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\DOI{10.5802/crchim.313}
\datereceived{2023-07-05}
\daterevised{2024-03-04}
\dateaccepted{2024-04-25}
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\dateposted{2024-08-27}
\begin{document}

\begin{noXML}

\makeatletter

%\makeatletter
%\def\TITREspecial{\relax}
%\def\cdr@specialtitle@english{GDR Prom\'eth\'ee - French Research Network on \textit{Hydrometallurgical Processes for Primary and Secondary Resources}}
%\def\cdr@specialtitle@french{GDR Prom\'eth\'ee - Groupement de Recherche sur les Proc\'ed\'es Hydrom\'etallurgiques pour la Gestion Int\'egr\'ee des Ressources Primaires ...}
%\makeatother


\CDRsetmeta{articletype}{research-article}

\shortrunauthors

\title{Lessons learned from the upscaling of an iron bio-oxidation
process to improve uranium recovery}

\alttitle{Enseignements tir\'{e}s des travaux men\'{e}s pour le
d\'{e}ploiement sur un site industriel d'un proc\'{e}d\'{e} de
bio-oxydation de fer ferreux visant \`{a} am\'{e}liorer la
r\'{e}cup\'{e}ration d'uranium}

\author{\firstname{Agathe} \lastname{Hubau}\CDRorcid{0000-0002-3896-7220}}
\address{BRGM, F-45060 Orl\'{e}ans, France}
\email[A. Hubau]{a.hubau@brgm.fr}

\author{\firstname{Kathy} \lastname{Bru}\CDRorcid{0000-0003-2502-0561}\IsCorresp}
\addressSameAs{1}{BRGM, F-45060 Orl\'{e}ans, France}
\email[K. Bru]{k.bru@brgm.fr}

\author{\firstname{Safiatou} \lastname{Idrissa Oumarou}}
\address{Soma\"{i}r, Arlit, Niger}
\email[S. Idrissa Oumarou]{safiatou.idrissa-oumarou@orano.group}

\author{\firstname{C\'{e}cile} \lastname{Leycuras}}
\address{Orano Mining, CIME, 2, route de Lavaugrasse, 87250 Bessines
sur Gartempe, France}
\email[C. Leycuras]{cecile.leycuras@orano.group}

\keywords{\kwd{Bio-oxidation}
\kwd{Ferrous iron}
\kwd{Ferric iron}
\kwd{Bioprocess}
\kwd{Uranium recovery}
\kwd{Industrial implementation}}

\altkeywords{\kwd{Bio-oxydation}
\kwd{Fer ferreux}
\kwd{Fer ferrique}
\kwd{Bioproc\'{e}d\'{e}}
\kwd{R\'{e}cup\'{e}ration d'uranium}
\kwd{Mise en \oe{}uvre industrielle}}

\thanks{Project IronBiox (``Bioaugmentation en fer ferrique'') funded
by Orano Mining}

\begin{abstract} 
This study presents the various steps performed for the industrial
implementation of a process dedicated to the bio-oxidation of the
ferrous iron contained in the solution used to leach uranium with the
objective of increasing its recovery. These steps included theoretical
works, laboratory-scale tests on synthetic effluents and laboratory-
and pilot-scale tests on industrial effluents at the mining site. The
main results from each of these steps are presented as well as the
challenges that have to be addressed for the integration of this
process into the existing flowsheet. They allowed to get insights into
the upscaling of a biotechnology at an industrial scale.
\end{abstract} 

\begin{altabstract} 
Cette \'{e}tude pr\'{e}sente les diff\'{e}rentes \'{e}tapes qui ont
\'{e}t\'{e} r\'{e}alis\'{e}es pour permettre la mise en \oe{}uvre
industrielle d'un proc\'{e}d\'{e} d\'{e}di\'{e} \`{a} la bio-oxydation
du fer ferreux. Ce fer \'{e}tait contenu dans une solution utilis\'{e}e
pour lixivier de l'uranium. L'objectif \'{e}tait ainsi d'augmenter sa
r\'{e}cup\'{e}ration. Les principales \'{e}tapes de cette \'{e}tude
comprenaient des travaux th\'{e}oriques, des essais en laboratoire sur
des effluents synth\'{e}tiques et des essais en laboratoire et en
pilote sur site minier avec des effluents industriels. Les principaux
r\'{e}sultats de chacune de ces \'{e}tapes sont pr\'{e}sent\'{e}s,
ainsi que les d\'{e}fis qui doivent \^{e}tre relev\'{e}s pour
l'int\'{e}gration de ce proc\'{e}d\'{e} dans le sch\'{e}ma de
traitement existant. Ces travaux ont permis d'acqu\'{e}rir des
connaissances li\'{e}es \`{a} la mont\'{e}e en \'{e}chelle d'une
biotechnologie \`{a} l'\'{e}chelle industrielle.
\end{altabstract} 

\maketitle

\vspace*{2pc}

\twocolumngrid

\end{noXML}

\section{Introduction} \label{sec1}

Over 200 different minerals have been reported in the literature to
host uranium, but only 20 of them are of economic importance (from
which uraninite, coffinite, brannerite and orthobrannerite)~\cite{1}.
Uranium is generally finely disseminated within the ore, in a
tetravalent or hexavalent form, meaning that concentration steps to
remove the gangue material is not possible since it would result in
loss of uranium in tailings. The ore is then usually completely leached
after crushing or grinding. In the leaching process, sulfuric acid is
mainly used, and the dissolution equations from U(IV) and U(VI) are
respectively listed in Equations~(\ref{eq1}) and~(\ref{eq2}).
{\begin{eqnarray}
&amp;&amp;\mathrm{UO}_{2}+4\mathrm{H}^{+}\rightarrow 
\mathrm{U}^{4+}+2\mathrm{H}_{2}\mathrm{O}\label{eq1}\Seqnsplit
&amp;&amp;\mathrm{UO}_{3}+2\mathrm{H}^{+}\rightarrow 
{\mathrm{UO}}_{2}^{2+}+\mathrm{H}_{2}\mathrm{O}\label{eq2}
\end{eqnarray}}\unskip
$\mathrm{UO}_{3}$ dissolution (Equation~(\ref{eq2})) occurs in dilute
sulfuric acid while $\mathrm{UO}_{2}$ {dissolution
(Equation~(\ref{eq1})) requires an oxidizing
reagent. The choice of the reagent depends on several factors such as
the composition of the solid, the economy of the process and the
availability of the reagents at the mining site. Ferric iron is one of
the cheapest oxidizing reagents and is therefore the principal oxidant
of U(IV) in acid leaching circuits~\cite{2}. The dissolution reaction
of tetravalent uranium with ferric iron leads to soluble hexavalent
uranium, as described in Equation~(\ref{eq3}).
{\begin{equation}\label{eq3}
\mathrm{UO}_{2}+2\mathrm{Fe}^{3+}\rightarrow {\mathrm{UO}}_{2}^{2+}+
2\mathrm{Fe}^{2+}
\end{equation}}\unskip
Iron can come directly from the ore since it can be present under
different mineral phases or it can be added as chemicals. Ferric iron
Fe$^{3+}$ can then be regenerated from ferrous iron Fe$^{2+}$ using an
oxidant, MnO$_2$ being often used for such purpose~\cite{2}, or using
microorganisms, following Equation~(\ref{eq4}).
{\begin{equation}\label{eq4}
4\mathrm{Fe}^{2+}+4\mathrm{H}^{+}+\mathrm{O}_{2}
\rightarrow 4\mathrm{Fe}^{3+}+2\mathrm{H}_{2}\mathrm{O}
\end{equation}}\unskip

