PbS biomineralization using cysteine: Bacillus cereus and the sulfur rush

Abstract

Bacillus sp. Abq, belonging to Bacillus cereus sensu lato, was isolated from an aquifer in New Mexico, USA and phylogenetically classified. The isolate possesses the unusual property of precipitating Pb(II) by using cysteine, which is degraded intracellularly to hydrogen sulfide (H2S). H2S is then exported to the extracellular environment to react with Pb(II), yielding PbS (galena). Biochemical and growth tests showed that other sulfur sources tested (sulfate, thiosulfate, and methionine) were not reduced to hydrogen sulfide. Using equimolar concentration of cysteine, 1 mM of soluble Pb(II) was removed from Lysogeny Broth (LB) medium within 120 h of aerobic incubation forming black, solid PbS, with a removal rate of 2.03 µg L-1 h-1 (∼8.7 µM L-1 h-1). The mineralogy of biogenic PbS was characterized and confirmed by XRD, HRTEM, and EDX. Electron microscopy and electron diffraction identified crystalline PbS nanoparticles with a diameter <10 nm, localized in the extracellular matrix and on the surface of the cells. This is the first study demonstrating the use of cysteine in Pb(II) precipitation as insoluble PbS and it may pave the way to PbS recovery from secondary resources, such as Pb-laden industrial effluents.

Full text

FEMS Microbiology Ecology, 96, 2020, aa151
doi: 10.1093/femsec/aa151
Advance Access Publication Date: 5 August 2020
Research Article
RESEARCH ARTICLE
PbS biomineralization using cysteine: Bacillus cereus
and the sulfur rush
Lucian C. Staicu1,*, Paulina J. Wojtowicz1, Mih´
aly P ´
osfai2,P
´
eter Pekker3,
Adrian Gorecki1, Fiona L. Jordan4and Larry L. Barton4
1Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland, 2Department of Earth and
Environmental Sciences, University of Pannonia, Egyetem u. 10, H-8200, Veszpr´
em, Hungary, 3Research
Institute of Biomolecular and Chemical Engineering, University of Pannonia, Egyetem u. 10, H-8200, Veszpr´
em,
Hungary and 4Department of Biology, University of New Mexico, MSCO3 2020, Albuquerque, NM 87131, USA
∗Corresponding author: University of Warsaw, Miecznikowa 1, Warsaw 02–096, Poland. Tel: +48-22-55-41-302; E-mail: staicu@biol.uw.edu.pl
One sentence summary: This article describes a novel strategy employed by bacteria to reduce the toxicity of lead by forming a mineral with limited
solubility.
Editor: John Stolz
ABSTRACT
Bacillus sp. Abq, belonging to Bacillus cereus sensu lato, was isolated from an aquifer in New Mexico, USA and
phylogenetically classied. The isolate possesses the unusual property of precipitating Pb(II) by using cysteine, which is
degraded intracellularly to hydrogen sulde (H2S). H2S is then exported to the extracellular environment to react with Pb(II),
yielding PbS (galena). Biochemical and growth tests showed that other sulfur sources tested (sulfate, thiosulfate, and
methionine) were not reduced to hydrogen sulde. Using equimolar concentration of cysteine, 1 mM of soluble Pb(II) was
removed from Lysogeny Broth (LB) medium within 120 h of aerobic incubation forming black, solid PbS, with a removal rate
of 2.03 μgL
−1h−1(∼8.7 μML
−1h−1). The mineralogy of biogenic PbS was characterized and conrmed by XRD, HRTEM and
EDX. Electron microscopy and electron diffraction identied crystalline PbS nanoparticles with a diameter <10 nm,
localized in the extracellular matrix and on the surface of the cells. This is the rst study demonstrating the use of cysteine
in Pb(II) precipitation as insoluble PbS and it may pave the way to PbS recovery from secondary resources, such as Pb-laden
industrial efuents.
