Bioresource Technology 261 (2018) 428–440

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Advances in bioleaching for recovery of metals and bioremediation of fuel T

ash and sewage sludge
⁎
Tingyue Gua,1, , Seyed Omid Rastegarb, Seyyed Mohammad Mousavic, Ming Lia, Minghua Zhoua
a
Key Laboratory of Pollution Process and Environmental Criteria, Ministry of Education, Tianjin Key Laboratory of Urban Ecology Environmental Remediation and
Pollution Control, College of Environmental Science and Engineering, Nankai University, Tianjin 300071, PR China
b
Department of Chemical Engineering, Faculty of Engineering, University of Kurdistan, Sanandaj, Iran
c
Biotechnology Group, Chemical Engineering Department, Tarbiat Modares University, Tehran, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: Bioleaching has been successfully used in commercial metal mining for decades. It uses microbes to biosolubilize
Bioleaching metal-containing inorganic compounds such as metal oxides and sulfides. There is a growing interest in using
Bioremediation bioleaching for bioremediation of solid wastes by removing heavy metals from ash and sewage sludge. This
Heavy metal review presents the state of the art in bioleaching research for recovery of metals and bioremediation of solid
Solid waste
wastes. Various process parameters such as reaction time, pH, temperature, mass transfer rate, nutrient re-
Biofilm
quirement, pulp density and particle size are discussed. Selections of more effective microbes are assessed.
Pretreatment methods that enhance bioleaching are also discussed. Critical issues in bioreactor scale-up are
analyzed. The potential impact of advances in biofilm and microbiome is explained.

1. Introduction investigated bioleaching for bioremediation of solid wastes, but this

Many industrial activities generate solid wastes that are toxic to the
environment. They pollute air, water and soil. Removing organic mat-
ters from solid wastes is relatively easy using biological, chemical and
physical means. However, the degradation of inorganic matters, espe-
cially those compounds containing heavy metals is usually difficult
(Kulshreshtha et al., 2014; Lee and Pandey, 2012). There are various
methods used to extract metals from metal oxides and sulfides such as
hydrometallurgy and pyrometallurgy. These classical methods for metal
extraction face problems such as environmental pollution, low recovery
yields and high operating costs (Pathak et al., 2009). Bioleaching uses
microorganisms to solubilize metal oxides and sulfides. This process is
sometimes called biosolubilization, which is simple and low cost (as
low as 1/3–1/2 of the costs in conventional processes) (Bosecker, 2001;
Watling, 2006, Motaghed et al., 2014). It is also environmentally
friendly as long as the leachate is contained and acidic wastewater is
neutralized before discharging (Kulshreshtha et al., 2014; Lee and
Pandey, 2012).
Bioleaching has been used to recover various metals from low-grade
mineral ores and tailings for many years at industrial scales using
processes such as dump bioleaching, heap bioleaching and in situ bio-
leaching (Watling, 2006; Cox and Bryan, 2017). Researchers have
application is not yet ready for deployment for practical bioremedia-
tion. The slow reaction kinetics is a major bottleneck (Pathak et al,
2017). Optimizations of operating parameters such as reaction time,
pH, temperature, mass transfer rate, nutrient requirement, pulp density
and particle size are needed to improve bioleaching process efficiency
(Rastegar et al., 2014b; Arshadi and Mousavi, 2014; Bosecker, 1997;
Ilyas et al., 2014). A proper pretreatment method is also an effective
way to improve bioleaching. This review discusses various issues in
bioleaching for the bioremediation of solid wastes with recovery of
useful metals as value-added products.
2. History of bioleaching
Since the 1940s many researchers have contributed to the clar-
ification of the mechanisms of specific microorganisms used in the
biosolubilization of metal oxides and sulfides (Mishra et al., 2005).
Most notably, sulfide ores have been bioleached in copper mining
commercially in several countries (Ehrlich, 2001). The term biohy-
drometallurgy is used to describe a subfield in hydrometallurgy that
involves biotechnology. It uses microbes to interact with insoluble
metal oxides/sulfides to convert them into soluble metal ions for further
recovery. It was first practiced in Rio Tinto mines in southwestern Spain

⁎
Corresponding author.
E-mail address: gu@ohio.edu (T. Gu).
1
Permanent address: Department of Chemical and Biomolecular Engineering, Ohio University, Athens, Ohio 45701, USA.