Bio-oxidation of iron uses chemolithotrophic microorganisms (various
Leptospirilli and Thiobacilli), which take their energy exclusively
from the oxidation of ferrous iron by oxygen, using dissolved CO$_2$ as
the unique source of carbon, and need for their growth a high level of
acidity in solution with pH values below 3. These microorganisms
participate in the natural biogeochemical cycles related to sulfide
metal deposits. Acidophilic bioleaching, i.e., catalysis of sulfur and
ferrous iron oxidation by microorganisms, is a recognized industrial
scale process used for the dissolution of Au, Cu, Ni or Co from
concentrates, ores, and mining waste (see BIOX or KCC
operations~\cite{3,4}). Through the oxidation of sulfides into sulfates
and ferrous into ferric iron, microorganisms enhance metal dissolution.
In the presence of a low sulfide content or in the absence of sulfides,
bioleaching can still be used, the main action of the microorganisms
being the bio-oxidation of ferrous iron (Equation~(\ref{eq4})).
Bioleaching presents the advantages of in situ generation of the
leaching agent (ferric iron and protons), mild operating conditions in
terms of pH (around 1--2) and temperature (20--50~\textdegree C), and
low operating costs~\cite{5}. The main drawback is a longer residence
time compared to chemical leaching~\cite{6}. The potential of
bioleaching for the recovery of uranium from ores was recognized since
the 1950s~\cite{7}. However, uranium was reported to inhibit
microorganisms growth and activity, the toxic threshold concentration
depending on the species and strains. This toxicity can be addressed
using an indirect leaching approach in which the production of the
acidic ferric iron solution and the leaching of the ore are separated
in two-stage processes~\cite{7}. This indirect approach is quite
unusual for conventional bioleaching operations, where the minerals to
leach often serve as a solid support for microorganisms. This means
that the use of the indirect approach requires overcoming several
scientific bottlenecks such as O$_2$ supply, attachment of the
microorganisms on a solid support, and management of the precipitates. 

This work aims to investigate the indirect bio-oxidation of ferrous
iron into ferric iron on two case studies, the KATCO mine in Kazakhstan
and the Soma\"{i}r mine in Niger, with the objective of increasing
uranium recovery yields and kinetics. This paper summarizes the
different steps undertaken to study the process and to perform its
scale-up on the two case studies. It also provides insights into the
main lessons learned in bringing a biotechnology process from
laboratory to industrial scale.

\section{Presentation of the case studies}\label{sec2}
The two case studies are mining sites predominantly owned by Orano
Mining: the KATCO mining site (Kazakhstan), owned at 51\% by Orano
Mining and 49\% by Kazatomprom; and the Soma\"{i}r mining site (Niger),
owned at 63.34\% by Orano Mining and 36.66\% by SOPAMIN
(SOci\'{e}t\'{e} du PAtrimoine des MINes du Niger). Table~\ref{tab1}
provides some general information about these two mining sites.

\begin{figure*}[t!]
\vspace*{3pt}
\includegraphics{fig01}
\caption{\label{fig1}Uranium leaching flowsheet for the KATCO case
study including the investigated bio-oxidation processes.}
\vspace*{3pt}
\end{figure*}

\begin{figure*}[t!]
\includegraphics{fig02}
\vspace*{3pt}
\caption{\label{fig2}Uranium leaching flowsheet for the Soma\"{i}r case
study including the investigated bio-oxidation processes.}
\vspace*{2pt}
\end{figure*}

\begin{table*}
\caption{\label{tab1}Case studies description (data from Orano Mining)}
\begin{tabular}{lcc}  
\thead
 &amp; KATCO  &amp; Soma\"{i}r \\
\endthead
Average content of U &amp; 0.2 to 0.5 kg U/t ore &amp; 1.8 kg U/t ore \\
Annual production capacity &amp; 4000 t/year &amp; 2000 t/year \\
Starting production year &amp; 2006 &amp; 1971 \\
Total uranium production &amp; 46,000 t &amp; ${&gt;}$75,000 t \\
\% of annual world production &amp; 7\% &amp; 3.5\%
\botline
\end{tabular}
\end{table*}

\subsection{KATCO case study (Kazakhstan)}\label{sec21}

KATCO is the world's largest in situ uranium mine, accounting for 15\%
of Kazakhstan's annual uranium production and 7\% of world production.
At this site, a leaching solution containing dilute sulfuric acid is
injected through wells into the uranium deposit. Uranium is dissolved
as the leaching solution passes through the deposit and is pumped to
the surface. Uranium is then extracted from the leachate with ion
exchange resins, followed by steps of refining, concentration, and
precipitation leading to the production of the so-called yellowcake.
The raffinate is re-injected into the wells after acidification,
continuing the closed circuit process.

An iron-oxidizing bioprocess was investigated in order to increase
uranium recovery. To allow the adequate integration of this process
into the current flowsheet (Figure~\ref{fig1}), the following
requirements were considered: a bio-oxidation kinetics of at least 
1~g${\cdot}$L$^{-1}{\cdot}$h$^{-1}$, since the ferrous iron concentration
in the leaching solution reaches around  1~g${\cdot}$L$^{-1}$ after U
recovery and the Hydraulic Residence Time (HRT) of the current
flowsheet is 1 h, no addition of nutrients to enhance microbial growth,
and no temperature regulation. The latter requirement is all the more
important that the temperature at the KATCO mining site varies from
${-}$5~\textdegree C to 20~\textdegree C (data from Suzak city, 60 km
away from the KATCO site).

\subsection{Soma\"{i}r case study (Niger)}
In Soma\"{i}r, the deposit is a horizontal sedimentary deposit at a
depth of around 50 to 70~m. \mbox{Extraction} is performed from an open pit
mine. The ore is then treated either by heap leaching or
pugging-and-curing leaching, depending on its uranium grade
(Figure~\ref{fig2}). The static treatment (acid heap leaching) is
operated on the low- or very low-grade ore (${&lt;}$1~kg U/t ore). It
consists in crushing the ore, agglomerating it with sulfuric acid and
then stacking it in sealed areas to be dripped with an acid solution
for several months. By percolating the ore, a leachate enriched in
uranium is obtained. For the higher grade ore, a dynamic treatment
(pugging and curing) is performed. In dynamic treatment, the ore is
crushed and ground before undergoing a sulfuric acid and nitrate
leaching for several hours. Uranium leachate is then recovered. After
both leaching steps, solvent extraction is performed, followed by
purification steps and precipitation. A concentrate of sodium uranate
called yellowcake containing 75~wt\% of uranium is produced.

\begin{figure*}[t!]
\includegraphics{fig03}
\vspace*{-3pt}
\caption{\label{fig3}Schedule of the study related to bio-oxidation of
ferrous iron to ferric iron to improve uranium recovery.}
\vspace*{-3pt}
\end{figure*}

In order to increase uranium recovery, MnO$_2$ is currently used as the
oxidizing reagent for heap leaching on-site. The use of iron-oxidizing
bacteria was investigated with the objective to reduce the consumption
of MnO$_2$ (Figure~\ref{fig2}). In particular, it was estimated that
implementing bio-oxidation would allow to save more than 43\% of
MnO$_2$, leading then to a reduction in the related operating costs of
more than 547 k{\texteuro}/year~\cite{8}. As for the KATCO case study,
the implementation of bio-oxidation should be performed without any
nutrients addition nor temperature regulation, the temperature at the
Soma\"{i}r mining site varying from 5~\textdegree C to 45~\textdegree
C. Regarding the processing conditions, the ferrous iron concentration
is much higher in Soma\"{i}r than in KATCO, reaching 10 to
20~g${\cdot}$L$^{-1}$, and the HRT is also higher with a value of 24 h. A
bio-oxidation kinetics of  0.5~g${\cdot}$L$^{-1}{\cdot}$h$^{-1}$ should
thus be reached. Another important parameter to consider is the use of
solvent extraction to recover U which may lead to traces of organic
solvents in the depleted effluent containing Fe$^{2+}$ as these traces
may inhibit microbial activities~\cite{9}.