Keywords: Bacillus cereus; lead (Pb); cysteine; galena (PbS); biominerals
INTRODUCTION
Lead (Pb) ranks as a major anthropogenic pollutant, with min-
ing industry and fossil fuel burning for energy production as
major contributors (Needleman 2004; Rumble 2018). Because Pb
is rarely found in nature as a pure element, its industrial pro-
duction is based on the mineral galena (PbS). Various indus-
trial activities, including the production of batteries, ammuni-
tion, plumbing, alloys, lead crystal glassware, shields for X-ray
equipment and nuclear reactors, employ Pb as a raw material
(Rumble 2018). All these products ultimately release lead to the
environment contributing to its mobilization through geo-
spheres. An important aspect adding to Pb pollution is the his-
torical heritage related to its use as an anti-knock additive in
gasoline and to mining activities (Mills, Simpson and Adderley
2014; Finlay et al. 2021). Although tetraethyllead, (CH3CH2)4Pb,
a petro-fuel additive used on large scale as an effective octane
rating booster, has been phased out, its legacy still persists in
terrestrial and aquatic ecosystems (Wedge 1999). Unlike organic
pollution, metals (including Pb) do not degrade (mineralize) in
the environment and thus accumulate over time.
Received: 17 May 2020; Accepted: 28 July 2020
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2FEMS Microbiology Ecology, 2020, Vol. 96, No. 9
Pb does not possess any known biological function and
the lead ion, Pb(II), is highly toxic. Pb has detrimental effects
on protein synthesis and leads to alteration of the osmotic
balance, enzyme inhibition, nucleic acid damage, disruption
of membrane functions and oxidative phosphorylation (Bru-
ins, Kapil and Oehme 2000). Lead is neurotoxic and is associ-
ated with learning disabilities (Caneld et al. 2003). In bacteria,
Pb(II) is suggested to cross the plasma membrane by nonspe-
cic uptake pathways for Mn(II) and Zn(II) (Bruins, Kapil and
Oehme 2000). In certain cases, free Pb(II) does not accumulate
in the cytoplasm because it is pumped out from the cell by
export systems using P-type ATPases (Rensing et al. 1998). These
transporters are distributed throughout Bacteria domain and
include CadA, ZntA, and PbrA (Jaroslawiecka and Piotrowska-
Seget 2014). Other intracellular proposed detoxication strate-
gies include binding of Pb(II) to metallothioneins and precipi-
tating it as insoluble phosphates (Dopson et al. 2003). However,
this strategy comes with important costs in terms of energy
(efux systems), materials, and the challenge to store and han-
dle solid inclusions intracellularly. A better alternative is to keep
Pb out of the cell in a non-bioavailable, low-solubility form,
such as phosphates, precipitated in extracellular polysaccha-
rides, and polymers naturally occurring in the cell wall (Dopson
et al. 2003).
From an environmental perspective, Pb biomineralization as
insoluble minerals is desirable because this process limits its
mobility (Roane 1999). Apart from phosphates, suldes (S2−)are
known to form highly insoluble minerals. For instance, PbS has
a low solubility constant product (Ksp)of3.2×10−28 (Rumble
2018), which renders it non bioavailable. Sulde precipitation of
metals is less pH sensitive than other metal precipitates, thus
being a process active under a wide range of geochemical con-
ditions (Fu and Wang 2011). Sulfate reducing bacteria (SRB) play
a key role in the conversion of high valence state sulfur (e.g.
SO42−)toS
2−(Muyzer and Stams 2008; Barton and Fauque 2009).
Understanding the behavior of Pb and the generation of sulde
by bacteria is critical not only for the environment but also for
human health. Such was the case of Pb poisoning in Flint, Michi-
gan, where the change in drinking water chemistry had a direct
impact on Pb release from the aging water supply system (Roy
and Edwards 2018). However, the use of low-valence state sulfur
for the precipitation of lead as PbS has been marginally inves-
tigated. This is particularly relevant since low-valence states
biomolecules (e.g. cysteine and methionine) are present in all
bacteria and are a readily available source of sulde for Pb
detoxication.
The aim of this study was to evaluate the immobilization of
Pb(II) by a bacterial strain able to produce hydrogen sulde from
cysteine and to characterize the PbS biomineralization product.
