoculum (such as  \textit{F.~mosseae}) in a
substrate (peat), a study conducted in 2019, showed that the
infectivity of AMF was negatively affected by parameters such as the
temperature of storage especially mild temperature 
(18~\textdegree C--25~\textdegree C)~\cite{53}. The results of our direct formulation
of biochar with spores confirmed the negative impact of biochar on
spores at short time but the same level of spores' viability was
maintained during storage. Therefore, to avoid these negative effects,
biochar and AMF mixing before application to soil has to be avoided, in
addition more research on the formulation need to be conducted to
better understand the effects of parameters such as biochar pH,
nutrients levels and particle sizes on\break AMF spores.

\subsection{Effects of biochar and AMF treatments on maize growth parameters and SPAD values}

   According to Figure~\ref{fig5}, all treatments using biochars and/or AMF
spores had no significant effect on the maize plants' growth compared
to the control. Maize growth was homogeneous throughout the 47 days of
cultivation, either on plant height (values between 60.4 and 67 cm, no
significant statistical effect) or shoot biomass (values between 1.41
and 1.6 cm, no significant statistical effect).

\begin{figure*}
\includegraphics{fig05}
\caption{\label{fig5} Effect of biochar and AMF on the maize growth (A)
and shoot production (B) (DW $=$ Dry weight) at the harvest. The error
bars indicate the standard deviation ($n = 4$). Different letters within
each parameter indicate significant differences at $p \leq 0.05$.
Sp30 and Sp125 $=$ 30 and 125 spores/kg of soil, ROP: Raw Olive pomace
biochar (2.5 g/kg of soil), ROP30 and ROP125 $=$ Raw biochar (2.5 g/kg of
soil) $+$ 30 or 125 spores/kg of soil, IROP: Impregnated olive pomace
biochar (2.5 g/kg of soil), IROP30 and IROP125 $=$ Impregnated biochar
(2.5 g/kg of soil) $+$ 30 or 125 spores/kg of soil).}
\end{figure*}

The use of ROP and IROP combined AMF did not affect significantly maize
plants growth (Figure~\ref{fig6}) and biomass production. The average growth
rate (cm${\cdot}$days$^{-1}$) between the treatments was similar
and varied by 1.2 to 1.4 cm${\cdot}$day$^{-1}$. 

\begin{figure}
\includegraphics{fig06}
\caption{\label{fig6} Plant growth rates (plant eight increment) at the
6 measurements times (data are means ($n=4$)).}
\end{figure}

Our results are in agreement with those reported by Mau and
Utami~\cite{54}, they investigated the effects of various doses of
cow dung-derived biochar amendment (0, 5, and 7.5 g/kg soil) and AMF
inoculation (0, 5, 10 and 15 spores/kg soil) on the growth of maize. No
effect of biochar on plant growth, with or without AMF inoculation, was
observed after a growing period of 8 weeks~\cite{54}. However, Liu
\etal~\cite{55} proved that the combination of \textit{Glomus
intraradices} BEG 141 (20~g/kg soil) with wheat straw biochar (20~g/kg
soil) increased maize plant biomass production by 70\% after 14 weeks,
while biochar or AMF alone have lower effects. Moreover, Zhuo
\etal~\cite{56} when studying the effect of biochar (20~g/kg) and
AMF (5\% v/v) adding on maize growth in contaminated soils, proved that
AMF$+$biochar had a positive impact on maize growth and biomass
production after 12 weeks, whereas biochar or AMF alone didn't\break impact
maize biomass (respectively 28 and 52\%). Abou El Seoud~\cite{57}
showed that biochar adding at doses of 0 and 10 g/kg soil) and AMF (250
spores/kg soil) has a positive impact on maize growth in a calcareous
soil after 6 weeks. Li and Cai~\cite{58} demonstrated that biochar
(50 g/kg soil) and AMF (700~spores/kg soil) in combination have a
positive impact on plant height after 12 weeks. All these experiments
seem to indicate a positive impact of biochar and AMF on maize growth
with high biochar rates (up to 50 g/kg soil) and with high AMF
inoculation (up to 700 spores/kg soil). In other side, the culture
duration could also impact the maize crops growth. Various field
experiments showed that biochars can have a time-delayed effect on
crops growth~\cite{59,60,61,62}.