\section{Main steps of the study}\label{sec3}

The study of the potential of an iron-oxidizing bioprocess for
improving uranium recovery and of its on-site implementation started in
2013. This study was performed in close collaboration between Orano
Mining and the BRGM (French Geological Survey), which has a great
expertise in bioleaching processes~\cite{4}. Figure~\ref{fig3} presents
the main steps of this study. Firstly, theoretical studies were
performed on both case studies in order to get valuable information on
the process such as the theoretical increase in uranium recovery, the
formation of precipitates, and the influence of the ambient temperature
on the evolution of the temperature within the bioprocess. Experimental
works were then performed using the KCC consortium, mainly composed of
the genera {\textit{Leptospirillum}}, {\textit{Acidithiobacillus}}
{and} {\textit{Sulfobacillus}}, which are either iron or sulfur
oxidizers or both. This consortium was chosen as it previously
demonstrated high efficiency for sulfides bioleaching and ferrous iron
bio-oxidation~\cite{14}. These first experimental works were performed
with a focus on the KATCO case study as it presents a higher potential
for the bio-oxidation process (details are given in the following
paragraphs). In 2018, due to an Orano Mining internal decision, it was
decided to perform the implementation of this innovative process on the
Soma\"{i}r case study and to no longer work on the KATCO case study.
All these steps are detailed in the following paragraphs.


\subsection{Preliminary studies}\label{sec31}
The first objective of the preliminary studies was to define the
intellectual property rights, by \mbox{reviewing} the commercial operations
and the associated patents. The Total Patent database was used with the
following key words: ``Iron oxidation AND ferrous AND bio'', ``Iron III
production AND bio'', ``Ferrous iron oxidizing bacteria'', ``Uranium
leaching AND bio AND oxidation''. Several patents were found on the use
of ferrous iron bio-oxidation, such as US4139456A 1979-02-13~\cite{10}
or US6043022A 2000-03-28~\cite{11}, but no patents were found on its
application for uranium recovery. Moreover, no commercial operation
related to such process was identified. 

Another objective of the preliminary studies was to perform
thermodynamic calculations in order to get valuable information, even
if theoretical, on the bio-oxidation process. These calculations aim at
(i)~describing the various reaction equilibria affecting the dissolution
of uranium when an acidic ferric iron solution is used, (ii) obtaining a
quantitative description of the compositions of the solutions, in terms
of dissolved species in particular, and (iii) predicting the formation
of precipitates during the bio-oxidation of ferrous iron to ferric
iron. Simulations were performed with the PhreeqC 7.3 software, which
uses the \textit{llnl} database developed for uranium speciation
complexation equilibria. Simulation results confirmed the increase in
U(IV) recovery by using acidic and oxidizing solutions and indicated
that precipitates of iron hydroxides or jarosite (K, Na) may occur.
These precipitates will have to be taken into account for the design of
the pilot and the industrial processes since they can lead to clogging
and then require the definition of a dedicated cleaning strategy.

The last objective of the preliminary studies was to evaluate the
influence of the ambient temperature on the evolution of the
temperature within the bioprocess and to check if it is compatible with
microbial activity. For the KATCO case study, the heat balance modeling
was carried out following the methodology used by Talati and
Stenstrom~\cite{12} and considering that the bio-oxidation is performed
in a pond of 3000~m$^3$ since this pond was used for
the recovery of leachates. The equipment chosen for performing the
bio-oxidation was a floating agitation system recently developed by
Milton Roy Mixing and Air Liquide for wastewater treatment
applications. This system allows both mixing and suspending solids in
the solution as well as injecting gases into the solution. It was
chosen as it was proven that it allows decreasing the costs of
bioleaching processes when implemented in stirred tank
reactors~\cite{13}. Results showed only small variations of the
temperature in the pond regardless of the atmospheric conditions (from
less than ${-}$5~\textdegree C in January to 20~\textdegree C in July),
the pond temperature varying between 24.2~\textdegree C and
24.8~\textdegree C (Figure~\ref{fig4}), and these temperatures are
compatible with the growth and activity of mesophilic or moderate
thermophilic microorganisms. This is due to the short HRT in the pond,
which was 1 h. Indeed, a longer HRT in the pond would have led to a
higher amplitude in the temperature evolution, as showed in
Figure~\ref{fig4}. This is because main thermal losses are linked to
the aeration of the pond and surface convection.

\begin{figure}[t!]
\includegraphics{fig04}
\caption{\label{fig4}Air and pond temperatures simulated for the KATCO
case study.}
\end{figure}

A heat balance model was also developed and implemented for the case
study of Soma\"{i}r. Similarly as for the KATCO case study, results
showed that the temperature of the pond is very close to the
temperature of the inlet effluent even if the HRT is 24 h. This is
because the main parameter controlling the temperature of the pond
considered in the Soma\"{i}r case study is the temperature of the inlet
effluent with only a minor influence of the aeration of the pond.

\begin{figure*}[t!]
\includegraphics{fig05}
\caption{\label{fig5}Schematic diagram of the experimental devices used
to assess bio-oxidation kinetics with KCC consortium, (a) STR and (b)
bubble column.}
\vspace*{2pt}
\end{figure*}

\subsection{Laboratory-scale tests at the BRGM and at Orano Mining's
Innovation Center for\newline Extractive Metallurgy (CIME)}\label{sec32}

\subsubsection{Laboratory-scale tests on the KATCO case study} \label{sec321}
\paragraph{Objectives} \label{sec3211}
The first experimental works were performed on the case study of KATCO
since it presents a higher potential for the bio-oxidation process
(considering the mineralogy of the deposit) and some operating
conditions are more favorable for such a process compared to the case
study of Soma\"{i}r. This is in particular the case of the lower Fe
concentration, which reduces the process piloting issues associated
with the formation of Fe-precipitates, and of the use of ion exchange
resins instead of solvent extraction for uranium recovery, therefore
avoiding the presence of traces of organic solvent which could inhibit
the microorganisms. 


The laboratory-scale tests were performed with a threefold objective:
\begin{enumerate}[(1)]
\item To assess the feasibility of reaching and maintaining
bio-oxidation rates with a well-known microbial consortium in
continuous mode at a temperature of 20~\textdegree C.  
\item To determine the influence of the HRT on bio-oxidation rates.
\item To demonstrate the increase of uranium recovery yield and rate in
the presence of biogenic ferric iron. 
\end{enumerate}

\paragraph{Methods} \label{sec3212}
Experiments were performed in two steps. First, 2~L stirred tank
reactor (STR) experiments were performed at the BRGM laboratory on a
synthetic solution with a composition in major elements similar to the
KATCO effluent (Fe, Ca, Mg, Al, $\mathrm{SO}_4^{2-}$ and Cl). 