The specic objectives were to (i) identify if cysteine is the sole
sulde-source for PbS precipitation, (ii) establish if the biominer-
alization process of PbS is intra- or extracellular and (iii) propose
a model for cysteine utilization and PbS biomineralization in the
investigated bacterial strain.
MATERIALS AND METHODS
Bacterial strain isolation
The bacterial strain was isolated from an aquifer in New Mex-
ico, USA (middle of the Rio Grande valley; Latitude: 35.106766 ◦N,
Longitude: −106.629181◦W) by Prof. Larry Barton and his team at
New Mexico University. The water sample was taken from well
water drawn at 381 m below the surface.
Phylogeny
Total DNA extraction of the isolate was done using a Genomic
Mini Kit (A & A Biotechnology, Gdynia, Poland) according to the
manufacturer’s instructions. The DNA concentration was deter-
mined using the QubitTM 2.0 Fluorometer (Invitrogen, Carls-
bad, CA, USA). About 10 ng of extracted DNA were used as
a template for amplication of 16S rRNA gene. Reaction was
prepared using KAPA HiFi polymerase in a nal concentration
of 0.5 U (KAPA Biosystems) and 0.3 μM of universal primers
(27F: AGAGTTTGATCMTGGCTCAG, 1492R: GGTTACCTTGTTAC-
GACTT). Annealing was performed at 63◦C and a total of 27
cycles were used (Mastercycler Nexus GX2 thermocycler, Eppen-
dorf). Three technical replicates were pooled, puried by Clean
up Purication Kit (EURx, Gdansk, Poland) and sent for sequenc-
ing (Oligo.pl, Polish Academy of Science, Warsaw, Poland).
For the phylogenetic classication of the isolate a molec-
ular approach utilizing sequencing of the 16S rRNA gene was
applied. A phylogenetic analysis based on the 16S rRNA gene
sequence of the isolate and 100 other reference Bacillus species
was performed using the Maximum Likelihood method and
Tamura-Nei model (Tamura and Nei 1993). Phylogenetic tree
was constructed using MEGA-X software (Kumar et al. 2018)
involving additional 99 16S rRNA originated from Bacillus spp.
strains deposited in NCBI database (as of 15 December 2019).
This analysis involved 101 nucleotide sequences including 16S
rRNA sequence of Clostridium botulinum 202F used as an outlier.
There were a total of 1367 positions in the nal dataset. Evolu-
tionary analyses were conducted in MEGA X (Kumar et al. 2018).
Initial tree(s) for the heuristic search were obtained automati-
cally by applying Neighbor-Join and BioNJ algorithms to a matrix
of pairwise distances estimated using the Maximum Composite
Likelihood (MCL) approach, and then selecting the topology with
superior log likelihood value. A discrete Gamma distribution was
used to model evolutionary rate differences among sites (ve
categories (+G, parameter =0.1132)).
Biochemical characterization
API 20E and 20NE strips were used to determine the biochemical
prole of the strain according to the manufacturer’s instructions
(BioM´
erieux). Triple Sugar Iron (TSI) Agar slants were prepared in
the lab and contained agar 1.2%, peptone 2%, meat extract 0.3%,
phenol red 25 mg L−1(a pH-sensitive dye), lactose 1%, sucrose
1%, glucose 0.1%, yeast extract 0.3%, NaCl 0.5%, sodium thiosul-
fate 0.3%, and ferrous sulfate 0.2%, pH 7.4 (adapted from Sigma
2020). API 20E and 20NE, and TSI were performed in triplicate. Oil
displacement activity assay was performed to assess the capac-
ity of the strain to produce surfactants (this technique measures
the diameter of clear zones on an oil-water surface resulted
from dropping of a solution containing biosurfactant) (Morikawa
2006). In short, 50 μL of diesel oil containing Sudan red dye were
added to a Petri dish containing distilled water. A drop of the
supernatant and the unltered culture of the strain grown in LB
and in LB +Pb +cysteine was added to the Petri dish, and the
spreading of the oil was observed. A drop of a Bacillus subtilis
ANT WA51 culture was used as control.