The results of chlorophyll content measurements represented by the SPAD
values at the 4th leaf stage showed significant effects for several
treated modalities compared to the control (Table~\ref{tab5}). When the spores
were applied alone (without biochar), there was a slight decrease in
the SPAD values (compared to the control). The same trend was observed
when the ROP biochar was applied alone, the SPAD value decreased by
6.4\% compared to the control. On the contrary, when AMF (Sp30 and
Sp125) were combined to ROP biochar, a positive effect occurred, i.e.,
a slight increase in the SPAD values compared to the control and the
treatments with AMF or ROP alone (Table~\ref{tab5}). Thus, IROP biochar is the
most effective product but only when applied alone. Otherwise, its
combination with the AMF caused a slight decrease in the SPAD value but
still higher than that of the control. \looseness=-1

\begin{table*}
\caption{\label{tab5} Effects of treatment on SPAD values at the
stages 4th and 7th-leaf of the maize plants\vspace*{-4pt}}
\fontsize{10}{11.3}\selectfont\begin{tabular}{lcccccc}  
\thead
\xmorerows{1}{Treatments}  & \multicolumn{6}{c}{SPAD Values} \\\cline{2-7}
 & 2-Leaf & 4th-Leaf & 5th-Leaf & 5--6th-Leaf & 6th-Leaf & 7th-leaf \\ 
\endthead
Control & 30 ${\pm}$ 3.4a & 24.9 ${\pm}$ 0.7bcd & 25 ${\pm}$ 1.8a & 23 ${\pm}$ 2.1ab & 18.6 ${\pm}$ 2.2b & 17.3 ${\pm}$ 1.0a \\ 
Sp30 & 29.5 ${\pm}$ 2.3a & 22.8 ${\pm}$ 3.8d & 23.7 ${\pm}$ 1.6a & 21.7 ${\pm}$ 1.6bc & 19.3 ${\pm}$ 1.4ab & 17.5 ${\pm}$ 1.3a \\ 
Sp125 & 28.2 ${\pm}$ 4.8a & 21.7 ${\pm}$ 1.6d & 23.2 ${\pm}$ 0.5a & 19.7 ${\pm}$ 1.6c & 19 ${\pm}$ 2ab & 16.8 ${\pm}$ 1.3a \\ 
ROP & 32.8 ${\pm}$ 3.1a & 23.3 ${\pm}$ 2.5cd & 23.9 ${\pm}$ 0.9a & 22 ${\pm}$ 1.7abc & 19.5 ${\pm}$ 1.7ab & 16.4 ${\pm}$ 0.3a \\ 
ROP30 & 30.6 ${\pm}$ 2.8a & 27.7 ${\pm}$ 3.6ab & 26.5 ${\pm}$ 2.2a & 24 ${\pm}$ 1.4ab & 21.6 ${\pm}$ 0.8a & 16.9 ${\pm}$ 0.9a \\ 
ROP125 & 28.2 ${\pm}$ 4.3a & 26.8 ${\pm}$ 3.8abc & 23.2 ${\pm}$ 1.6a & 21.4 ${\pm}$ 2.1bc & 21.4 ${\pm}$ 2a & 16.6 ${\pm}$ 1.3a \\ 
IROP & 31.7 ${\pm}$ 5.9a & 30.3 ${\pm}$ 1.3a & 23.8 ${\pm}$ 7.6a & 25 ${\pm}$ 2.1a & 21.6 ${\pm}$ 0.9a & 17.9 ${\pm}$ 1.5a \\ 
IROP30 & 30.6 ${\pm}$ 3.7a & 27.2 ${\pm}$ 3.1abc & 25.5 ${\pm}$ 3.1a & 23.2 ${\pm}$ 2.6ab & 19.6 ${\pm}$ 1.7ab & 16.6 ${\pm}$ 0.6a \\ 
IROP125 & 29.2 ${\pm}$ 2.9a & 27.8 ${\pm}$ 1.8ab & 26.5 ${\pm}$ 3.6a & 23.6 ${\pm}$ 2.1ab & 21.1 ${\pm}$ 2.8ab & 16.7 ${\pm}$ 2.1a
\botline
\end{tabular}
\tabnote{Values obtained are expressed as mean ${\pm}$ SE ($n = 4$). 
Different letters indicate the least significant difference (LSD test,
$p < 0.05$).}
\vspace*{-7pt}
\end{table*}

All SPAD values vary over time but decrease at the end (Table~\ref{tab5}), and
at the 7th leaf stage of maize, the chlorophyll contents of the plants
were homogeneous among the treatments indicating that there was no
significant effect ($p < 0.05$) of AMF or/and biochars
at the end of the cycle, despite some variations during growth. Effects
of biochar and/or AMF observed on SPAD values seem to be therefore\break
transitory.