As mentioned before, the KCC consortium was chosen for these tests.~It
is important to mention that similar acidophilic microorganisms (same
genera and species) were previously found in Kazakhstan~\cite{15,16,17}.
One test was also performed in abiotic conditions in order to assess
the influence of the biomass on the bio-oxidation of the ferrous iron.
A solid support made with coal particles was introduced into the STR in
a basket (Figure~\ref{fig5}) in order to allow
the biomass to create biofilms. Indeed, attached bacteria are often
more efficient than planktonic cells due to higher resilience of
biofilms towards potential inhibitions~\cite{18}. As O$_2$ and CO$_2$
{supply are key parameters for the bio-oxidation of ferrous iron
(Equation~(\ref{eq4})),  STRs were equipped with baffles and a Rushton
turbine to ensure high mass transfers.
The temperature was regulated to 25~\textdegree C as the preliminary
study showed that this would be the operation temperature in KATCO. The
reactor was first launched in batch mode to allow the fixation of the
microorganisms on the solid support and then operated in continuous
mode by progressively reducing the HRT. HRT is related to the liquid
phase in all the works performed in this study since the solid support
was kept within the reactor in order to allow the biomass to grow. 

\begin{figure*}[t!]
\includegraphics{fig06}
\vspace*{-6pt}
\caption{\label{fig6}Bio-oxidation rates and yields in the STR
experiments (left) and bubble column (right) with a KATCO synthetic
effluent and for various operating conditions (from~\cite{22}).}
\vspace*{-6pt}
\end{figure*}

Secondly, tests coupling the ferrous iron bio-oxidation and the KATCO
ore leaching were performed. A bubble column, considered as fluidized
bed reactor, was considered for the bio-oxidation reactor. This choice
was motivated by the small \mbox{volume} of the bubble column which allows
continuous \mbox{coupling} with the uranium leaching test since its input flow
rate was 100~mL${\cdot}$h$^{-1}$
(Figure~\ref{fig5}). The bubble column was
operated at 20~\textdegree C with coal as solid support. The reactor
was first launched in batch mode to allow fixation of the
microorganisms on the solid support and then operated in continuous
mode by progressively reducing the HRT. Bio-oxidation in bubble columns
was optimized at the BRGM laboratory and then operated in close loop by
Orano Mining in their laboratory. Tests were performed with a real
effluent from KATCO. Uranium leaching was performed in columns, each
column containing about ten kg of ore each. Fe$^{3+}$-enriched
solution was injected at a flow rate of 100 mL${\cdot}$h$^{-1}$ allowing
an ascending} {volumetric flow rate per unit area} {of 
4~L${\cdot}$h$^{-1}{\cdot}$m$^{-2}$, close to the one used at the KATCO
mine. Effluents were acidified to pH 1.6 with dilute sulfuric acid,
before passing through anionic resins to recover uranium.
Uranium-depleted solutions were then treated in the bio-oxidation
column for ferrous iron oxidation.


Each reactor was monitored daily for pH, redox potential and
temperature. Sampling was performed daily in the Fe$^{3+}$-enriched
solution in order to analyze the total Fe concentrations---by Atomic
Absorption Spectroscopy (AAS)---and the Fe$^{2+}$ concentrations---by
titration with Ce(IV) sulfate 0.001 M. Bacterial cells were also
counted occasionally in the Fe$^{3+}$-enriched solution. In order to
determine the dynamics of microbial \mbox{populations}, DNA extraction
followed by CE-SSCP (capillary \mbox{electrophoresis} with single-strand
conformation polymorphism) was performed at the end of each test.
Results related to the biomass are not given here due to
confidentiality issues.


\paragraph{Results} \label{sec3213}

STR experiments performed in abiotic conditions, i.e., without
microorganisms, led to an oxidation rate of about
0.1~g${\cdot}$L$^{-1}{\cdot}$h$^{-1}$, showing that the air oxidation of
ferrous iron does not allow to reach industrial requirements (here set
at 1~g${\cdot}$L$^{-1}{\cdot}$h$^{-1}$}). When using the KCC consortium,
an oxidation rate of about 1.1~g${\cdot}$L$^{-1}{\cdot}$h$^{-1}$ {was
obtained at a HRT of 1 h, confirming the potential of the biomass to
perform the bio-oxidation of ferrous iron into ferric iron. It should
be mentioned that this performance was obtained at 25~\textdegree C,
whereas the optimal temperature for the KCC consortium is
40--42~\textdegree C~\cite{14}, confirming the robustness of this
consortium (Figure~\ref{fig6}, left). Similar results were obtained
with the bubble column (Figure~\ref{fig6}, right). It is then
interesting to highlight that it was possible to adapt the biomass for
oxidizing 100\% of Fe$^{2+}$ at a HRT of 1 h and at 20~\textdegree C,
as shown in Figure~\ref{fig6}. Moreover, no washout of the biomass was
observed, confirming that the use of a solid support enables the
biomass to be maintained in the reactor even if the HRT was lower than
its doubling time since, for example, the doubling time of \textit{L.
ferriphilum}  is around 3.1 h~\cite{19,20,21}. During these tests, the
clogging of the biomass support by a biofilm growth and/or by
precipitates remained moderate. 


A high reproducibility was observed between the results obtained with
the STR and with the bubble column, confirming the robustness of the
process. Moreover, the bio-oxidation rate showed only small variations
when the oxidation yield reached its maximum value. For example, at a
HRT of 3 h in the STR, the relative standard deviation of the
bio-oxidation rate was 1.2\% during 6 days, which corresponds to 48 HRT
and 6 measurements of the kinetics. At a HRT of 1 h in the bubble
column, the bio-oxidation rate varied by 2.4\% during 14 days, which
corresponds to 336 HRT and 10 measurements of the\break kinetics. 


When the bio-oxidation of ferrous iron was coupled with uranium
leaching, an increase in uranium recovery was observed compared to acid
leaching only (Figure~\ref{fig7}).

\begin{figure}
{\vspace*{4pt}}
\includegraphics{fig07}
{\vspace*{4pt}}
\caption{\label{fig7}Uranium dissolution yields and acid consumption as
a function of L/S ratio (from~\cite{22}).}
\end{figure}

This was accompanied by a decrease in acid consumption of 0.6 kg/t ore,
which represents a reduction of 6.4\% in the total acid consumption.
These tests were performed on a real effluent from the KATCO case study
and therefore showed that the presence of traces of radioelements did
not significantly affect the bio-oxidation performances.

\subsubsection{Laboratory-scale tests on the Soma\"{i}r case
study}\label{sec322}

\paragraph{Objectives}\label{sec3221}

As mentioned previously, a change in the case study occurred in 2018
due to an Orano Mining internal decision and the Soma\"{i}r case study
was chosen for the on-site implementation of the bio-oxidation of
ferrous iron. Since the composition of the Soma\"{i}r effluent markedly
differs from the KATCO one, in particular regarding the Fe$^{2+}$
concentration with values up to 20~g${\cdot}$L$^{-1}$,
complementary laboratory-scale tests in bubble columns were performed
in Orano Mining's Innovation Center for Extractive Metallurgy (CIME).
The objectives of these tests were to assess the influence of the
change in effluent composition on the bio-oxidation performances and to
collect reliable data to design the pilot-scale experiments. 

\paragraph{Methods}\label{sec3222}

Tests were performed in a 5~L STR similar to the one described in
Section~\ref{sec3212} with a similar monitoring. A synthetic solution with a
composition similar to that of the Soma\"{i}r effluent was used, in
particular regarding the content in iron, nutrients (K, Mg, P,
$\mathrm{NH}_{4}^{+}$) and potential inhibitor elements (Al,
$\mathrm{NO}_{3}^{-}$, Cl$^{-}$). Continuous tests were performed at
30~\textdegree C, the mean temperature of the effluent, and the pH was
regulated at 1.2. The reactor was first launched in batch mode to allow
the fixation of the microorganisms on the solid support and then
operated in continuous mode by progressively reducing the HRT.

\begin{figure*}[t!]
\includegraphics{fig08}
\vspace*{-3pt}
\caption{\label{fig8}Bio-oxidation yield and rate during
laboratory-scale tests with the synthetic effluent from the Soma\"{i}r
case study.}
\vspace*{-3pt}
\end{figure*}