Incubations and reagents
Aerobic incubations:
Investigations on the capacity of the strain to metabolize lead
and cysteine were carried out in Lysogeny Broth (LB). LB medium
(from BioMaxima) contained tryptone 10 g L−1, yeast extract,
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Staicu et al. 3
5gL
−1, NaCl 5 g L−1. LB medium was adjusted to pH 5 and
autoclaved. Stock solutions of lead acetate trihydrate—hereafter
Pb(II)—and of L-cysteine-HCl monohydrate (Sigma) were lter-
sterilized using 0.2 μm pore diameter Acrodisc PF lters (Gelman
Sciences, USA) and added aseptically to the growth medium as
indicated to give nal concentrations of total 1 mM of each.
Due to the nature of the LB medium, an organically-rich solu-
tion, the soluble Pb(II) concentration can vary from one incuba-
tion to another. However, the results indicated a limited varia-
tion between independent experiments (±10–15%). To circum-
vent this limitation, the initial Pb(II) concentration (t0) was mea-
sured for all experiments and was used to calculate the Pb(II)
removal rates. The asks were inoculated with 1% of a stock
culture of Bacillus sp. Abq and the incubation was carried out
at 30◦C on a rotary shaker at 150 rpm, in the dark. The initial
pHvaluewassetto5.0using1NHClacid.Thereasonwasthe
tendency of the pH to increase by around two units during incu-
bation, reaching around 7.5 at the end of the experiment. Based
on a pilot experiment, above pH 8 lead starts to precipitate from
solution.
Anaerobic incubations:
Anaerobic incubations were performed in LB. 80 mL of sterile
LB medium were added aseptically to serum bottles (120 mL),
which were then crimp-sealed with butyl rubber septa and alu-
minum caps, and the headspace was ushed with nitrogen gas
for 5 min through a 0.22 μm lter to ensure sterility. The incu-
bations were performed at 30◦C, pH 7.0, in the dark on a rotary
shaker at 150 rpm. In order to better visualize the formation of
the precipitate, after 48 h of incubation homogenous samples
were taken and centrifuged in microcentrifuge tubes.
Electron microscopy
Samples for transmission electron microscopy (TEM) were pre-
pared by depositing a drop of the cell suspension on cop-
per TEM grids covered by an ultrathin amorphous carbon lm.
TEM analyses were performed using a Talos F200X G2 instru-
ment (Thermo Fisher), operated at 200 kV accelerating volt-
age, equipped with a eld-emission gun and a four-detector
Super-X energy-dispersive X-ray spectrometer, and capable of
working in both conventional TEM and scanning transmission
(STEM) modes. Low-magnication bright-eld (BF) images, high-
resolution (HRTEM) images and selected-area electron diffrac-
tion (SAED) patterns were obtained in TEM mode. In addi-
tion, bright- and dark-eld images were also obtained in STEM
mode. Elemental compositions were determined using energy-
dispersive X-ray spectrometry (EDS).
Lead (Pb) measurement and PbS analysis
Bacillus sp. Abq was grown in LB medium supplemented with
1 mM lead acetate and 1 mM L-cysteine or lead-containing LB
medium without L-cysteine. The lead concentration was cho-
sen based on literature reporting concentrations up to 1.5 mM
Pb(II) in environmental samples associated with acid mine
drainage (Campaner et al. 2014). We used equimolar and 3:1
concentrations of cysteine to Pb(II) in order to obtain stoichio-
metric removal and to demonstrate the impact of cysteine
on increasing Pb(II) removal rates, respectively. Control exper-
iments were performed to determine the potential abiotic inter-
action between Pb and cysteine. Another experiment assessed
whether the degradation of cysteine was intra- or extracellu-
lar. For this, the supernatant of a Bacillus sp. Abq culture grown
using the same incubation conditions in LB medium amended
with 1 mM cysteine was recovered, lter-sterilized and inocu-
lated with 1 mM Pb(II), and with 1 mM Pb(II) +1mMcysteine.