At the end of the culture, the treatments did not have a significant
effect on the chlorophyll contents, as indicated by the measurements of
SPAD values (Table~\ref{tab5}). 
The SPAD measurements followed a particular
pattern of change over time, increasing early in the growth up to the
4th leaf stage and declining afterward until the 7th leaf-stage. The
decrease in SPAD values with the plant age regardless of the treatments
was also observed in previous studies on
maize~\cite{63,64,65}. It was demonstrated that there is a
correlation between chlorophyll content in leaves and the plant's
nutrient status~\cite{66,67,68} as N status. A study by Hammad
\etal~\cite{69}  showed a significant decrease in maize yield and
quality without nitrogen application. Since chlorophyll contents
(measured SPAD values) are proportional to the amount of nitrogen
present in the leaves~\cite{70,71}, the decrease in SPAD values
over time can be due to nitrogen deficiency in plants. When maize is
cultivated in optimal conditions, the plant tends to take up nitrogen
(N) during vegetative growth, and this N is later remobilized from
leaves to reproductive organs to support kernel
formation~\cite{72,73}. In the case of low availability of soil
N, this remobilization happens sooner in the growth stages~\cite{73}
which can explain the decrease of SPAD values.

\subsection{Effect of biochar and AMF treatments on nutrients uptake}

After 46 days of culture, total nitrogen uptake by the maize crop
increased overall with AMF treatments and biochar application (Tables~\ref{tab6}
and~\ref{tab7}). A minimum N concentration of around 1.19\% was observed in the
control assays while the highest N concentrations were found in the
plants treated with AMF (Sp30) and AMF combined with IROP biochar
(IROP30). Biochar addition slightly enhanced N concentration. Moreover,
modalities with biochar and spores were not significantly different
from those with biochar alone concerning N concentration. Sp30
Modalities were all higher than corresponding modalities without
spores. Some trends were observed for N content (general diminution of
N content with biochar compared to modalities without biochar), but
they were not significant. As for N concentration, modalities with 30
spores/kg soil were all higher than corresponding modalities without
spores, but this effect was significant only for control and Sp30
modalities. The statistical analysis of the P concentrations results in
maize plants showed few significant effects of treatments (Table~\ref{tab6}).
Sp125 showed a significant effect of AMF addition on P concentration
(but not on P content). Biochar treatments have no effect on P
concentration or content when comparing modalities with the same
spores' treatment.

\begin{table*}
\caption{\label{tab6} Effect of treatment on shoot N, P, K, and C
content}
\begin{tabular}{lcccc}  
\thead
\xmorerows{1}{Treatments}  & \multicolumn{4}{c}{{Concentration (\%)} } \\\cline{2-5}
 & N & P & K & C \\ 
\endthead
{Control}  & 1.19 ${\pm}$ 0.17c & 0.49 ${\pm}$ 0.03a & 3.53 ${\pm}$ 0.21abc & 44.48 ${\pm}$ 0.25b \\ 
{Sp30}  & 1.72 ${\pm}$ 0.52a & 0.48 ${\pm}$ 0.01a & 3.75 ${\pm}$ 0.12ab & 45.63 ${\pm}$ 0.57a \\ 
{Sp125}  & 1.24 ${\pm}$ 0.08bc & 0.42 ${\pm}$ 0.03b & 3.22 ${\pm}$ 0.14bc & 45.83 ${\pm}$ 0.15a \\ 
{ROP}  & 1.36 ${\pm}$ 0.06abc & 0.47 ${\pm}$ 0.02ab & 3.60 ${\pm}$ 0.12abc & 45.93 ${\pm}$ 0.23a \\ 
{ROP30} & 1.50 ${\pm}$ 0.3abc & 0.44 ${\pm}$ 0.01ab & 3.55 ${\pm}$ 0.15abc & 45.88 ${\pm}$ 0.53a \\ 
{ROP125}  & 1.24 ${\pm}$ 0.23bc & 0.43 ${\pm}$ 0.03b & 3.08 ${\pm}$ 0.13c & 45.75 ${\pm}$ 0.53a \\ 
{IROP}  & 1.23 ${\pm}$ 0.15bc & 0.48 ${\pm}$ 0.02a & 3.84 ${\pm}$ 0.12a & 45.80 ${\pm}$ 0.22a \\ 
{IROP30}  & 1.58 ${\pm}$ 0.2ab & 0.46 ${\pm}$ 0.03ab & 3.44 ${\pm}$ 0.24abc & 45.98 ${\pm}$ 0.33a \\ 
{IROP125}  & 1.35 ${\pm}$ 0.13bc & 0.45 ${\pm}$ 0.05ab & 3.38 ${\pm}$ 0.4abc & 46.13 ${\pm}$ 0.53a 
\botline
\end{tabular}
\xxtabnote{Values obtained are expressed as mean ${\pm}$ SE ($n = 3$). 
Different letters indicate the least significant difference (LSD test, $p < 0.05$).}
\vspace*{5pt}
\end{table*}