https://doi.org/10.1016/j.biortech.2018.04.033
Received 14 February 2018; Received in revised form 1 April 2018; Accepted 7 April 2018
Available online 21 April 2018
0960-8524/ © 2018 Elsevier Ltd. All rights reserved.
T. Gu et al.
in the beginning of the 1890s (Mishra et al., 2005). Bioleaching has
been used for decades at industrial scales without operators knowing
the exact roles of microorganisms in metal solubilization until 1961
when Acidithiobacillus ferrooxidans was discovered in the leachates
(Brandl, 2008). This bacterium oxidizes Fe2+ to Fe3+ and then using
Fe3+ as an electron acceptor (i.e., oxidant) to solubilize metal sulfides.
In 1965, Acidianus brierleyi as the first iron and sulfur-oxidizing ar-
chaeon was discovered (Brierley, 2007). Since 1980, bioleaching has
been effectively practiced at industrial scales for mining in various lo-
cations owing to the better understanding of the microbes involved
(Mishra et al., 2005). Bioleaching of numerous copper ores such as
chalcopyrite concentrates has been carried out since 1997 in Chili,
Mexico, USA, Australia and South Africa. Dump bioleaching at a high
altitude was found to be a very low cost process for extracting copper
from ores (Watling, 2015). Other successful commercial operations
including bioheap leaching and in situ bioleaching have been reported
as well (Watling, 2006, 2015).
To increase bioleaching efficiency, some pretreatment methods
were used. For example, the Fairview Mine in South Africa has the
longest history of using the BIOX process for biooxidation pretreatment
of sulfidic gold ores (Kaksonen et al., 2014). The Youanmi project in
Australia utilized moderately thermophilic bacteria including Sulfoba-
cillus thermosulfidooxidans and biooxidation with operating tempera-
tures between 45 and 55 °C . Another notable process was developed by
GeoBiotics. It is called GEOCOAT for the biooxidation of refractory gold
ores by coating the concentrate slurry onto a support rock or a substrate
material, and stacking this coated material in a biooxidation heap
(Mishra et al., 2005; Rawlings, 2013).
3. Microbes used in bioleaching
3.1. Mesophilic bacteria
Most industrial microorganisms used in the bioleaching are meso-
philic bacteria. Microbes in the Acidithiobacillus and Leptospirillum
genera are mesophilic that prefer temperatures between 25 and 35 °C
for growth.
3.1.1. Acidithiobacillus
Bacteria in the Acidithiobacillus genus are rod-shaped, Gram-nega-
tive and non-spore forming. They can grow under aerobic condition.
They derive energy by oxidizing sulfur compounds such as elemental
sulfur, sulfides and thiosulfate that are either reduced or partially re-
duced electron donors. Sulfate is usually the final oxidation product.
Most Acidithiobacillus species are chemolithotrophic species that use
CO2 in the air as the carbon source. This means that they do not need an
organic carbon supplement in bioleaching operations, which is very
advantageous. Acidithiobacillus ferrooxidans, Acidithiobacillus thioox-
idans, Acidithiobacillus caldus, Acidithiobacillus albertis, Acidithiobacillus
acidophilus, Acidithiobacillus concretivorus, Acidithiobacillus prosperus are
the most important Acidithiobacillus species in bioleaching. All of them
are rod shaped. These bacteria can oxidize elemental sulfur and gen-
erate sulfuric acid that can reduce the culture medium pH to as low as
1, making them appropriate in the bioleaching of metal oxides and
sulfides to recover metals (Hoque and Philip, 2011).
A. ferrooxidans, A. thiooxidans bacteria can grow well at an acidic pH
between 1 and 3, while other Acidithiobacillus genus require a higher pH
that is not sufficiently acidic for bioleaching (Hoque and Philip, 2011).
Thus, these two microbes are the most popular Acidithiobacillus species
in bioleaching. A. ferrooxidans is a more important species in bio-
leaching because in addition to harvesting energy by oxidizing reduced
sulfur compounds, Fe2+ is used as an energy source. In the absence of
oxygen, A. ferrooxidans is able to grow on reduced inorganic sulfur
compounds using ferric ion as an alternative electron acceptor with CO2
as the carbon source. Like A. thiooxidans, A. caldus does not use Fe2+ as
an electron donor in its metabolism.
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Bioresource Technology 261 (2018) 428–440
3.1.2. Leptospirillum
Leptospirillum ferrooxidans is an acidophilic obligatory chemolitho-
troph that uses Fe2+ as an energy source. This bacterium can tolerate a
lower pH (about 1.2), and higher concentrations of uranium, mo-
lybdenum and silver than what A. ferrooxidans can, but it is more
sensitive to copper and is also unable to oxidize sulfur compounds. L.
ferrooxidans has a lower growth rate than A. ferrooxidans, but it can be
accelerated by the addition of Zn2+. Furthermore, L. ferrooxidans is less
widespread than A. ferrooxidans, and this may reflect an inability to
compete in the natural environment (Ewart and Martin, 1991). Because
of this, a co-culture of L. ferrooxidans with A. ferrooxidans or A.
thiooxidans is sometimes used to solubilize metals in a sulfidized solid
matrix (Bosecker, 1997; Hoque and Philip, 2011). With regard to
growth temperature, Leptospirillum sp. have an upper limit of around
45 °C and a lower limit of around 20 °C (Rawlings, 2013).
3.2. Thermophilic bacteria and archaea
Due to high microbial activities with heat generation in exothermic
biooxidation in stirred tank reactors and in heaps, thermophiles are
desired in bioleaching to tolerate the high temperatures. Increased
operating temperatures and the use of thermophilic bacteria improve
not only reaction rates but also yields of extracted metals from some
minerals. Moderately thermophilic bacteria grow at a temperature
around 50 °C. Extreme thermophiles which are usually archaea
(Kaksonen et al., 2017) can grow at a temperature above 60 °C. A.
brierleyi is an archaeon formerly classified under genus Sulfolobus. Sul-
folobus species are capable of utilizing Fe2+, S0 and sulfides as energy
sources that are also used by Sulfobacillus thermosulfidooxidans, which is
a spore-forming facultative autotrophic bacterium. However, in lab
tests, its growth will only occur in a culture medium enriched with
yeast extract (Bosecker, 1997; Rastegar et al., 2014a). This hampers its
use in the field unless it is grown in a mixed culture with other microbes
that supply its nutritional needs.
3.3. Heterotrophic bacteria and fungi
Heterotrophic bacteria and fungi rely on organic compounds as
energy sources in their metabolism. In the growth phase, they secrete
different organic acids such as lactic, citric, oxalic and gluconic acids as
well as enzymes. These compounds can solubilize metals from a solid
matrix by forming soluble metal complexes and chelates. Bacillus spe-
cies are found to be the most effective bacteria, while some fungal
species in the Aspergillus and Penicillium genera are often used in bio-
leaching (Rastegar et al., 2014a, Rasoulnia et al., 2016; Vakilchap et al.,
2016).
3.4. Mixed culture consortia
It is simple to operate a pure-culture bioreactor in a lab because
there is no shift in the microbial population structure over time. It is
also less complex to study the intrinsic bioleaching mechanisms in such
a bioreactor. Due to the strong acidity produced by bioleaching mi-
crobes, often one microbe continuously thrive in the bioreactor even
without sterilization of the culture medium and solids, unless another
organism can tolerate the low pH. In recent years, some researchers
used mixed culture communities for bioleaching (Feng et al., 2013,
Latorre et al., 2016). In one study, the use of a co-culture of A.
thiooxidans and A. ferrooxidans considerably increased the recovery
yields of Cu, Ni, Pb and Zn from waste printed circuit boards compared
with results using the single cultures of these bacteria. Much more re-
search will be done using mixed cultures in the future due to its po-
tential to make bioleaching more practical in the field that often har-
bors mixed culture communities (Li et al., 2014, Ma et al., 2017).
T. Gu et al.
2H++SO42-
Fe2+
S2O32-
Fe3+ Fe2+
Fe3+
QS molecule FeS2
Biofilm attachment
Pyrite
Chemotaxis Quorum sensing (QS) Mature biofilm
4. Bioleaching mechanisms
4.1. Contact bioleaching
In contact leaching, cells attach to the surface of solid particles.
Sessile cells in biofilms are embedded in the extracellular polymeric
substances (EPS) secreted by the sessile cells (Schmidt and Schaechter,
2012). The cells suspended in the bulk fluid are called planktonic cells.
Microbes in nature often form synergistic biofilm consortia (Costerton,
2007). Some microbial members in a community produce metabolites
to benefit other members. Some other members can protect sessile cells
against harmful environmental factors such as antimicrobials (Li et al.,
2016).
Fig. 1 is a schematic illustration of the mechanism of bioleaching of
FeS2 by A. ferrooxidans. The mechanism is mainly found to be mediated
by microbial enzymes (oxidases or reductases). In the presence of
oxygen, A. ferrooxidans oxidizes FeS2 and uses O2 as the terminal
electron acceptor in the following reactions (Schmidt and Schaechter,
2012; Mishra et al., 2005; Rastegar et al., 2016),
FeS2 + 3.5O2 + H2 O → Fe 2 + + 2H+ + 2SO42 − (1)
There is still a lack of experimental evidence to show exactly how
contact leaching takes place, in which microorganisms break the metal
sulfide bond in metal sulfides (Watling, 2006).
4.2. Non-contact bioleaching
With the non-contact bioleaching mechanism, physical contact is
not required between solids and microbes. Solubilization of metals from
insoluble solids using bacteria and fungi relies on the three methods
below (Bosecker, 1997):
(1) Secretion of organic and inorganic acids (proton formation),
(2) redox reactions, and
(3) secretion of complexing agents for ligand formation.
Sulfuric acid is a popular inorganic acid for chemical leaching. In
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Bioresource Technology 261 (2018) 428–440
Fig. 1. A schematic illustration of steps of
bacterial cell - mineral interactions with de-
tails on the actions of A. ferrooxidans on
pyrite. The sessile cells in a biofilm con-
sortium generate ferrous ion and thiosulfate
with the latter finally oxidized to sulfuric
acid. [Figure redrawn with modifications
after Schmidt and Schaechter (2012)].
O2
Detached cells
Time
bioleaching, it is produced by different Acidithiobacillus bacteria. Ferric
ion is also a strong oxidizing agent that is reduced during the solubi-
lization of metal sulfides. Oxalate, malonate, citrate and succinate are
the most important organic ligands produced by different bacteria and
fungi. In the following reactions the formation of Fe3+ from oxidization
of Fe2+ is shown. Fe3+ dissolution of metal sulfides (MeS) is also shown