\paragraph{Results}\label{sec3223}

Some of the results obtained during the experiments are given in
Figure~\ref{fig8}. As showed in} Figure~\ref{fig8}, it is possible to
reach and maintain the bio-oxidation rate targeted for the Soma\"{i}r
case study, which is around 500~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$. This
bio-oxidation rate was obtained for a bio-oxidation yield lower than
100\% and did not change regardless of the decrease in the HRT, which
seems to indicate that the maximum oxidative performances of the
biomass have been reached. Moreover, the solid support concentration
seems to be a limiting factor since it was not possible to reach a
bio-oxidation of 500~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$ with a solid
support concentration of 40~g${\cdot}$L$^{-1}$. Figure~\ref{fig8} also
shows a decrease in the oxidation rate after several days of operation.
This could be explained by the formation of iron precipitates, which
clogged the solid support leading to a reduction in the bio-oxidation
performances, since the biomass was fixed on the solid support. This
assumption was supported by the observation of a very large quantity of
red--orange precipitates when the reactor was stopped and opened. This
precipitate is most \mbox{probably} mainly composed of jarosite, which is the
predominant precipitate detected in bioleaching systems, even at low
pH~\cite{19}. A similar effect of the iron precipitates when coal
particles are used as solid support for bio-oxidation was reported by
Hubau et~al.~\cite{19}. In particular, they demonstrated that
jarosite formation clogged the solid support pores, inducing very low
mass transfers and a reduced microbial activity. This clogging effect
was somehow mitigated after some time when using a bubble column due to
the attrition occurring within the fluidized bed which opened the coal
pores, leading to a new increase in the bio-oxidation rate~\cite{19}.
These phenomena, observed at microscopic scale, create high variations
in kinetics at macroscopic scale and should then be considered
carefully when designing a bioreactor. In particular, it seems that
other solid supports could be more appropriate for the bio-oxidation of
Fe$^{2+}$-rich effluents, such as woven nylon~\cite{23}.\looseness=-1


The results obtained with these works performed at laboratory scale on
a synthetic effluent from the Soma\"{i}r case study confirmed the
potential of the bio-oxidation to improve uranium recovery. A
prefeasibility study and a design to cost analysis were performed based
on these first results, including in particular the comparison of the
two designs for the bioreactor which are the STR and the bubble column,
the latter being similar to a fluidized reactor, with no mechanical
agitation. First estimations of the capital expenditures (CAPEX) and
operational expenditures (OPEX) as well as a risk assessment were
performed. In particular, results showed that CAPEX and OPEX are
reduced by more than 30\% and 40\%, respectively, when using a
fluidized reactor. The main drawback identified for such reactor is
related to mass transfers, meaning that works will have to be performed
on its design to optimize them. A planning for on-site implementation
and up-scaling of the process was then defined.\looseness=-1

\subsection{On-site tests performed at the Soma\"{i}r
mine}\label{sec33}

First of all, the transfer of microorganisms on-site followed Nagoya
protocol on Access to Genetic Resources and the Fair and Equitable
Sharing of Benefits Arising from their Utilization to the Convention on
Biological Diversity. This protocol, adopted in 2010 and ratified by
137 parties, deals with the use of genetic resources, in order to share
fairly and equitably the benefits and thus to contribute to the
conservation and sustainable use of biodiversity.

Moreover, a dedicated training of the mining operators was conducted
allowing them to efficiently monitor and operate bioprocesses. This
training also included information related to the risks associated to
handling the bio-oxidation solutions that would be produced, the main
one being the acidity of the solutions. It should be mentioned that the
mine operators already had several acid effluents to manage on-site and
had therefore defined the appropriate safety measures. In addition, a
laboratory equipped for bioprocesses development and monitoring was
implemented on-site (Figure~\ref{fig9}). In particular, it included a
microscope for cell counting with Thoma plates. These were a
prerequisite for the transfer to the \mbox{Soma\"{i}r} mine of the process
development, operation and up-scaling works.

\begin{figure}
{\vspace*{4pt}}
\includegraphics{fig09}
{\vspace*{4pt}}
\caption{\label{fig9}Photo of the laboratory developed at the
Soma\"{i}r mine (@Orano Mining).}
{\vspace*{4pt}}
\end{figure}

A method for the continuous monitoring of the main operating parameters
was established on-site and was used at each scale. The reactors were
equipped with pH, redox potential, dissolved O$_2$ and temperature
probes with continuous records (time step around 2h30). Total Fe
concentrations were determined daily through AAS, while an empirical
correlation was used to evaluate Fe$^{2+}$ concentrations from redox
potential values~\cite{24}. In order to determine the biomass
concentration, Thoma cell counting was performed. Regarding the biomass
attached to the solid, a detachment protocol based on the use of
ultrasounds was established on-site (Thoma cell counting after
detachment) but its execution remained limited due to difficulties in
sampling the solid support.

\subsubsection{Laboratory-scale tests in shake flasks}\label{sec331}

\paragraph{Objectives}\label{sec3311}
The main objectives of the tests in shake flasks performed on-site were to adapt
the microbial consortium to the industrial effluents and to define how
the bio-oxidation process could be integrated in the current flowsheet.
In particular, two integration options were investigated:
\begin{itemize}
\item In the first one (Figure~\ref{fig10}), bio-oxidation is performed
on the leachate from the heap leaching (called 	``production
effluents''). In this case, uranium-enriched leachate is recirculated
in the heap after its bio-oxidation in order to concentrate uranium in
the leachate before its recovery in the solvent extraction unit. The
main drawback of this option is the potential toxicity of uranium
towards bio-oxidation microorganisms.

\item In the second one (Figure~\ref{fig11}), bio-oxidation is
conducted on the raffinate, i.e., the uranium-depleted effluent after
the solvent extraction unit. The advantage of this option is that
radioelements concentration in the raffinate is very low. However, the
raffinate may contain traces of organic solvents due to its treatment
in the solvent extraction unit.\looseness=-1 
\end{itemize}

\begin{figure*}[t!]
\includegraphics{fig10}
\caption{\label{fig10}First bio-oxidation processing option for the
Soma\"{i}r case study.}
\end{figure*}

\begin{figure*}[t!]
\includegraphics{fig11}
\caption{\label{fig11}Second bio-oxidation processing option for the
Soma\"{i}r case study.}
\vspace*{-2pt}
\end{figure*}

This step also aimed to check that all the equipment planned to be used
in the pilot and industrial operations do not present any toxicity
towards the microorganisms.~Last but not least, this step aimed to
produce the biomass that will be used to inoculate the 6~L bioreactors.

\paragraph{Methods}\label{sec3312}
Subcultures were performed in 1~L bottles with a working volume of 
250~mL, placed on a shaking table. The temperature was not regulated and
could therefore vary from 17~\textdegree C to 43~\textdegree C in a
24-h period (evolution of temperature in the laboratory). In order to
ensure a good adaptation of the microorganisms to the industrial
effluents (both from the production effluent and the raffinate), they
were first diluted at 50 vol\% with the synthetic effluent and their
content was gradually increased up to 100 vol\%. The protocol used to
subculture and scale up the process consisted in inoculating 10\% of
both solid and liquid phase. pH and redox potentials were measured on
a daily basis.


\paragraph{Results}\label{sec3313}
Results showed that the bio-oxidation rate strongly depended on the
effluent composition, with values varying from 
10~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$ to 
120~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$. These results have to be considered
carefully, since the tests were performed in batch mode with no control
of some of the operating conditions, such as temperature and aeration,
and with a monitoring carried out only once or twice a day,
whereas Fe$^{2+}$ was sometimes completely oxidized in few hours. It
was therefore not possible to use the obtained values of the
bio-oxidation rate to predict the performances of a continuous
operation. However, these results gave interesting insights into the
adaptation of the microorganisms to the two industrial effluents. In
particular, it was observed that the use of uranium-enriched effluent
led to far lower bio-oxidation rates compared to the ones obtained with
the uranium-depleted effluent. Bio-oxidation should therefore be
performed on the uranium-depleted effluent, which corresponds to the
processing option presented in Figure~\ref{fig11} even if the effluent
may contain traces of solvent. The influence of traces of solvent on
bio-oxidation performances was investigated and results showed a
detrimental effect, consistent with the study from Mazuelos 
et~al.~\cite{9}. In order to limit this issue, it was recommended to
add a decantation step before the bio-oxidation process and to pump the
effluent from the bottom of the decantation system since the solvent
will stay at the surface. Another solution could be to add a treatment
step with activated charcoal since it is usually used to remove organic
solvents but this treatment is quite expensive and complex to operate
(in particular for the regeneration of the activated charcoal) at a
remote industrial site.