\begin{table*}
\caption{\label{tab7} Effect of treatment on shoot N, P, K, and C
content}
\begin{tabular}{lcccc}  
\thead
\xmorerows{1}{Treatments}  & \multicolumn{4}{c}{Content (mg)} \\\cline{2-5}
 & N & P & K & C \\ 
\endthead
{Control} & 21.71 ${\pm}$ 1.73b & 8.97 ${\pm}$ 1.28a & 65.14 ${\pm}$ 7.25a & 825.37 ${\pm}$ 127.71a \\ 
{Sp30} & 29.17 ${\pm}$ 12.4a & 7.93 ${\pm}$ 1.35ab & 61.85 ${\pm}$ 10.53ab & 755.68 ${\pm}$ 143.68a \\ 
{Sp125} & 21.74 ${\pm}$ 4.2ab & 7.32 ${\pm}$ 1.16ab & 56.35 ${\pm}$ 10.49abc & 808.14 ${\pm}$ 181.4a \\ 
{ROP} & 20.42 ${\pm}$ 6.07ab & 7.00 ${\pm}$ 2.05ab & 53.82 ${\pm}$ 14.8abc & 686.82 ${\pm}$ 191.19a \\ 
{ROP30} & 24.10 ${\pm}$ 6.44ab & 7.10 ${\pm}$ 0.81ab & 57 ${\pm}$ 7.16abc & 734.79 ${\pm}$ 71.55a \\ 
{ROP125} & 20.22 ${\pm}$ 7.47ab & 6.84 ${\pm}$ 0.92b & 49.11 ${\pm}$ 7.75bc & 734.12 ${\pm}$ 146.89a \\ 
{IROP} & 18.74 ${\pm}$ 2.1b & 7.32 ${\pm}$ 1.11ab & 58.77 ${\pm}$ 8.05abc & 703.36 ${\pm}$ 112.19a \\ 
{IROP30} & 23.39 ${\pm}$ 7.38ab & 6.75 ${\pm}$ 1.61b & 50.33 ${\pm}$ 10.54abc & 679 ${\pm}$ 174.37a \\ 
{IROP125} & 18.33 ${\pm}$ 6.81b & 6.09 ${\pm}$ 2.26b & 45.68 ${\pm}$ 16.71c & 647.64 ${\pm}$ 289.67a 
\botline
\end{tabular}
\xxxtabnote{Values obtained are expressed as mean ${\pm}$ SE ($n = 3$). 
Different letters indicate the least significant difference (LSD test, $p < 0.05$).}
%\vspace*{4pt}
\end{table*}

   The statistical analysis of the results of the K concentrations in
maize plants showed few significant effects of treatments (Table~\ref{tab6}).
Comparing modalities with the same spores' treatment or the same
biochar effect showed no significant effect of these treatments on K
concentration or content. 

  The results of the C concentrations in maize plants showed no
significant effects of treatments on C\break uptake and had a minimal impact
on the C concentration of the maize plants. All the concentrations were
significantly different ($p < 0.05$) from the control and were
very similar. C content was similar for all modalities.