below (Mishra et al., 2005; Rastegar et al., 2016).
2Fe 2 + + 2H+ + 0.5O2 → 2Fe3 + + H2 O (2)
MeS + 2Fe3 + → Me 2 + + S 0 + 2Fe 2 + (3)
Although no contact is required in this type of bioleaching, biofilm
formation on solid particles can nonetheless improve bioleaching per-
formances because biofilms provide a diffusional barrier that leads to
locally concentrated metabolites such as organic acids on particle sur-
faces. This will be further discussed in Section 9.
4.3. Thiosulfate and polysulfide mechanisms
Recently, the dissolution of metal sulfides was categorized de-
pending on their solubility in acids. These are (i) acid-insoluble metal
sulfides (thiosulfate mechanism), and (ii) acid-soluble metal sulfides
(polysulfide mechanism) (Schippers and Sand, 1999).
In the thiosulfate mechanism, metal sulfides such as FeS2, MoS2,
WS2 are exclusively oxidized by Fe3+. The reactions in the thiosulfate
mechanism are shown below:
FeS2 + 6Fe3 + + 3H2 O → S2 O32 − + 7Fe 2 + + 6H+ (4)
S2 O32 − + 8Fe3 + + 5H2 O → 2SO4 2 − + 8Fe 2 + + 10H+ (5)
In the polysulfide mechanism, some metal sulfides such as ZnS, PbS,
FeAsS, CuFeS2 and MnS2 are dissolved by the combined effects of Fe3+
and H+ ions. Due to proton attack, the chemical bonds between metal
and sulfur moiety are broken and H2S·+ radical is formed and is sub-
sequently converted to H2S2. It is further oxidized via higher poly-
sulfides and polysulfide radicals to elemental sulfur. Hence, this me-
chanism is called the “polysulfide mechanism.” The reactions for this
mechanism are shown below:
T. Gu et al.
2MS + 2Fe3 + + 2H+ → 2M 2 + + H2 S2 + 2Fe 2 + (6)
H2 S2 + 2Fe3 + → 0. 25S8 + 2Fe 2 + + 2H+ (7)
0.25S8 + 3O2 + 2H2 O → 2SO4 2 − + 4H+ (8)
5. Designs of bioleaching reactors and processes
The design of a bioleaching process depends on microbial activities,
chemicals or minerals in ores or solid wastes. Thus, a process proven for
a particular solid may not be suitable for another solid. In the following,
some of the most popular process setups in bioleaching are explained.
5.1. Laboratory bioleaching
Several different experimental approaches have been used to eval-
uate bioleaching. They include airlift percolator, shake flask, airlift
reactor, stirred-tank reactor, aerated column and ore column (Borja
et al., 2016). Experiments were performed by researchers using airlift
percolators consisting of a glass tube with a sieve-plate at the bottom. It
was filled with particles suspended in a leaching solution inoculated
with bacteria. The leaching solution was recirculated back to the
column top by compressed air just like in other air-lift bioreactors for
fermentation. This percolator reactor suffered from low dissolved O2
and slow mass transfer, leading to low metal recovery yields. Sub-
merged leaching using shake flasks, stirred-tank reactors have been
evaluated by researchers. Shaking and stirring increase the rate of
aeration. This shortens reaction times considerably with increased
metal extraction yields (Bosecker, 1997; Borja et al., 2016). Column
leaching involves passing leaching solutions through a solid stationary
phase to model the dissolution of contaminants from the sample in the
field. It is used to model a heap or dump bioleaching processes with a
scale as large as several kilograms and up to a few tons of solids
(Bosecker, 1997; Borja et al., 2016).
5.2. Industrial leaching processes
Currently, industrial scales of bioleaching for the extraction of
metals from different low grade ores are being practiced. Dump
leaching has been used industrially to recover precious metals and
copper from ores. In this method, an acidified water or acidified ferric
sulfate solution is sprayed on solid particles in the dump and the lea-
chate solution is collected in ditches at the base of the dump in a
continuous operation (Pradhan et al., 2008).
Dump leaching of copper sulfide ores to recover copper has been
practiced in several countries and this technique has been expanded for
other commodity metals as well, including gold, nickel, uranium and
zinc (Watling, 2015). The metal extraction efficiency is low and the
duration of the operation lasts many days. In order to increase effi-
ciency, the leachate solution is controlled before going into an oxida-
tion basin with microorganisms that produce Fe3+. This method faces a
problem which is the possible escape of the leachate solution into
natural water supplies causing water pollution. Thus, measures must be
taken to prevent runoff.
Heap leaching is another industrial bioleaching method that has
advantages over traditional methods, especially when other econom-
ically feasible options are limited (Fig. 2) (Rawlings, 2002). It is similar
to dump leaching. It is mostly used for ores that have fine sizes, but still
too coarse for flotation. Pipes are inserted to supply sufficient O2 in
different locations in the heap.
Another industrial leaching method is the so-called underground
leaching method used to mine tailings that are too poor for in situ
leaching (Rawlings, 2002; Watling, 2015). In underground leaching,
the deposit is mined by several wells arranged in series, polygons, or
rings. A liquid containing bacteria is introduced into the wells. It filters
through the stratum and leaches the useful metal components. The
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Bioresource Technology 261 (2018) 428–440
working solution is pumped out through other wells or collected in
drifts. The solutions are collected downhole and pumped to the surface
for recovery. Underground leaching is used for mining nonferrous
metals and precious elements, and it is feasible for mining phosphates
and borates as well (Petersen, 2016). A major drawback of underground
leaching is the slow rate of chemical reactions.
Tank leaching is an industrial bioleaching technique that has much
higher reaction rates than other techniques but it is more expensive due
to increased capital costs (Watling, 2015; Petersen, 2016). It is popular
for gold and zinc ore concentrates. Pilot plants for bioleaching of Cu, Ni
and Zn from metal sulfide concentrates utilizing mesophilic and ther-
mophilic microbes have been set up and a commercial process to ex-
tract Co from a pyrite concentrate has been reported (Bosecker, 1997;
Watling, 2015; Petersen, 2016).
6. Operating parameters and performances
There are various parameters in the bioleaching process that can be
divided into chemical, microbiological and mineralogical categories. To
improve bioleaching performances, various operating conditions need
to be optimized.
6.1. pH
An appropriate pH of the culture medium can enhance the activities
of microbes in bioleaching. It also affects solubilization of metals and
the stability of metal ions in the liquid phase (Asghari and Mousavi,
2014; Fonti et al., 2016; Yu et al, 2014). pH in the range of 2–2.5 was
the optimal value for the commercial bioleaching of a metal sulfide by
A. ferrooxidans and A. thiooxidans. When pH was greater than 2.5, the
bioleaching rate decreased due to: (1) a decreasing Fe3+ concentration
as a result of Fe3+ precipitation in jarosites such as ammonium jarosite,
and (2) the attachment of bacteria to the jarosite, leading to decreased
bioleaching performance. For Penicillium, the optimum pH range for
growth was much wider, between 2 and 8 (Asghari and Mousavi, 2014;
Fonti et al., 2016).
Different strategies to maintain a low pH have been applied in-
cluding: (1) a pre-acidification stage of the sediment slurry (to neu-
tralize acid-consuming substances), (2) addition of Fe3+ ions, (3)
biostimulation of indigenous S-oxidizers in a low-grade ore or a solid
waste, and (4) bioaugmentation with Fe-oxidizing and S-oxidizing
bacterial strains with Fe2+ and S0 as energy sources (Fonti et al., 2016).
When Fe2+ is added to a culture medium, S0 appears to have a buf-
fering effect that prevents excessive pH shift (Asghari and Mousavi,
2014). Some studies controlled the culture medium pH. In this way the
variation of pH due to the bioleaching processes was not assessed
correctly. In general, the recovery of metals in a bioleaching process
represents the sum of bioleaching plus chemical leaching due to the low
initial pH in the medium (Fonti et al., 2016).
6.2. Redox potential
Owing to the biooxidation of Fe2+ to Fe3+ in the culture medium,
the redox potential of the medium increases (redox potential up to
+700 mV or more vs. Ag/AgCl). The high redox potential is suitable for
leaching of metals in an ore or solid waste. The culture medium redox
potential increases with the bacterial activity which benefits bio-
leaching of the metal. However, the redox potential during bioleaching
is not significantly affected by the amount of S0 provided (Asghari and
Mousavi, 2014; Fonti et al., 2016).
There are some reported studies that controlled redox potential to
accelerate bioleaching at the initial stage of bioleaching. Some re-
searcher showed that a high redox potential favors the bioleaching of
pyrite while a low redox potential favors the bioleaching of chalco-
pyrite. Chalcopyrite leaching stops almost completely when the redox
potential slips beyond the critical potential at a ferric to ferrous molar
T. Gu et al.
Fig. 2. A schematic illustration of a heap leaching process for copper ore. [Figure redrawn with modifications after Rawlings (2002)].
ratio of about 1 (Gericke et al., 2010; Petersen and Dixon, 2006).
Electro-bioreactor was also used to control the solution’s redox poten-
tial in the bioleaching of chalcopyrite, and it was shown that with a
redox potential range of 400–425 mV (vs. Ag/AgCl), chalcopyrite could
be effectively extracted (Ahmadi et al., 2010). The Nernst equation
below can be used to adjust the redox potential (E) by manipulating
operating temperature and ion concentrations (Zhao et al., 2015a),
RT [Fe3 +]
E= E∘ + ln
F [Fe2 +]
where E° is the standard reduction potential (at 25 °C, 1 M solutes or
1 bar partial pressure for gases), R the universal gas constant, T absolute
temperature and F Faraday constant.
6.3. Pulp density
The pulp density (i.e., the solid mass to liquid volume ratio) is one
the most important factors in bioleaching. In general, the bioleaching
efficiency decreases when pulp density increases because of: (1) in-
creased toxicity of heavy metals in the solid waste that inhibits meta-
bolic activities of microbes, (2) the adsorption of metal ions in the so-
lution to the solid matrix in the medium and therefore decreasing the
solubility of the metal in the bioleaching medium, and (3) inadequate
mixing that leads to insufficient O2 supply. In several published studies,
1% (w/v) was found to be the optimal pulp density for petroleum re-
finery waste catalysts (Pradhan et al., 2010; Asghari and Mousavi,
2014). In a different study, 1% (w/v) of fuel-oil ash was also the op-
timum for bioleaching of oil-fuel ash (Rastegar et al., 2016, 2015a)