Cultures were inoculated and incubated on the shaker at 30◦C
for 7 days. Samples were removed from the asks at various
time points (12, 24, 48, 72, 96 and 120 h), centrifuged at 6000 ×g
for 10 min, and the supernatant was analyzed for lead after it
was ltered through a Nucleopore lter with a pore diameter of
0.22 μm. The specic Pb(II) reduction rate was calculated from
the slope of the linearized time course. The experiments were
performed in triplicate.
Pb was measured by Flame Atomic Absorption Spectroscopy
(FAAS) using a Thermo Scientic—SOLAAR M Series and a gas
mixture of air and acetylene. The calibration curve range was 0–
10 mg L−1and the lower limit of quantication was 0.01 mg L−1.
PbS: The mineral composition of the investigated sam-
ples was obtained via powder X-ray diffraction (XRD) by
using a SmartLab RIGAKU diffractometer with graphite-
monochromatized CuK radiation operating at 9 kV. The
measurements were conducted at 2–75◦2θ(depending on the
measurement) with a measuring step of 0.05◦2θ/s. The XRD
patterns were interpreted using the XRayan program (Version
4.2.2).
RESULTS AND DISCUSSION
Phylogenetic classication of the isolate
Based on these results, the isolate was classied as Bacillus
sp. Abq (Abq from Albuquerque, New Mexico). The accession
number obtained for Bacillus sp. Abq is MT072324. The results
(Fig. 1) indicate that the isolate is most closely related to Bacil-
lus cereus (99.79% similarity) and to B. thuringiensis (99.79% sim-
ilarity), which cluster together and belong to the Bacillus cereus
sensu lato group (Jensen et al. 2003).
Morphology and biochemical characterization
On LB medium, Bacillus sp. Abq forms round, off-white colonies,
with clear edges, ∼5 mm across. The strain is aerobic, showing
limited anaerobic growth (Fig. S1, Supporting Information).
The use of API test kits (20E and 20 NE) revealed that the
strain can use a wide range of substrates such as citrate, argi-
nine, tryptophan, gelatin, esculin, and glucose (aerobically and
via fermentation) (Table S1, Supporting Information). In addi-
tion, Bacillus sp. Abq reduces nitrate to nitrite, and uses various
carbohydrates including maltose, gluconate and maltose. On the
other hand, it cannot degrade urea, does not produce indole or
H2S (from thiosulfate, S2O3) and it cannot use a number of car-
bohydrates such as mannitol, inositol and saccharose.
The biochemical analysis was complemented by perform-
ing the Triple Sugar Iron (TSI) test (Fig. S1, Supporting Informa-
tion). The bottom of the tube turned yellow, indicative of glu-
cose fermentation. The fact that no darkening appeared in the
inoculated tube revealed the incapacity of Bacillus sp. Abq to
reduce sulfate and thiosulfate to hydrogen sulde, that would
have reacted with the Fe(II) present in the medium by forming a
black iron sulde mineral precipitate.
Numerous Bacillus species have been reported for their
capacity to produce various surfactants such as surfactin, iturin
A, polymixins and lipopeptides (Jahan et al. 2020). In order to
check the possibility of an extracellular interaction of bacterial
products with cysteine and Pb(II), the production of surfactants
by the isolate was tested using the oil displacement activity
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4FEMS Microbiology Ecology, 2020, Vol. 96, No. 9
Figure 1. Phylogenetic tree for 16S rRNA sequence of Bacillus spp. GenBank accession number of the 16S rRNA sequences used for the phylogenetic analysis are given
in parentheses. The tree with the highest log likelihood (-9369.02) is shown. Statistical support for the internal nodes was determined by 1000 bootstrap replicates and
values of ≥50% are shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The 16S rRNAof the Bacillus sp. Abq,
analyzed in this study, is highlighted by a red rectangle.
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Staicu et al. 5
Figure 2. Lead removal by Bacillus sp.Abq under aerobic conditions. (A), Removal
of 1 mM Pb(II) in incubations amended with 1 mM and 3 mM cysteine. The abi-
otic control contains 1 mM Pb(II) and 1 mM cysteine. The biotic control without
cysteine contains 1 mM Pb(II). The results represent the average of three repli-
cates, δ<5%; (B), Removal rates of 1 mM Pb(II) in solutions containing 1 mM and
3 mM cysteine. The removal rate of Pb(II) is the slope (a) of the equation y =ax
+b. Growth conditions for (A) and (B): LB, oxic, initial pH 5.0, nal pH ∼7.5, 30◦C,
150 rpm, dark.
assay and was compared to Bacillus subtilis ANT WA51, a strain
capable of surfactant production. Bacillus sp. Abq did not pro-
duce surfactants in any of the test conditions: in LB, in LB +Pb,
and in LB +Pb +cysteine.