The results of analysis of plants' nutrients concentrations and
contents showed that the Sp30 and IROP30 treatments enhance the N
uptake respectively by 45\% and 33\% compared to control. On the other
hand, there was no significant effect of the treatments on the C and K
uptake while in P concentrations, a negative effect of treatments was
observed. This finding can be explained by the low doses of applied
biochar and AMF. Mau and Utami~\cite{54} observed an increase in P
uptake with all treatments: biochar, AMF and combined treatment under
similar experimental conditions. Wang \etal~\cite{74} demonstrated
that biochar application to soil increased C sequestration, and the
same result was observed with the combination of biochars with
mycorrhizal fungi~\cite{75,76}.

AMF is known to improve P uptake in the plant \cite{77}. Under
higher doses of biochar, its application could directly or indirectly
improve the nutrients uptake and bioavailability of P~\cite{78} due
to a less binding to non-soluble forms~\cite{79}. An increase of P
uptake by maize has been observed in some studies as an effect of
biochar and/or AMF addition in soil~\cite{56,57,58,69}.
Thus, it is well proved that biochar and AMF can improve nutrients
uptake depending on their applied doses as well as the culture\unskip\break
duration.


\subsection{Effect of biochar and AMF treatments on\break mycorrhizal colonization}

The mycorrhizal colonization frequency (F\%) indicates the number of
roots where mycorrhizae are observed. The mycorrhizal intensity (M\%)
reflects the abundance of mycorrhizae observed on each mycorrhized
fragment. Mycorrhizal frequencies F\% of treated plants under described
treatments (Table~\ref{tab8}) were almost similar, except for the control. This
indicates that all root systems were colonized by AMF, even the
modalities without AMF treatment. During the inoculation of the
treatments, cross-contamination was avoided by not using the same
instruments for the differents pots, which can suggest then the
presence of indigenous AMF in soil. A single treatment of ROP or IROP
did not enhance the plant's colonization by indigenous AMF even if the
F\% increase compared to the control. The inoculation of AMF
significantly improved mycorrhizal colonization intensity with a
dose-dependent relationship (Table~\ref{tab8}). A single inoculation of AMF at a
minimum level of 30 spores per kg of soil results in an increase of AMF
colonization by 113.9\% compared to the control. Biochar treatment has
no effect on mycorrhizal intensity of maize roots.

\begin{table}
\caption{\label{tab8} Effects of treatment on mycorrhizal colonization
frequency (F\%) and intensity (M\%)}
\begin{tabular}{lcc}                     
\thead
\xmorerows{1}{{Treatments}}  & 
{\parbox[t]{1.7cm}{\centering Mycorrhizal frequency}}  
& {\parbox[t]{1.7cm}{\centering Mycorrhizal intensity}}
\vspace*{2pt}  \\\cline{2-3}
 & F\% & M\% \\ 
\endthead
{Control} & 90.83 ${\pm}$ 6.87b & 21.5 ${\pm}$ 13.09c \\ 
{Sp30} & 100 ${\pm}$ 0.0a & 46.03 ${\pm}$ 11.23b \\ 
{Sp125} & 98.89 ${\pm}$ 1.92a & 74.24 ${\pm}$ 16.56a \\ 
{ROP} & 100 ${\pm}$ 0.0a & 22.69 ${\pm}$ 4.99c \\ 
{ROP30} & 100 ${\pm}$ 0.0a & 57.51 ${\pm}$ 12.65ab \\ 
{ROP125} & 100 ${\pm}$ 0.0a & 64.07 ${\pm}$ 15.23ab \\ 
{IROP} & 99.17 ${\pm}$ 1.67a & 23.79 ${\pm}$ 4.14c \\ 
{IROP30} & 100 ${\pm}$ 0.0a & 55.62 ${\pm}$ 18.89ab \\ 
{IROP125} & 99.17 ${\pm}$ 1.67a & 64.17 ${\pm}$ 13.85ab
\botline
\end{tabular}
\tabnote{Values obtained are expressed as mean ${\pm}$ SE ($n = 3$).
Different letters (a, ab or b) indicate a least significant difference
(LSD test, $p < 0.05$).{\vspace*{-3pt}}}
\end{table}