6.4. Particle size
The solid waste particle size in the bioleaching medium is another
important factor in metal solubilization. The bioleaching of fine parti-
cles in ash and sludge has a huge advantage. The large surface to vo-
lume ratios accelerate bioleaching reaction rate. However, in the bio-
leaching of ores, energy is needed to create small particles. A particle
size smaller than 45 µm is usually preferred. It should be noted that,
sometimes, a very small particle size can damage the structure of fungal
cells in bioleaching microbial communities and results in a lower bio-
mass, leading to decreased bioleaching efficiency (Islam and Ting,
2009; Asghari and Mousavi, 2014).
Bioresource Technology 261 (2018) 428–440
6.5. Temperature
The optimum temperature to have high metabolic activities from
mesophilic microorganisms in bioleaching is around 30–35 °C. At a
higher temperature between 50 and 80 °C, thermophilic bacteria should
be used. When a temperature is much higher than the suitable tem-
perature for growth, microbes do not grow well or even die. Because
metal oxide and metal sulfide solubilization processes are exothermic,
heat can accumulate which increases the culture medium temperature
(9) undesirably and thus it should be controlled (Bosecker, 1997; Pradhan
et al., 2008; Fonti et al., 2016).
6.6. Nutrients
Microorganisms used for bioleaching need different nutrients.
Chemolithotrophic microbes can derive energy from reduced inorganic
compounds like sulfur and Fe (II) ions as electron donors. They use CO2
instead of organic carbon as the carbon source. Usually a mineral
supplement is added in lab tests if the feed particles do not have them
(Rastegar et al., 2016).
6.6.1. Elemental sulfur
S0 is an electron donor (i.e., energy source). As shown in the me-
chanism discussion above, elemental sulfur may be an important in-
termediate in the oxidation of sulfide minerals. Increasing the amount
of available S0 can improve bacterial growth and the rate of acidifica-
tion and therefore the biosolubilization efficiency. However, in some
studies, too much S0 showed a negative impact on biosolubilization of
some minerals such as MgO, CaO, Al2O3 (Asghari and Mousavi, 2014;
Fonti et al., 2016). S0 powder is the main commercial S0 source in
bioleaching. Its very low solubility is a disadvantage. S0 pastilles pro-
duced by power plants are favored for bioleaching due to low cost
(Asghari and Mousavi, 2014; Fonti et al., 2016).
6.6.2. Ferrous ion
The main source of Fe in the culture medium for bioleaching is
usually in the form of FeSO4 with a concentration of 0.3–16 g/L. An
increased amount of Fe2+ in the medium increases energy supply be-
cause this electron donor is oxidized to Fe3+. In one study, the bio-
leaching rate for iron oxidizing bacteria increased after adding 3 g/L of
Fe2+ in the culture medium. However, the rate decreased at higher
Fe2+ concentrations because of: (1) lowered metabolic activity, (2)
elevated Fe3+ precipitation rate that hampered diffusional mass
432
T. Gu et al.
transfer for bioleaching, (3) improper bacterial adaptation in the cul-
ture medium, and (4) metabolite toxicity including Fe3+ toxicity
(Asghari and Mousavi, 2014; Fonti et al., 2016; Wong et al., 2015).
6.6.3. Dissolved O2 and CO2
Oxygen has a fundamental impact on the overall bioleaching pro-
cess (Gleisner et al., 2006). The dissolved O2 supply comes from aera-
tion, stirring or shaking. Under O2 limitation, the rates of S0 and Fe2+
oxidization reactions decrease, resulting in a delay in sulfuric acid
production and metal solubilization. A dissolved O2 concentration be-
tween 1.5 and 4 mg/L is often required for optimal microbial activities
in bioleaching (Kock et al., 2004). Some conflicting results in the lit-
erature could to attributed to whether the oxygen effects are detected in
the bacterial culture with and without the presence of sulfide minerals
(Mandl et al., 2014).
In addition to aerobic conditions for the oxidation of sulfur, some
bioleaching microorganisms are also able to use Fe3+ as an oxidant in
anaerobic respiration when cells are deprived of oxygen (e.g., under-
neath an aerobic biofilm that serves as an oxygen barrier through
oxygen consumption and O2 diffusional limitation). The reduction of
Fe3+ regenerates Fe2+ that is used by those aerobic cells with access to
oxygen within the same biofilm structure (Schmidt and Schaechter,
2012). This anaerobic process is important to explain sulfuric acid
formation even without oxygen which often is a limiting factor in in situ
bioleaching. A. ferrooxidans anaerobically reduces ferric ion using S0 as
the electron donor in the following reaction,
S 0 + 6Fe3 + + 4H2 O → HSO4− + 6Fe 2 + + 7H+ (10)
3+ 0 leaching conducted with ultrasonic is given in Table 1. Some ultrasonic
Fe oxidation of S is one of the two steps in sulfur metabolism in
bioleaching microbes. In the first step, sulfur:ferric ion oxidoreductase
converts sulfur to sulfite and in the second step sulfite:ferric ion oxi-
doreductase converts sulfite to sulfate in the periplasmic space (Sugio
et al., 1992). Different from the two-step mechanism above, Corbett and
Ingledew (1987) proposed that electrons donated by S0 enter the re-
spiratory chain via the bc1 complex. The electrons are transported
through the periplasmic membrane and are finally used for the reduc-
tion of Fe3+. More work is needed to elucidate the mechanism for
electron transport from S0 to Fe3+.
In addition to O2, CO2 is critically important because it provides the
carbon requirements for the growth of autotrophic bacteria. However,
the effect of CO2 supplementation on microbial growth was probably
overlooked in many instances because dissolved CO2 was more difficult
to measure than dissolved O2. The optimal inlet CO2 concentration for
ferrous ion oxidation was found to be between 7% and 17% (v/v) in
several studies (Bosecker, 1997; Kock et al., 2004; Fonti et al., 2016).
7. Pretreatment in bioleaching
Some pretreatment methods have been increasingly used to increase
metal extraction efficiency in bioleaching. Pretreatment can be used for
either microorganisms or solid particles.
7.1. Pretreatment of microorganisms and solid wastes
In the bioleaching process, selecting and breeding efficient bacteria
for solid waste bioleaching are becoming more and more important.
Mutation breeding is a frequently-used and effective method to obtain
high-performance microorganisms (Xie et al., 2013). Mutation of mi-
croorganisms was used to increase the tolerance of microorganisms to
solid wastes with high toxicity. Mutation of cyanogenic bacteria was
used to promote gold recovery with bacteria tolerant of alkaline con-
dition (Natarajan and Ting, 2014). There are different methods to
mutate microorganisms to increase bioleaching efficiency. The effects
of different pretreatment methods on bioleaching are summarized in
Table 1 and discussed below.
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Bioresource Technology 261 (2018) 428–440
7.1.1. UV
One research group used UV mutation to treat A. ferrooxidans GF
and Acidiphilium cryptum DX1-1 for chalcopyrite bioleaching (Xu et al.,
2010). They obtained high-performance bacteria that increased the
dissolution of copper (Xu et al., 2010). Another group of researchers
treated mixed microorganisms with mutagen diethyl sulfate (DES), UV,
and their combinations. It was found that the best one increased the
content of Cu2+ by 101% in 20 days of bioleaching compared with the
control culture (Kang et al., 2009).
7.1.2. Microwave
The complexities and the intergrown nature of ores result in a poor
liberation of valuable minerals (Olubambi et al., 2007; Ola-Omole et al.,
2016). It has been shown that bioleaching efficiency can be improved
after microwave treatment. Microwave has been investigated for mi-
neral processing for many years (Ola-Omole et al., 2016). It can reduce
processing costs owing to the reduction in the energy required for
grinding. Metal extraction yields can be improved with a shorter pro-
cessing time due to accelerated chemical reaction rates (Olubambi
et al., 2007). The drawback is an extra processing step that consumes
energy (Pathak et al., 2017). For ash and sludge with sufficiently small
particle sizes, this pretreatment is not needed.
7.1.3. Ultrasonication
Microbes and ores or solid wastes can be sonicated before bio-
leaching to improve metal extraction efficiency. The application of ul-
trasound expands the advantages of bioleaching while avoiding or
lessening its shortcomings to an acceptable level. A summary of bio-
irradiation effects are discussed below (Capote and de Castro, 2007).
7.1.3.1. Stimulation of microbes. The effects of ultrasound on the
bacterial metabolic activity depend on some factors such as the type
of microbes (Gram negative or Gram positive), their shape (rod or
coccus) and size (big or small) (Vyas and Ting, 2018). The underlining
mechanisms are not clear (Erriu et al., 2014). Recently, ultrasound has
been reported to enhance enzymatic/microbial activities by improving
stirring at microscopic and macroscopic levels and changing the
permeability of cell membranes (Vyas and Ting, 2018). Vargas et al.
(2004) observed that using ultrasonic treatment of more than 4 min
caused an increase of around 28% in metabolic activity due to the
disruption of A. niger and release of invertase enzymes (Vargas et al.,
2004). It should be noted that sonication of extracellular enzymes in a
solution only leads to a reduction in bioactivity due to enzyme
denaturation and thus sonication should be optimized and controled.
7.1.3.2. Improved bioleaching efficiency. The effects of ultrasound on
bioleaching efficiency are related to the type of bioleaching process
including: one-step bioleaching (the ore/solid waste is added to
inoculum at the beginning of inoculation), two-step bioleaching (a
high cell count or metabolite concentration is obtained first and then
ore/solid waste is added), and spent medium leaching (the
microorganisms are removed before the addition of ore/solid waste)
(Vyas and Ting, 2018). The mechanical effects from ultrasonication are
significant where the reactions in a bioleaching process are
heterogeneous. Although in one way increasing sonication intensity is
desired to enhance mechanical effects in order to increase metal
extraction, but it also increases the membrane permeability of
microorganisms that leads to increased toxic effect of the metals to
the microorganisms. An optimum intensity (1.5 W/cm2) was found for
A. niger bioleaching.
7.1.3.3. Enhanced selectivity in bioleaching. Ultrasound pretreatment
increased the selectivity in extraction favoring leaching of metals like
Ni. One study showed that up to 92% Ni and 12.5% Fe were extracted
in a one-step bioleaching process within 20 days (Vyas and Ting, 2018)
T. Gu et al. Bioresource Technology 261 (2018) 428–440