Lead (Pb) removal
Figure 2A presents the aerobic removal of lead by Bacillus sp.
Abq in the presence of 1 mM and 3 mM cysteine. Using equimo-
lar cysteine to Pb (1:1 ratio), the strain started to remove Pb
from solution after the rst 12 h of incubation (∼4%), at which
time point the removal increased with incubation time. 14% Pb
was removed after 24 h, 44% after 48h, 59% after 72 h, 81%
after 96 h, while at the last sampling point, 120 h, no Pb was
detected in solution. Interestingly, using a 1:3 ratio Pb to cys-
teine, the Pb removal kinetics were accelerated, 56% removal
after 24 h and no Pb measured in solution after 48 h. The start
of Pb removal coincides with the mid-exponential phase of the
bacterial growth (Fig. 3). The abiotic control using equimolar cys-
teine to Pb shows that lead is not removed during incubation,
thus excluding the potential abiotic interactions leading to Pb(II)
precipitation. On the other hand, the biotic control containing
Pb(II) but without cysteine might indicate a slight Pb(II) removal
that could be attributed to measurement errors or to the lim-
ited interaction of Pb(II) with bacterial biomass. As for removal
rates, in the case of 1:1 ratio, Pb was removed at 2.03 μgL
−1h−1
(∼8.7 μML
−1h−1), while in the case of 1:3 ratio, the removal
rate increased to 4.78 μgL
−1h−1(∼20.4 μML
−1h−1)(Fig.2B).
By tripling the ratio of Pb to cysteine, the Pb removal rate only
Figure 3. Growth of Bacillus sp. Abq under various conditions. Control represents
the strain grown in LB; Control +cysteine contains 1 mM cysteine; control +
Pb(II) contains 1 mM Pb(II); cysteine +Pb(II) represents the incubation containing
1 mM Pb(II) and 1 mM cysteine. Growth conditions: as described in Fig. 2.
increased 2.35-fold (2.03 μgL
−1h−1vs 4.78 μgL
−1h−1). This indi-
cates that the increase of the removal rate is not a linear process,
leading to saturation for concentrations exceeding the equimo-
lar ratio. In the case of the abiotic control (Pb +cysteine), Pb con-
centration remained constant in solution as a function of time.
Overall, this set of results identies cysteine as the causative
agent of Pb removal from solution and Bacillus sp. Abq as the
agent capable of using cysteine for this process.
The growth of Bacillus sp. Abq was measured under var-
ious conditions: the culture in LB, the culture in LB supple-
mented with 1 mM cysteine, the culture in LB supplemented
with 1 mM Pb(II), and the culture in LB supplemented with 1 mM
cysteine and 1 mM Pb(II). As a general observation, the culture
supplemented with 1 mM cysteine and 1 mM Pb(II) displayed
the highest optical density (OD) relative to the other three con-
ditions that gave comparable results (Fig. 3). During the rst
12 h of growth, the cultures produced similar optical densities;
however, after this time point, the Pb(II) +cysteine incubation
started to exhibit a higher OD than the other treatments. These
results should be interpreted with caution because the forma-
tion of black PbS interferes with the proper OD measurement,
thus not indicating a better growth of Bacillus sp. Abq when
exposed to Pb(II) and cysteine. The important point is that the
removal of Pb from solution is well correlated with the bacterial
growth, conrming the biotically-driven lead transformation in
this system. In order to exclude the possible chemical interac-
tion of cysteine with Pb, we performed a series of experiments
in LB and sterile distilled water at pH values relevant for the cur-
rent study (pH 5, 7 and 9). The results did not indicate any for-
mation of black PbS, nor the removal of Pb from solution (data
not shown).