 The results of the root staining assay showed colonization of all
plants roots by AMF (Figure~\ref{fig7}). According to the results 
(Table~\ref{tab8}),
biochar addition to the soil did not significantly enhance AMF root
colonization and did not have a negative impact on it either. However,
AMF treatments provided new propagules and enhance maize root
colonization. It is important to underline that biochar made from woody
biomass addition to agricultural soils could increase the AMF spore
number in the soil~\cite{70,71}. Indeed, when studying the
effects of olive waste-based compost, a wood-based biochar, and their
combination on soil fertility and maize growth, these authors found
that a single application of biochar had a negative impact on
mycorrhizal symbiosis after 3 months while the\break compost gave the best
results in terms of mycorrhizal frequency (F\%) and intensity of
mycorrhizal colonization (M\%) and F\%. 

\begin{figure*}
{\vspace*{3pt}}
\includegraphics{fig07}
{\vspace*{4pt}}
\caption{\label{fig7} Root sections stained with trypan blue showing
arbuscular mycorrhizal structures. Roots were observed at a
magnification of ${\times}$100 (A,~C) and ${\times}$400 (B). (A) A
section of maize root colonized by AMF. (B) Vesicles and hyphae. (C)
Darkly cells containing arbuscules.}
\vspace*{3pt}
\end{figure*}

Moreover, Mau and Utami~\cite{54} observed an initial decrease of
mycorrhization in biochar modalities, but after 8 weeks, mycorrhization
in modalities treated with biochar was higher than control. Likewise,
Li and Cai~\cite{58} observed a positive effect of biochar addition
on AMF colonization after 12 weeks in a sterilized soil. All these
results showed the complexity of the interactions between biochars and
AMF which can be dependent on factors like soil microorganisms activity
or biochar porosity and particles sizes~\cite{80}. Biochar contains
pores of varying sizes, which can affect the retention and availability
of water, nutrients, and microorganisms. Large biochar particles with
large pores may allow water to drain quickly, reducing the moisture
available for mycorrhizal fungi to grow and colonize plant roots. On
the other hand, small biochar particles with smaller pores may retain
more water and provide a more favorable microenvironment for AMF. In
another study, Vicia faba pre-germinated seeds were seedeed in soils
amended with OMWW at 0, 10, and 30 days after OMWW
treatments~\cite{81}. It was found that OMWW application reduced but
did not eliminate AMF root colonization only when the seeding was done
right after the treatments (Day 0).\break This suggests that applying biochar
at the same time as AMF is not an interesting practice. Biochar can be
used as a growth support for mycorrhiza. However,  soil amendment and a
sufficient period of  incubation are required.

\section{Conclusions}\label{sec4}

   This study was performed in order to evaluate the interest to use
olive pomace-based biochar as an amendment and its impact on AMF spore
viability and plant colonization. Preliminary results showed that the
use of ROP-biochars and IROP-biochars significantly decreased spores'
viability. The application of biochars and AMF treatments to soil did
not have a significant effect on maize plants growth, biomass
production and shoot chlorophyll content. Moreover,\unskip\break AMF treatments did
not significantly affect the nutrients uptake except for the Sp30 and
IROP30 treatments which improved N uptake. The application of biochars
(ROP and IROP) didn't change the maize root colonization by mycorrhiza,
which is probably due to the direct effect of biochar on spores'
germination and on the short time of plant growth. Considering above
results, longer experiments should be performed in order to better
understand and to precisely assess the long-term influence of those
biochars and AMF on crops. 

   The current study confirms that the usefulness of olive mill wastes
derived biochars applied alone or in combination with mycorrhiza is a
complex process and depends on several parameters. Indeed, even if
biochar can be used as a support for mycorrhizae growth, it is
important to control the level of\unskip\break colonization of the formulated
biochar before soil application. The results obtained in this work have
shown that the direct application of mycorrhiza spores on biochar's can
cause an immediate negative effect that may be due to the chemical
characteristics of biochar (pH, element content\,\ldots). While data on
biochar effects on mycorrhiza are accumulating, there are several
important gaps in the knowledge on these interactions. It is possible
that negative or neutral effects have been under-reported.

The potential synergism between biochar and mycorrhizal for soil
management practices depend on the environmental circumstances (e.g.,
soil nutrient content, plants species) and biochar parameters (e.g.,
quality and application rate). 

\section*{Conflicts of interest}
Authors have no conflict of interest to declare.

\section*{Acknowledgements} 

This work was funded by FERTICHAR project, through the ARIMNet2 Joint
Call by the following funding agencies: MHESRT (Tunisia), ANR (France),
and HAO DEMETER (Greece). The authors gratefully acknowledge these
funding organisms for their support.

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