Table 1
Pretreatment to improve metal extraction.
Ore/waste Bioleaching process Pretreatment condition Outcome Ref.

Lateritic nickel ore One-step 30 min sonication of broth + spores + ore at 20 kHz, Ni: 95% Swamy et al.
1.5 W/cm2 Fe: 0.2% both in 14 days (1995)

Lateritic nickel ore One-step 30 min sonication of broth + spores + ore at 43 kHz, Ni: 95%, 14 days Sukla et al.
1.5 W/cm2 Fe: 0%, 14 days (1995)

Lateritic nickel ore One-step 30 min sonication of broth + spores + ore at 43 kHz, Ni: 95%, 14 days Kar et al. (1996)
1.5 W/cm2 Fe: 0%, 14 days

Black shale Two-step 7 min sonication, 40 kHz, during 15 days of growth and Cu: 92%, Zn: 87% , Co: 71% over 24 days Anjum et al.
36 days of bioleaching Al: 92%, 24 days (2010)
Fe: 83%, 21 days

Hospital waste incinerator Spent medium 10 min sonication of spent medium + waste at 40 kHz, Increase of around 20 mg/kg Al, 15 mg/kg Anjum et al.
bottom ash leaching 18 days of bioleaching Fe and 5 mg/kg Zn on sonication (2014)

Olivine ore Two-step 15 min sonication, 37 kHz, 0.2 W/cm2, during 7 days of Ni: 15.7%, 17 days marginal increase in Mg, Chiang et al.
growth and 17 days of bioleaching Al, Si & Cr (2014)

gold ore One-step 30 min Ultrasonic of L. ferriphilum YXW at 40 kHz, As: 93.2, Fe: 58.7, 10 days Yuan et al.
500 W (2013)

gold ore One-step 10 s microwave, of L. ferriphilum YXW As: 99.7, Fe: 76.0, 10 days Yuan et al.
(2013)

gold ore One-step 120 s UV of L. ferriphilum YXW As: 95.2, Fe: 66.8, 10 days Yuan et al.
(2013)

Arsenic-rich gold One-step 180 s UV of bacterial flora Fe2+oxidation rate (%): 99.8 Xie et al. (2013)
concentrates 120 s UV of bacterial flora Fe2+ oxidation rate (%): 98
60 s UV of bacterial flora Fe2+ oxidation rate (%): 81

Arsenic-rich gold One-step ultrasound 10 min Fe2+ oxidation rate (%): 99.8 Xie et al. (2013)
concentrates ultrasound 20 min Fe2+ oxidation rate (%): 99.8
ultrasound 30 min Fe2+ oxidation rate (%): 99.8

Arsenic-rich gold One-step microwave 10 s Fe2+ oxidation rate (%): 98 Xie et al. (2013)
concentrates microwave 20 s Fe2+ oxidation rate (%): 99.6
microwave 30 s Fe2+ oxidation rate (%): 99.6

without pretreatment. With ultrasonic pretreatment, the reaction rate
and recovery rate were both increased with 95% Ni extracted in 14 days
while the leaching of Fe was negligible. The negligible leaching of Fe
was due to the oxidation of Fe2+ to Fe3+ during sonication that led to
the formation of peroxide and free radicals.
7.1.3.4. Acceleration of metal extraction reactions. The maximum
extraction efficiency was achieved within 24 days when a solid waste
was treated ultrasonically, leading to higher metal recovery yields than
those achieved over 36 days without sonication pretreatment. With
ultrasonication longer than 24 days, metal recovery yields started to
decline, probably due to precipitation because of leachate saturation or
undesirable reactions such as complexation reactions.
7.2. Solid waste pretreatment
The main aim in the pretreatment of a solid waste is to increase the
accessibility of valuable metals for subsequent bioleaching to improve
metal extraction efficiency. This is done to: i) extract some metals that
can compete with the main target metals in the bioleaching stage (e.g.,
the extraction of Cu, Fe and Al can compete undesirably with the ex-
traction of gold, silver and some other rare and expensive elements), ii)
remove toxic compounds that have negative effects on microorganisms
by using water or acid wash to neutralize the culture medium which can
counteract the release of carbonate and oxidization that increase pH,
affecting acidophilic bacteria adversely, and iii) make accessible valu-
able metals with increased concentrations in the solid and be able to use
flotation or magnetic separation to remove less valuable metals
(Arshadi and Mousavi, 2015).
Thermal pretreatment of fuel ash by incineration prior to bio-
leaching removes carbonaceous compounds (Bharadwaj and Ting,
2013) and concentrates inorganic matters. This pretreatment is
beneficial to bioleaching because of: (1) increased metal solubilization
efficiency due to promotion of chemolithotrophic microbes used in
bioleaching, (2) increased metal concentrations after bioleaching, thus
facilitating the subsequent metal precipitation stage, (3) decreased use
of chemical reagents in the bioleaching process due to reduced feed,
and (4) harvest of the heating value stored in the carbon content in the
ash (Rasoulnia et al., 2016). Burning temperature should be controlled
and optimized to avoid the fusion of ashes and metal volatilization.
8. Bioleaching for bioremediation
Many thermal power plants in developing countries burn coal or
fuel oil because the much cleaner natural gas is not always available or
affordable. Coal and fuel oil produce large amounts of fly ash and
bottom ash. The residual organic matters in the ashes can be removed
using different biological, chemical and physical methods easily
(Reijnders, 2005). However, inorganic materials which are different
compounds containing metals, some of them heavy metals, often in the
form of metal oxides and sulfides, are not degraded by these methods
(Bosecker, 2001; Lee and Pandey, 2012). In the following sections, the
characteristics of different solid wastes including fuel-oil ash, coal ash
and sewage sludge and their bioleaching for the recovery of metals as
value-added products are discussed.
8.1. Fuel-oil ash
8.1.1. Chemical characteristics
The fly ash collected by a cyclone collector is called CY fly ash. It is
called EP fly ash if it is collected by an electrostatic precipitator (Tsai
and Tsai, 1998). Bottom ash is collected at the bottom of a furnace. It is
heavier than fly ash. Fuel oils contain Fe, Ni, V, and Zn, in addition to
Al, Ca, Mg, Si, and Na. These metals exist in the crude oil supply. They