\subsubsection{Laboratory-scale tests in 6~L reactors}\label{sec332}

\paragraph{Objectives}\label{sec3321}
The laboratory-scale tests performed in 6~L reactors had three
objectives. The first one was to assess the ability of the
microorganisms to oxidize industrial effluents at the targeted rate
(500~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$) and to test the influence of the
operating conditions on the performances. The second one was to
investigate the efficiency of woven polypropylene as solid support for
biomass. This choice was motivated by previous works showing that
bio-oxidation performances with woven polypropylene are not as affected by
the formation of precipitates as when using coal particles~\cite{23}.
The last objective was to produce enough biomass to inoculate
larger-scale bioreactors.

\begin{figure*}[t!]
{\vspace*{2pt}}
\includegraphics{fig12}
{\vspace*{1pt}}
\caption{\label{fig12}Bio-oxidation yields and rates in the 5~L STR
operated on the Soma\"{i}r site.}
{\vspace*{1pt}}
\end{figure*}

\paragraph{Methods}\label{sec3322}
Similarly to the laboratory-scale tests performed at the BRGM and at
Orano Mining's CIME, two types of reactors were used: (i) a 5~L STR,
similar to the one described in Section~\ref{sec3212}.
Figure~\ref{fig5} was used from September 2019
to March 2020 and (ii) a 6~L bubble column was operated from April 2020
to March 2022. In both reactors, the input effluent was injected at the
bottom of the reactor and overflowed from the top. Air was also
injected at the bottom through a fritted cylinder. For the supply of
CO$_2$, gas bottles were mostly used but Na$_2$CO$_3$
was also considered as it is already used on-site for uranium
production, which would then simplify the logistics at this remote
site. Some tests were also \mbox{performed} without any enrichment of the air
in CO$_2$. In the 5~L STR, the woven polypropylene was placed in the
basket and was ``crumpled'' to reach the targeted solid support
concentration. In the bubble column, the solid support was fixed in the
middle of the column: woven polypropylene support was either crumpled
in a basket or stretched across the height, i.e., in the same direction
as the liquid and bubbles flow. The reactors were first launched in
batch mode to allow fixation of the microorganisms on the solid support
and then operated in continuous mode by progressively reducing the HRT.
The tests were first conducted on an effluent made with 50 vol\% of
industrial effluent and 50 vol\% of synthetic effluent, the content of
industrial effluent being then increased to 75 vol\% and to 100 vol\%
when the biomass was considered robust enough to be adapted to such
effluent. In order to monitor the biomass, DNA extraction and CE-SSCP
were performed on the solid phase from time to time. Results related to
the biomass are not given here due to confidentiality issues.
%\vspace*{4pt}

\paragraph{Results}\label{sec3323}

The first tests were performed in the 5~L STR with a temperature
maintained at 40~\textdegree C. Results showed that it is possible to
reach and maintain a Fe}$^{2+}$ bio-oxidation rate of at least 
500~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$, a bio-oxidation rate of about 
600~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$ being even reached and maintained in some
of the operating conditions investigated (Figure~\ref{fig12}). As shown
in Figure~\ref{fig12}, the HRT was first reduced from 48~h to 18~h,
each reduction performed after \mbox{stabilizing} the bio-oxidation yield at
100\%. These good \mbox{bio-oxidation} performances obtained with the woven
\mbox{polypropylene} showed that this material can be used as solid support for
biomass.

The CO$_2$ supply was changed from gaseous CO$_2$ to Na$_2$CO$_{3}$
after 70 days of operation without affecting the performances. However,
it affected the acid consumption by increasing the pH. H$_2$SO$_{4}$
addition was finely monitored to estimate the additional cost
associated to the use of Na$_2$CO$_{3}$. 

\begin{figure*}[t!]
\includegraphics{fig13}
\caption{\label{fig13}Bio-oxidation yields and rates in the 6~L bubble
column operated on the Soma\"{i}r site (from December 2021 to March
2022).}
\end{figure*}

The concentration of industrial effluent was increased from 50 vol\% to
75 vol\% after about 85 days. A decrease in the bio-oxidation yield was
observed with no change in the bio-oxidation rate. This can be due to a
limited bio-oxidation capacity of the biomass with the amount of solid
support used, since an increase in HRT allowed to reach again a
bio-oxidation yield of 100\%. Similar results were obtained with the 6
L bubble column. 

After more than 60 days of operation, no precipitates were observed on
the solid support. This confirmed the benefits of using woven polypropylene
as solid support instead of coal particles which tend to be clogged
quite easily (see Section~\ref{sec322}). Moreover, it was observed that the
mode of handling the solid support had a strong effect on the decrease
in \mbox{bio-oxidation} performances due to the formation of precipitates:
stretching the woven polypropylene across the height allowed to more
than double the time of operation before a cleaning is required
compared to crumpling the solid support in a central basket.


The potential of the biomass to bio-oxidize the Fe$^{2+}$ contained in
the industrial effluent without any dilution, i.e., with a
concentration of 100 vol\%, was tested in the 6~L bubble column. A
bio-oxidation yield of 100\% was obtained for a HRT of 25 h, which
corresponds to a bio-oxidation rate of about
350~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$. 

The 6~L bubble column reactor was also used to investigate the
influence of temperature regulation and CO$_2$ enrichment of air on the
bio-oxidation performances. Tests were performed from July 2021 to
March 2022 on an industrial effluent concentrated at 50 vol\%. Results
obtained from December 2021 to March 2022 are given in
Figure~\ref{fig13}. The relative standard deviation of the
bio-oxidation rates during each operating condition was low (less than
5\%). These relative standard deviations were in the same order of
magnitude than the ones observed during the laboratory-scale tests at
the BRGM laboratory  (Section~\ref{sec321}) despite the higher
difficulty in maintaining stable operating conditions on-site. It is
therefore possible to compare the results obtained at different
operating conditions. Good performances were obtained at temperatures
far lower than 40~\textdegree C, which is really promising from an
industrial perspective. Regarding the CO$_2$ partial pressure, a
bio-oxidation yield of almost 100\% was obtained even when the air was
not enriched in CO$_2$. These results should be confirmed at a larger
scale, especially since in the 6~L bubble column the air flow rate was
in large excess to guarantee a good homogenization and mass transfers
within the bioreactor, and biomass had time to colonize the solid
support before the enrichment in CO$_2$ was turned off.


The 6~L bubble column was operated for several months (from April 2020
to November 2022). \mbox{During} this long period of operation, clogging of
the solid support by precipitates was observed. Even if the quantity of
precipitates was highly reduced compared to when using coal particles,
it raised the issue of maintenance. A protocol based on partial
cleaning of the solid support on a regular basis was therefore
developed and validated.

Based on these results, 9 Key Performances Indicators (KPI) were
defined in order to better evaluate and compare the performances of the
bio-oxidation process at each step of the scale-up. They are therefore
critical milestones to reach for the industrial implementation of this
bioprocess. Some examples of KPIs considered here are the following:
concentration of industrial effluent, bio-oxidation rate, frequency of
maintenance of the solid support and time to reach the optimal
performances again after a maintenance operation. 

\subsubsection{Pilot-scale tests in 30~L reactors}\label{sec333}

\paragraph{Objectives}\label{sec3331}

The pilot-scale tests performed in 30~L reactors aimed to optimize the
operating \mbox{parameters,} in particular for the start and the restart of
the bioreactor after a maintenance operation. They also aimed to
produce enough biomass to inoculate the larger-scale bioreactor.