Pb(II) is suggested to cross the bacterial plasma mem-
brane through non-specic uptake pathways for Mn(II) and
Zn(II) (Jaroslawiecka and Piotrowska-Seget 2014). Bacteria have
evolved efcient extracellular and intracellular defense mecha-
nisms against lead toxicity. Lead can be sequestered outside the
bacterial cell through its precipitation as insoluble phosphates
or adsorption onto extracellular polysaccharides (Dopson et al.
2003). Lead sulde has a very low solubility, Ksp =3.2 ×10−28,
second to phosphates, e.g. lead phosphate, Ksp =3×10−44 (Rum-
ble 2018). However, in view of the scarcity and essentiality of
phosphorous for bacterial metabolism, the use of phosphates to
sequester Pb(II) has marginally been reported (Chen et al. 2016).
In some instances, Pb(II) is pumped out from the cell by
export systems using P-type ATPases which are distributed
throughout bacteria (Rensing et al. 1998). These transporters
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6FEMS Microbiology Ecology, 2020, Vol. 96, No. 9
Figure 4. PbS formation by Bacillus sp. Abq under aerobic and anaerobic conditions. (A), Aerobic incubation after 48 h in LB medium. The incubations were conducted
under the same conditions as described in Fig. 2. The abiotic control contains LB, 1 mM Pb(II) and 1 mM cysteine, but without bacterial inoculum. (B), Anaerobic
incubation after 48 h in LB medium. The incubations were conducted at initial pH 7.0, nal pH 7.5–8.0, 30◦C, 150 rpm, dark. The abiotic control contains LB, 1 mM Pb(II)
and 1 mM cysteine, but without bacterial inoculum. 1 mM SO4, sulfate, S2O3, thiosulfate, and methionine were used under anoxic conditions.
include CadA, ZntA, and PbrA (Jaroslawiecka and Piotrowska-
Seget 2014). Interestingly, these transporters appear to have
an efcient crosstalk. For instance, when PbrA transporter was
inactivated in Cupriavidus metallidurans CH34, the zntA and cadA
increased their activity to complement for the loss of the former
(Taghavi et al. 2009). Hynninen et al. (2009) documented a mixed
detoxication mechanism where Pb(II) is rst exported from the
cytoplasm by PbrA and then is sequestered extracellularly as a
phosphate salt using the inorganic phosphate produced by PbrB.
PbS formation under aerobic and anaerobic conditions
The formation of PbS under aerobic conditions in LB starts after
12 h of incubation and increases during incubation, accompa-
nied by a progressive darkening of the solution (Fig. 4A). This
indicates the progressive increase in PbS concentration with
time, a result supported by the decrease in concentration of sol-
uble Pb(II) (Fig. 2A). A separate experiment aimed to establish if
cysteine can be degraded extracellularly by collecting the super-
natant from a Bacillus sp. Abq culture grown using the same
incubation conditions in LB medium amended with 1 mM cys-
teine was recovered. The supernatant was lter-sterilized and
inoculated with 1 mM Pb(II), and with 1 mM Pb(II) +1mMcys-
teine. The incubations did not turn black and the Pb measure-
ment did not indicate any Pb removal during the 7-day incu-
bation period (data not shown). The abiotic control containing
1 mM Pb(II) and 1 mM cysteine indicates PbS cannot be formed.
Since we could not perform the analysis on cysteine degradation
as a function of incubation time, the decrease in Pb(II) concen-
tration, the formation of PbS (characterized in detail below), as
well as the control experiments, collectively indicate the con-
tribution of cysteine as the source of H2S and its intracellular
degradation by Bacillus sp. Abq.
In a separate experiment, Bacillus sp. Abq was incubated in
LB anaerobically (Fig. 4B). An abiotic control experiment con-
taining LB medium amended with cysteine and Pb(II) was per-
formed to exclude the possibility that the components of the
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Staicu et al. 7
Figure 5. Bright-eld TEM image showing dark clusters of PbS nanoparticles sur-
rounding Bacillus sp. Abq formed under aerobic conditions. PbS nanoparticles
are present in the extracellular environment and some are tightly attached to
bacterial cells.
medium can react with Pb(II) forming PbS. The anaerobic exper-
iment concluded that sulde can only be formed by Bacillus sp.