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Table 2
Summary of bioleaching of fuel-oil ash in the literature.
Microorganism Condition Type of reactor Recovery Ref.

A. ferrooxidans pH = 1 Shake flask V 82% Rastegar et al. (2015b)

inoculum percentage = 1% Ni 86%
initial Fe2+ concentration = 1 g/L Cu 87%
pulp density = 4% (w/v)
Time = 10 days

A. ferrooxidans pH = 1 Shake flask V 74% Rastegar et al. (2015a)

inoculum percentage = 1% Ni 95%
initial Fe2+ concentration = 2.6 g/L Cu 88%
pulp density = 1% (w/v)
Time = 4 days
A. thiooxidans pH = 1 Shake flask V 96.4% Rastegar et al. (2016)
initial sulfur = 9 g/L Ni 100%
pulp density = 1% (w/v) Cu 99.2%
Time = 7 days

A. thiooxidans pH = 1 Reactor V 77.6% Rastegar et al. (2016)

initial sulfur = 9 g/L Ni 89.7%
pulp density = 1% (w/v) Cu 65.7%
Time = 8 days

P. simplicissimum pH = 3 Shake flask V 100% Rasoulnia et al. (2016)

pulp density = 1% (w/L) Ni 40%
Time = 10 days Fe 48.3%

A. niger aeration rate = 763 mL/min, sucrose concentration = 102 g/L Reactor V 83% Rasoulnia and Mousavi (2016)
inoculum size = 40 mL/L Ni 30%

are enriched in the ash after incineration. Transition metals (Fe, Mn,
and Co) and alkaline-earth metals (Ba, Ca, and Mg) may also be added
for the suppression of soot or for corrosion control (Al-Malack et al.,
2013). Thus, they can be found in the ash as well.
8.1.2. Bioleaching of fuel-oil ash
Several studies for the recovery of heavy metals from fuel-oil fly ash
are summarized in Table 2. For instance, V, Ni and Cu were bioleached
from fuel-oil ashes with operating conditions of pH 1, 1% (v/v) in-
oculum, 1 g/L initial Fe2+ concentration, 4% (w/v) pulp density and
incubation time of 10 days in shake flasks. At these conditions the
maximum recovery yields were 82% V, 86% Ni and 87% Cu, reflecting
improvements of 20%, 16% and 8%, respectively when compared with
chemical leaching. Using a modified shrinking core model, it was found
that the bioleaching process was controlled by diffusional mass transfer
(Rastegar et al., 2015b). A recent work statistically optimized different
parameters on the bioleaching of fuel-oil ash by A. ferrooxidans using
the response surface methodology (RSM) (Rastegar et al., 2015a). With
initial conditions of pH 1.3, 2.6 g/L Fe2+ and 1% (w/v) pulp density,
maximum recovery yields of 74% V, 95% Ni, and 88% Cu were ob-
tained in shake flasks with 100 mL culture medium. V was then re-
covered using NaOH for pH adjustment to form NaVO3, which was
precipitated using NH4Cl from the bioleachate liquor. After calcination,
V2O5 was produced. It was subsequently used to make highly ordered
single crystalline NaV6O15 nanorods with high purity, which are useful
in many industrial applications (Rastegar et al., 2015a).
Another work studied Cu, Ni and V recoveries from fuel-oil ash
using A. thiooxidans in small shake flasks with 50 mL culture medium,
which was then scaled up to a 2.4 L bubble column bioreactor (Rastegar
et al., 2016). The optimal bioleaching initial conditions were found to
be pH 1, 9 g/L sulfur and 1% (w/v) pulp density. The highest recovery
yields after 9 days of incubation were 99.2% Cu, 100% Ni and 96.4% V,
respectively for the shake flasks. Risk assessments using various toxicity
assessments indicated that the Cu, Ni and V levels in the ash after
bioleaching were all well below US EPA regulatory limits for safe dis-
posal. In the 2.4 L bubble column bioreactor, the highest recovery
yields were 65.7% Cu, 89.7% Ni and 77.6% V, respectively with an
initial pulp density of 1% (w/v) after an 8-day incubation period. These
efficiencies were lower than those from the shake flasks which were
incubated for 9 days. With an increased incubation time and more ef-
ficient mass transfer (e.g., by using an airlift column instead of a bubble
column), it is expected that the recovery yields could be improved
considerably.
In another work (Rasoulnia and Mousavi, 2016), fuel-oil ash was
bioleached using Penicillium simplicissimum with an initial pulp density
of 1% (w/v). Several different bioleaching methods including one-step,
two-step, and spent-liquor methods were tested. The highest extraction
yields of V and Ni were bioleached with the spent-liquor method. Al-
most 100% V, 40% Ni and 48% Fe were recovered. Using simulated
chemical leaching with a mixture of 5237 ppm (w/w) citric acid,
3666 ppm gluconic acid, 1287 ppm oxalic acid and 188 ppm malic acid
as comparison, it was found that bioleaching increased Ni and V re-
coveries by 12% and 19%, respectively.
8.2. Coal ash
8.2.1. Chemical characteristics
Coal is widely used for electricity generation. Coal fly ash remains a
serious environmental problem. The chemical characteristics of coal fly
ash differ significantly from those of fuel-oil fly ash (Reijnders, 2005).
The major metal oxides in coal are SiO2, Al2O3, FeO3, CaO, MgO, Na2O
and K2O (Meawad et al., 2010). Coal fly ash is considerably richer in
various valuable elements such as Ni, Zn and Cu. The chemical ele-
ments in fly ash are listed in Table 3 (Jadhav and Hocheng, 2015). Finer
fly ash particles tend to be richer in these elements because of the
condensation-volatilization process (Sahoo et al., 2016).
8.2.2. Bioleaching of coal ash
Table 4 lists several organisms that have been used for the bio-
leaching of coal ash. The bioleaching of fly ash using newly isolated
fungi Fusarium oxysporum and Penicillium glabrum were investigated in
one-step and two-step bioremediation processes. Using F. oxysporum in
bioleaching, the bioremediation process recovered Mo (100%), S
(64.4%) Ni (50%) and Cu (33.3%). P. glabrum recovered Mo (100%), S
(57.4%), Ni (25%), Si (24.7%), V (12.5%), Ti (5%) and Sr (3.2%) from
the fly ash (Taştan, 2017). After 4 h of bioleaching, An Aspergillus niger
broth removed almost 100% metals except Cr (93% recovered), Ni
(83%), As (78%), and Pb (70%) (Jadhav and Hocheng, 2015). The