\paragraph{Methods}\label{sec3332}
3 bubble columns, similar to the 6~L bubble column (with woven
polypropylene used as solid support and stretched across the height of
the columns), were operated from August 2020 to November 2022. Air was
initially enriched with gaseous CO$_2$ (1\%) before switching to
Na$_2$CO$_{3}$ supply. Temperature was set at 35~\textdegree C. The
reactors were first launched in batch mode to allow fixation of the
microorganisms on the solid support and then operated in continuous
mode by progressively reducing the HRT. O$_2$ and CO$_2$ partial
pressures were occasionally measured at the top of the reactors. In
order to monitor the biomass, DNA extraction and CE-SSCP were performed
on the solid phase from time to time.

\paragraph{Results}\label{sec3333}
Similarly to the 6~L bubble column tests, the bio-oxidation
performances were not affected when the temperature was no longer
regulated and when the air was not enriched in CO$_2$. This result was
observed when the industrial effluent was concentrated at 100 vol\%.
However, it was observed that a CO$_2$ enrichment of the air is
requested when the operation is being started or during a batch mode
operation in order to ensure an efficient microbial growth. It was also
observed that a temperature of around 20~\textdegree C is a limiting
factor in such phases.

The experiments performed with the 30~L reactors also allowed to
determine the quantity of solid support required to maintain a biomass
concentration allowing to reach the targeted bio-oxidation
performances. The initial ratio of woven polypropylene, set to 
40~m$^{2}{\cdot}$m$^{-3}$, led to a bio-oxidation rate of about
350~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$ when the industrial effluent was
used (i.e., concentration of 100 vol\%). This ratio was progressively
reduced to 9~m$^{2}{\cdot}$m$^{-3}$ and results showed that this ratio
still allowed reaching and maintaining a bio-oxidation rate of about
350 mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$.

Moreover, this testing campaign demonstrated the importance of O$_2$
transfer. In particular, when a clogging of the fritted cylinder was
observed, leading then to a reduction of the air flow rate, the
bio-oxidation performances were strongly decreased. It was observed
that the declogging of the fritted cylinder allowed reaching again the
bio-oxidation performances within a short period, confirming the
robustness of the biomass. Finally, it should be mentioned that a
bio-oxidation rate of about 300--350~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$
was obtained for more than 4 months of continuous operation of the
bioreactors.

\begin{figure*}[t!]
\includegraphics{fig14}
\caption{\label{fig14}Evolution of the colonization of one piece of the
solid support (woven polypropylene) by the biomass
({\textcopyright}Orano Mining).}
\end{figure*}


\subsubsection{Pilot-scale tests in a 700~L reactor}\label{sec334}
\paragraph{Objectives}\label{sec3341}
The 700~L bioreactor was operated in order to continue the scale-up of
the bioprocess and in particular to define the operating protocol to
inoculate a bioreactor with a scale factor higher than the usual one.
Indeed, upscaling in bioprocesses is usually performed with a scale
factor of 10 but here it was planned to go from 30~L to 700~L, i.e., a
scale factor of 23. This step also aimed to define additional KPIs and
to get more reliable data for the design of the industrial system.

\paragraph{Methods}\label{sec3342}

A tank of 700~L was designed in which the industrial effluent was
injected at the bottom of the reactor and overflowed from the top. An
air diffuser tube bound in a spiral at the bottom of the reactor was
used to inject air. Na$_2${CO}$_{3}$ was used for the CO$_2$ supply,
with a CO$_2$ content in the air of 0.4--0.6\%. No regulation of
temperature was used. Woven polypropylene was used as solid support and
a dedicated system was designed in order to stretch it across the
height of the tank. The volumetric ratio of solid support was the same
as for the 30~L bioreactor in order to keep the same concentration of
biomass. K$_{L}$a (Volumetric Mass Transfer Coefficient) was determined
for different air flow rates in order to ensure sufficient O$_2$ mass
transfer by comparison to O$_2$ uptake rate and stoichiometry of the
reaction.

This reactor was operated between April 2022 and November 2022. It was
first launched in batch mode to allow fixation of the microorganisms on
the solid support and then operated in continuous mode by progressively
reducing the HRT. O$_2$ and CO$_2$ partial pressures were occasionally
measured at the top of the reactor, as well as O$_2$ uptake rate
(through dissolved O$_2$ probe).

\paragraph{Results}\label{sec3343}
The batch phase and the first continuous steps went quite fine even if
the inoculation ratio was low (3.5\% for the solid support). In
particular, a bio-oxidation yield of 80\% was obtained with a HRT of 
48~h, which corresponds to a bio-oxidation rate of 
160~mg${\cdot}$L$^{-1}{\cdot}$h$^{-1}$. The effluent was then changed to
another one produced on the mining site (details not communicated here
due to confidentiality issues), which led to a strong decrease in
bio-oxidation performances. This may be caused by the presence of
elements which inhibit the microorganisms. However, it was not possible
to validate this assumption since Orano Mining decided to stop the
study.

Even if it was not possible to reach good bio-oxidation performances
with this 700~L bioreactor, its piloting still allowed defining a
protocol to observe the colonization of the solid support by the
biomass, as showed in Figure~\ref{fig14}. This
protocol and the feedback of its use could be used in another case
study as they bring insightful information on the colonization of the
solid support by the biomass, which is a critical parameter for a
successful scale-up of a bioprocess.

\begin{figure*}[t!]
{\vspace*{1pt}}
\includegraphics{fig15}
{\vspace*{1pt}}
\caption{\label{fig15}Cryo-SEM (A and B) and STEM (C and D) photographs
of biofilms on woven polypropylene support (new support A and C; old
support B and D).}
{\vspace*{1pt}}
\end{figure*}

\section{Discussion}\label{sec4}


These works carried out at different scales highlighted that the main
critical parameters for the implementation of the process of
bio-oxidation of ferrous iron to ferric iron are related to biomass and
to mass transfers. Specific works were then performed both at the
mining site and at the BRGM and CIME laboratories.

First of all, it is critical to understand the mechanisms related to
biofilm formation and growth in order to reach good performances of the
bioprocess. Due to limited access to characterization equipment at the
Soma\"{i}r mine, the in-depth characterization works were performed at
BRGM, on a 150~mL bubble column operated in continuous mode fed with a
model solution (ferrous sulfate in a nutrient medium). The aim was to
better understand the biofilm formation and its influence on the
\mbox{bio-oxidation} rate of the bioreactor. In particular, cryogenic scanning
electron microscopy (Cryo-SEM) was used to observe the attachment of
cells onto the solid support without prior preparation steps (the
methodology is described in~\cite{19}). A comparison of biofilms
between new and old woven \mbox{polypropylenes} (used as solid support for 1.5
month and 1.75 year respectively) was performed (Figure~\ref{fig15}).
These works showed that even though the woven polypropylene had large
holes (around 400~$\upmu$m, as used on-site), biofilms
completely clogged the solid support, \mbox{probably} affecting the mass
transfer. Thin sections were also observed with a scanning transmission
electron microscopy (STEM) (the methodology was adapted
from~\cite{25}). It enabled to see that the microorganisms did not seem
to be directly in contact with the polypropylene, a layer of
extracellular polymeric substances secreted by the bacteria being
in-between. It was also observed that precipitates were included in the
biofilms and that microorganisms did not seem to be located in the same
areas than precipitates, as if their inclusion in the biofilm was not
simultaneous. Monitoring of the biofilm growth was performed on-site
with the development of specific methodologies. In particular, a
methodology was developed in order to estimate the amount of biomass
attached to the solid support, this methodology including a dedicated
protocol for recovering the attached cells followed by counting in
Thoma cells. A methodology was also developed to estimate the growth of
the biofilms on the woven polypropylene used in the 700~L reactor. This
methodology is based on taking pictures of several given pieces of the
solid support, at the same distance and with the same light on a
regular basis (see Figure~\ref{fig14}).