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Table 3 Table 5
Elements in coal fly ash (ppm by mass). Physicochemical and biological properties of sewage sludge.
[Reproduced from Pathak et al. (2009) with permission from Elsevier].
Element Jadhav and Hocheng (2015) Meawad et al. (2010)
Property Value
Aluminum 10.7 ND*
Antimony ND 4.6 pH 5–8
Arsenic 0.86 43.4 Total solids (%) 0.83–12
Barium ND 807 Volatile solids (% TS) 30–88
Beryllium ND 5 Moisture (%) up to 95
Boron 20.3 311 Protein (% TS) 15–41
Cadmium 0.35 3.4 Nitrogen (% TS) 1.5–6
Calcium 24.31 ND Phosphorus (ppm) 0.8–11
Chromium 18.2 136 Potassium (ppm) 0.4–3
Cobalt 9.08 35.9 Silica (% TS) 10–20
Copper 40.2 112 Organic acids (mg/L HAc) 200–2000
Fluorine ND 29 Total coliforms (numbers/g dry) 1.2 × 108
Iron 29 ND Fecal coliforms (numbers/g dry) 2 × 107
Lead 10.1 56.8 Fecal Streptococci (numbers/g dry) 2.1 × 105
Magnesium 50.3 ND Salmonella sp. (numbers/g dry) 7.9 × 102
Manganese 66 250 Enteric virus (numbers/g dry) 3.6 × 102
Mercury ND 0.1 Cu (ppm) 112–2300
Nickel 22.5 77.6 Ni (ppm) 23.3–622
Potassium 17 ND Zn (ppm) 354–3096
Selenium 10.4 7.7 Cr (ppm) 45.8–8110
Silver ND 3.2 Cd (ppm) 1.5–54
Sodium 31.4 ND Pb (ppm) 26–465
Strontium ND 775
Thallium ND 9
Titanium 0.8 ND
8.3. Sewage sludge
Vanadium 1.07 252
Zinc 36.3 148
8.3.1. Chemical characteristics
* ND: not determined. The treatment of municipal wastewater produces huge amounts of
sewage sludge. Different types of sludge are produced, including the
operating initial pulp density, shaking speed and temperature were primary sludge from mechanical treatment and secondary sludge from
optimized to maximize metal recovery. Toxicity tests were carried out biological treatment. Incineration is often used to dispose of municipal
for the fly ash before bioleaching on the germination of Vigna radiata sewage sludge. The process produces fly ash in the off-gas and bottom
(mung bean). It was found that the mung bean failed to germinate with ash. The fly ash compositions differ depending on the locality. It usually
20% (w/v) pulp density. In comparison, after the bioleaching treat- contains Al, Cd, Cr, Cu, Fe, Mn, Ni, Pb, Zn, As and Hg. Metals such as Al,
ment, a 78% germination rate was observed at the same pulp density. Cu and Zn may be abundant enough in sewage sludge and their in-
The bioleaching of Al and Fe elements from coal fly ash using cineration ash, making their recoveries economical (Ishigaki et al.,
Thiobacillus thiooxidans (now reclassified as A. thiooxidans) was in- 2005; Lee and Pandey, 2012). Table 5 shows physicochemical proper-
vestigated. A batch bioleaching experiment was carried out at an initial ties and heavy metal contents of sewage sledge.
pulp density of 10% (w/v) in the inoculated culture medium. After
3 weeks of incubation, it resulted in extraction recovery yields of 25% 8.3.2. Bioleaching of sewage sludge
Al and 22% Fe (Seidel et al., 2001). Table 6 shows a summary of sewage sludge bioleached with dif-
These initial investigations on bioleaching provided encouraging ferent methods. The recovery yields of metals depend on the miner-
results. There is a growing interest in using bioleaching to treat coal alogy of metals, several different factors and the method applied for
ash. Coal ash is considered by the US Department of Energy as a good bioleaching. For example, in different studies for the bioleaching of
source of valuable rare earth elements for recovery. The value in the anaerobic sludge using A. ferrooxidans and A. thiooxidans, the recovery
recovery of useful metals will incentivize more stringent fly ash cap- yields of different metals were 34–100% Cu, 18–80% Cr, 18–69% Cd,
ture, resulting in less emission. It is anticipated that intensive bio- 10–58% Pb, 58–99% Mn, 42–100% Ni, and 38–100% Zn. Results
leaching research will be carried out in this area in the near future. showed that most metals tend to dissolve easily from metal sulfides in
the sludge (Kim et al., 2005; Pathak et al., 2009). Batch and continuous
modes of sewage sludge bioleaching using Thiobacillus sp. with Fe2+

Table 4
The bioleaching of coal ash in the literature.
Microorganism Conditions Efficiency Reactor type Ref.

F. oxysporum pH 5.0 Mo (100%), S (64.4%) Ni (50%) and Cu (33.33%) Shake flask Taştan (2017)
Biomass concentration = 1 g/L
Pulp density = 0.5–1%

P. glabrum pH 6.0 Mo (100%), S (57.43%), Ni (25%), Si (24.7%), V Shake flask Taştan (2017)
Biomass concentration = 1 g/L (12.5%), Ti (5%) and Sr (3.2%)
Pulp density = 0.5–1%

A. niger Time 4 h Cr 93%, Ni83%, As 78%, Pb 70%. Shake flask Jadhav and Hocheng
shaking speed: 50-rpm, temperature: 30 °C, solid/liquid (2015)
ratio: 1 g/100 mL

T. thiooxidans 3 weeks 25% for Al and 22% for Fe Shake flask Seidel et al. (2001)
Pulp density = 10%

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T. Gu et al. Bioresource Technology 261 (2018) 428–440

and S0 in the culture medium have been reported in the literature (Kim

Pathak et al. (2009), Seth et al.

Bosshard et al. (1996), Wu and

Kim et al. (2005), Pathak et al.
et al., 2005; Pathak et al., 2009). A continuously stirred tank reactor

Pathak et al. (2009), Filali-

(CSTR) inoculated with A. ferrooxidans offered higher recovery yields of

Meknassi et al. (2000)

Ishigaki et al. (2005)

Cd (50–93%), Cu (33–91%), Ni (48–98%) and Zn (62–94%) within a

Wang et al. (2009)

Krebs et al. (2001)

Chen et al. (2016)

Jain et al. (2010)

Zhu et al. (2013)

relatively short incubation time compared to batch bioleaching (Seth
et al., 2006; Pathak et al., 2009). In some reported studies, the si-

Ting (2006)
multaneous sludge digestion and metal leaching (SSDML) was carried
(2009)

(2006)
out in bioleaching reactors. The bioleaching for SSDML with A. fer-
Ref.

rooxidans led to recovery yields of 41–100% Cu, 48–100% Zn, 25–100%

Cd and 15–84% Ni (Filali-Meknassi et al., 2000; Pathak et al., 2009).
The bioleaching of secondary activated sludge in a dual-stage pro-

Shake flask in one-/

cess was reported in the literature (Jain et al., 2010). The sludge was
Continuous mode

Two-stage rector
Type of reactors

first treated using less acidophilic thermophilic sulfur oxidizing mi-

Shake flask

Shake flask
Shake flask

Shake flask

Shake flask

Shake flask

Shake flask
crobes in an auto-heated aerobic digester at a temperature between 55
Bioreactor
bioreactor

two-step
and 60 °C. It was then fed to a CSTR reactor for bioleaching at 30 °C by
mesophilic sulfur oxidizing microbes, leading to recovery yields of 76%
Cu, 78.2% Mn, 79.5% Ni and 84.2% Zn. A mixed culture containing A.
Cu: 33–91%, Ni 48–98%, Zn 62–94%, Cr 6–8%, Cd-50–93%, Pb 3%, Mn-

Cu 67%, Zn 78%, Cr and Cd 100% at 10 g/L pulp density in the presence

Cu and Pb 60–70%, Al, Mn, Zn 80–100%, Fe 30% in 2 days at 10 g/L pulp

thiooxidans was used to treat fly ash collected from burned sewage

Cu, Cd, Zn 80%, Al 60%, Ni, Fe 30% in the presence of sewage sludge

Cd 96%, Mn 91%, Pb 73%, Zn 68%, Cr 35%, Fe 30% from 1% water-

Cu 34–100%, Ni 42–100%, Zn 38–100%, Cr 18–80%, Cd 18–69%, Pb
10–58%, Mn 58–99% with S 30–100 g/L in 1–16 and 10 days (in one