Another important feature of the biomass is related to its inhibition
by elements possibly found in the industrial effluents. Indeed, even if
some potential inhibitory elements were identified before performing
the tests, such as uranium in the production effluent
(Figure~\ref{fig10}) or traces of organic solvents in the raffinate
(Figure~\ref{fig11}), others elements may have an inhibitory effect on
the metabolic activity of the \mbox{bacteria.} An ecotoxicity approach was
then developed on-site. In this approach, detailed analysis of the
effluents was combined to laboratory-scale tests in shake flasks. Such
approach was implemented on a regular basis on the industrial effluent
used for the tests since some variations in its chemical composition
can be found.

Another key parameter of the performances of this bio-oxidation process
is the formation of precipitates, as they play a significant role in
the mass transfers. In this study, precipitates were produced even
though pH was most of the time regulated to 1.2. These precipitates
were not characterized due to the difficulty in collecting and
analysing them, since they are associated to radioelements. However,
some information could be obtained from Hubau et~al.~\cite{19} who
performed bio-oxidation tests in a similar context with SEM-EDS
(Scanning Electron Microscope-Energy Dispersive Spectroscopy)
characterization of the precipitates~\cite{19}. They showed that
precipitates were a mixture of jarosite and ammoniojarosite, which are
often reported in bioleaching systems. These precipitates came from
ferric iron and sulfates together with cationic 
nutrients  ($\mathrm{K}^{+},\mathrm{NH}_{4}^{+},\ldots$).\unskip\break
Understanding the precipitation mechanisms is crucial to decrease their
production allowing then improving mass transfers and optimizing
maintenance of the bioreactors.

Last but not least, it is essential to optimize gas--liquid mass
transfers all along the upscaling of the process. Gas--liquid mass
transfers were studied at each step of the on-site tests (except for
the laboratory-scale tests in shake flasks) by analyzing gas
composition in the outlet flow of the bioreactor, by measuring O$_2$
uptake rate during bio-oxidation and by estimating k$_{L}$a. It should
be mentioned that there is a need to develop monitoring instruments
dedicated to acidophilic bioleaching environments in order to optimize
gas--liquid mass transfers. For example, it would have been very useful
to measure the dissolved CO$_2$ content. However, the related probes
available on the market are, to our knowledge, not adapted to this
application.

\section{Main lessons learned}\label{sec5}

Even if the study was stopped before industrial implementation of the
investigated bioprocess, several lessons were learned from the tests
performed at laboratory- and pilot-scale. This work also allowed
identifying opportunities of process improvement. 

In particular, the conception of the bioreactor (dimensions, stirring,
air injection, mode of handling the solid support within the reactor)
was shown to be a key parameter for the industrial implementation of
the ferrous iron bio-oxidation. This is all the more important that
some critical requirements are somehow antagonist. For example, it is
required to avoid bacterial washout from the reactor in order to
maintain high bio-oxidation performances so fluid turbulences should be
limited to avoid biofilm detachment from the solid support. However, a
good homogenization is a prerequisite to ensure high mass transfer, in
particular for gas, and then high bio-oxidation performances. It is
also essential to consider the management of precipitates when
designing the bioreactor in order to maintain the performances over
long periods of time and to limit the effects of the maintenance phase.
Last but not least, the design of the bioreactor has a strong impact on
the CAPEX and on the OPEX. For example, a Stirred Tank Reactor allows
high mass transfer but leads to high CAPEX and OPEX. Using a pond with
air diffusers (which could be considered as the industrial equivalent
of the bubble column) has the advantages of low CAPEX and OPEX but may
lead to hydrodynamic short circuits and then to low mass\break transfers. 

Regarding the operating conditions, results showed that the
microorganisms used in the bioprocess have a strong robustness, which
is a really positive feature since it means that they can be adapted to
changes in operating conditions without decreasing their performances,
and that a brutal change in operating conditions (due to an unexpected
issue for example) does not lead to a loss in biomass. In particular,
regarding the effluent composition, it was observed that it is possible
to adapt the biomass to high concentrations of elements by first
diluting the effluent and progressively decreasing the dilution level.
This parameter is easier to evaluate in continuous mode rather than in
batch mode, as the change in effluent composition is less abrupt.
Moreover, results showed that the temperature and the enrichment of air
in CO$_2$, which highly determines the biomass formation, are very
important during batch phases such as the start-up or the restart after
a maintenance operation. They are not so important during the
bio-oxidation phase. These phases should then be carefully considered
during the scale-up and later for the industrial exploitation of the
process, as they are decisive for the on-site implementation, not only
from a technical point of view but also from an economic point of view.

Results obtained in this study highlighted the need to have and to
maintain shake flasks for checking the toxicity of any new effluent or
material that will have to be placed within the bioreactors. These
shake flasks could also be used to assess the composition of the
effluent towards the biomass resistance in case of an unexpected change
in the mine process. 

Another lesson learned from this study is that it is essential to have
a good knowledge of the biofilm properties. Monitoring the overall
process parameters (oxidation yield, total Fe and Fe$^{2+}$
concentrations, etc.) does not allow understanding the mechanisms
related to the biofilms (such as formation, growth, clogging,
detachment), which are nevertheless a pivotal point of this bioprocess.
Changes in biomass at the microscopic scale may results in high
variations of the behavior and performances of the whole process at
macroscopic scale. Better understanding the biofilm properties often
requires access to cutting-edge characterization equipment not
available on the industrial sites, so strong collaboration with
research laboratories should be encouraged.

Finally, the definition of Key Performance Indicators (KPIs) quite
early in the implementation strategy was proven to be very effective to
guide the planning of the experimental testwork for the upscaling of
the bioprocess. They also constitutes the basis of an iterative
approach for the update of the techno-economic feasibility of the
process, considering the information obtained at the different
development steps, allowing the identification of technical and
economic hot spots as soon as they appear, which need then more
research work for their\break optimization.


\section{Conclusions}\label{sec6}

The development of the bio-oxidation of ferrous iron to improve uranium
recovery was initiated with theoretical works and with laboratory-scale
tests \mbox{performed} on synthetic effluent to evaluate its potential. From
these first results, the main steps for its upscaling and its
implementation on a mining site were defined. The main results from
each of these steps are presented, discussed and put into perspective
in order to support an iterative approach for the design of the
bioreactor and for the update of the techno-economic feasibility of the
process with the final objective to define the most appropriate
operational technology and flowsheet.

Even if this study was stopped at the 700~L piloting stage due to an
internal decision of Orano Mining, it still allowed improving process
know-how and operational experience that could then be used for the
industrial implementation of any bioprocess. In particular, several
lessons can be learned and allow identifying opportunities to improve
the investigated bioprocess and the approach used for its upscaling. 


\section*{Declaration of interests}

The authors do not work for, advise, own shares in, or receive funds
from any organization that could benefit from this article, and have
declared no affiliations other than their research organizations.


\section*{Funding} 

This study was performed under the project IronBiox (``Bioaugmentation
en fer ferrique'') funded by Orano Mining. 


\section*{Acknowledgments} 

The authors thank all the
persons who have contributed to this project and in particular Mohamed
Kariman, Allasane Issaka Oumarou and Moussa Sabiou from Soma\"{i}r;
Dominique Morin, Yannick M\'{e}nard, Christopher Bryan, J\'{e}r\^{o}me
Jacob, Catherine Joulian, Pierre Gall\'{e}, Dominique Breeze and
Patrick d'Hugues from BRGM; Jean-Luc Rey, V\'{e}ronique Dutheil,
Alexandre Michaut, Magali Celier and Pascal Nardoux from Orano Mining.

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