Cu 41–100%, Ni 15–84%, Zn 48– 100%, Cr 9–50%, Cd 25–100%, Pb

sludge. The culture medium was mixed with 4% (w/v) of the fly ash
from an anoxic sewage sludge as nutrients and 10 g/L S0 for incubation
Cd, Ni, Zn 60–80% in batch culture with 200 g/L pulp density

lasting 14 to 21 days. The process leached almost 80% of Cu, Cd, Zn,
and 60% Al, and 30% Fe, Ni with low Pb dissolution (Krebs et al., 1997,
Cu 57–85%, Cd 53–68.5%, Zn 39–56.4%, Pb 2–19.6%

2001). With the utilization of a mixed culture consortium of iron- and

sulfur-oxidizing bacteria at an initial S0 concentration of 2–5 g/L, the
Cu 76%, Ni 79.5%, Zn 84.2%, Mn 78.2% in 24 h

process leached almost all Cr and Cd, plus 67% Cu and78% Zn (Ishigaki
Cu 73.1%, Zn 78.7%, Pb 24.7%, Cd 79.5%

Cu 75.3%, Zn 72.6%, Pb 34.5%, Cd 65.4%

et al., 2005). Similar levels of leached metals from fly ash were also
7–93% in 1–4 and 14 days (in one study)

obtained at a higher pulp density of 40 g/L in a semi-continuous tank

Leaching efficiency and conditions

reactor with a Thiobacillus sp. strain when pH was lowered by adding

10 g/L S0 and sulfuric acid (Krebs et al., 2001). When Pseudomonas
putida was grown with 24–42 g/L glucose at a pulp density of 20 g/L for
washed sample in 20 days
suspension in 2–3 weeks

7 days recovery yields were between 60 and 80% for most bioleached
5–69%, Mn 77–100%

metals. When the fly ash loading was greater than 30 g/L, the yields fell
density in 30 days

below 20% because of the lack of carbon source (Krebs et al., 1997).
The effect of gluconic acid produced by A. niger instead of citric acid
of 2–5 g/L S

has been examined because the metals inhibited the accumulation of

case)

citric acid. The operating conditions were 2–3 weeks of incubation

time, 100 g/L sucrose concentration at 40 g/L pulp density, which led to
recovery yields greater than 80% Cd and Zn, roughly 60% Cu, 5–10%
(iii) A. thiooxidans, A. ferrooxidans, (mixed culture of
(i) A. ferrooxidans (with ferrous sulfate/S addition)
A. ferrooxidans, A. thiooxidans (Fe and S oxidizers)

Ni and Cr and 95% Pb (Bosshard et al., 1996). A similar study on the

bioleaching of metals in the fly ash from a sewage sludge incinerator
(i) A. ferrooxidans (aerobic sludge digestion-

Less-acidophilic thermophilic sulfur-oxidizer

using A. niger was reported (Wu and Ting, 2006). With a pulp density of
10 g/L over a period of 30 days, the recovery yields were 80–100% for
S and iron oxidizing bacterium)

Al, Mn, Zn; 60–70% for Cu, Pb and roughly 30% for Fe in both one-step
(i) Mixed culture of A. thiooxidans
(ii) Thiobacillus sp. strain TM-32

and two-step bioleaching processes. The bioleaching of fly ash pre-

treated with water wash using A. niger for 20 days at 10 g/L pulp den-
sity in one-step or two-step processes was also reported. Pretreatment
due to removed alkaline chlorides (K, Na, Ca, etc.) resulted in the en-
hanced metal recovery with yields of 96% Cd, 35% Cr, 30% Fe, 91%
bioleaching)

A. ferrooxidans

A. ferrooxidans

Mn, 73% Pb and 68% Zn (Wang et al., 2009).

Microbe used

(i) P. putida
(ii) A. niger

The benefits of bioleaching on the pyrolysis of sewage sludge have

been reported in the literature. Published data indicated that after
bioleaching, the sewage sludge had different physicochemical proper-
ties that enhanced later metal removal. One study (Chen et al., 2016)
Sewage sludge bioleaching in the literature.

Sewage sludge (anaerobically digested sewage

Specific type and source of waste/by-product

used an initial pulp density of 6% (w/v). It achieved optimal metal

removal yields of 79.5% Cd, 73.1% Cu, 24.7% Pb and 78.7% Zn. The H/
Fly ash (municipal solid waste) from

C, N/C ratios in the sewage sludge after bioleaching were reduced but
electrostatic precipitator unit

the O/C ratio increased favorably. The fulvic acid, humic acid, and the
Fly ash of municipal solid waste

total humic contents of the sewage sludge after bioleaching treatment

Secondary activated sludge

were higher in comparison with sewage sludge before bioleaching.

Thermogravimetric analyses showed that there was less residue after
the pyrolysis of the bioleached sewage sludge in comparison with the
sewage sludge before the bioleaching treatment.
Sewage sludge

Sewage sludge

Sewage sludge
sludge)

9. Biofilms and microbiome in bioleaching

Table 6

The majority of microbes in nature live in microbial communities

437
T. Gu et al.
known as biofilms (Costerton, 2007; Zhao et al., 2015b). Different
microbial species in a biofilm consortium play different roles, making
biofilms robust and resistant to harmful environmental factors. In the
past, bioleaching researchers did not pay enough attention to the role
biofilms played in bioleaching. Most studies used a pure strain microbe
and they did not differentiate the effects of sessile cells from those of
planktonic cells. pH is often a key operating parameter in bioleaching.
Only the culture medium pH was reported in the literature. However, it
is known to the biofilm research communities that the local conditions
underneath a biofilm can be very different from those in the bulk-fluid
phase. The pH values even in two neighboring spots within the same
biofilm can differ by two units or more (Vroom et al., 1999). The sessile
cell’s volumetric density in a biofilm is easily 100 times or 1000 times
higher than planktonic cell density in the same microbial system. This
alone can make the pH values differ by 2 or 3 units because of increased
local secretion of organic acids. A dense biofilm may harbor its acidity
underneath the biofilm by exerting a diffusional mass transfer barrier.
The local conditions underneath a biofilm are what matter because
bioleaching reactions occur right below the biofilm on a solid particle
surface. Thus, to evaluate the acidity provided by microbes, the pH
underneath a biofilm is more relevant than that in the bulk-fluid phase.
Microelectrode pH sensors are available to detect the pH at a precise
location in a biofilm. Another method is to use nanoparticles coated
with pH-sensitive fluorescent molecules that can be viewed under a
microscope (Shi et al., 2016) on a cross-section for precise pH dis-
tribution inside a biofilm.
Metagenomics have been used to identify phylogenetic composi-
tions in biofilms. The cost for next-generation sequencing has come
down greatly in recent years, from thousands of dollars per sample to
just hundreds. It is now widely used in environmental microbiology,
gradually replacing PCR (polymerase chain reaction) that requires
primers (Friedland et al., 1994) to detect specific organisms. In recent
years, the scientific community has started using this technique to study
microbiomes in biofilms used in bioleaching (Cárdenas et al., 2016).
The US government and some leading scientists in the US started the so-
called microbiome initiative in May 2016 to study various interactions
of different microbial components in a microbial community for various
applications including medical, environmental and industrial applica-
tions. Bioleaching is one of the research areas that will definitely benefit
from the advances in microbiomes. With a better understanding of
microbial interactions in biofilms, more efficient and robust biofilms
can be developed or engineered to decrease bioleaching time and to
increase bioleaching yields. This is likely a key to the future economic
success of bioleaching of heavy metals from ash and sewage sludge.
10. Conclusion
Bioleaching is an environmentally friendly technology for com-
mercial metal recovery from metal ores in mining. There is a growing
interest in adapting the technology to remove heavy metals from solid
wastes such as fuel ash and sewage sludge. However, bioleaching is not
ready for such applications yet because of some technical hurdles. This
review summarizes field applications and laboratory research activities
in bioleaching with an emphasis on the optimization of process para-
meters. With advances in microbiome and better understanding of
biofilm ecology, it is anticipated that bioleaching technology will see
some significant breakthroughs in the not so distant future.
Acknowledgements
M. Zhou and M. Li were financially supported by Grant Nos.
91545126 and 21328602 of the National Natural Science Foundation of
China.
438
Bioresource Technology 261 (2018) 428–440
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.biortech.2018.04.033.
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