Abq in the presence of cysteine, as documented by the formation
of a black precipitate (Fig. 4B). All other sulfur sources could not
be used to generate H2S. The formation of whitish precipitates
in all other treatments, including the abiotic control experiment,
indicates the formation of Pb(II) oxides at pH ∼8.0. These results
indicate that this strain was able to degrade cysteine anaerobi-
cally to H2S. On the other hand, the possible use of sulfate and
thiosulfate by Bacillus sp. Abq for anaerobic respiration can be
ruled out (Barton and Fauque 2009).
PbS characterization
Low-magnication TEM images show cells surrounded by very
ne-grained, crystalline material, producing dark contrast in
bright-eld images (Fig. 5). The nanoparticles occur extracellu-
larly and attach to the cell membrane. In addition, particles also
occur in some distance from the cells. According to EDS spectra
obtained from clusters of the nanoparticles, they clearly contain
Pb and S (Fig. 6C). Although the S K-peak overlaps with the Pb
M-peaks, precluding an accurate quantitative evaluation of the
composition, the presence of S is unambiguously shown by an
analysis of the intensity prole (Fig. S2), additionally supporting
the formation of extracellular PbS.
The spherical bacterial inclusions in Fig. 5could be poly-
hydroxyalkanoate (PHA) granules. PHA serve as both a source
of energy and as a carbon store (Shively et al. 2011), and var-
ious members of the genus Bacillus were shown to produce
these inclusions (Singh, Patel and Kalia 2009). EDS analysis did
not indicate the presence of Pb or S in the areas where these
spots are present, thus excluding the possibility of Pb-containing
intracellular accumulations. Due to the development of a black
color during incubation, as a result of PbS formation, various
staining techniques such as Sudan Black B, Nile Blue A or Nile
Red could not have been applied to screen for the presence of
PHA (Legat et al. 2010). Alternatively, these inclusions could be
spores. The species belonging to the genus Bacillus can form
spores under stressful conditions such as when exposed to toxic
metals (Selenska-Pobell et al. 1999). The major limitation in eval-
uating the formation of spores during Pb(II) exposure was the
production of black PbS which makes the use of various spore
staining techniques (e.g. Schaeffer-Fulton stain using malachite
green) inefcient.
Unequivocal identication of the Pb-bearing phase is pos-
sible by an analysis of electron diffraction patterns and high-
resolution TEM images. SAED patterns obtained from clusters of
nanoparticles show rings, suggesting that the material is crys-
talline but has a very ne (nm-scale) grain size, and the grains
occur in random crystallographic orientations (Fig. S3, Support-
ing Information). The d-spacings corresponding to the rings are
consistent with the structure of galena, PbS, and their relative
intensities also match the features expected for galena (the most
intense peak being at ∼3˚
A, the next strongest at ∼2.1 ˚
A, and
the others being all very weak). HRTEM images of the nanoparti-
cles show periodic fringes in most of them, indicating their crys-
talline character (Fig. 6A). The sizes of galena nanocrystals range
from about 4 to 10 nm in this image. A Fourier transform (FT) cal-
culated for the image shown in Fig. 6A (inset) displays only the
rings corresponding to the structure of galena. A single nanopar-
ticle (about 3 by 6 nm in size) is shown in Fig. 6B along with its
FT; this particle is viewed along a major crystallographic axis,
resulting in distinct spots in the FT that can be indexed on the
basis of the galena structure. The composition of a cluster of PbS
particles is conrmed by the EDS spectrum in Fig. 6C.
According to XRD data, the sample is dominated by lead sul-
de, PbS (Fig. 7), with all detected peaks belonging to galena. The
peaks on the diffractogram are slightly broad, which indicates
the small (nm-scale) crystal size of the precipitate.
Model of cysteine degradation and extracellular PbS
formation
Cysteine is the source of sulfur for the biosynthesis of a numer-
ous cofactors such biotin, lipoic acid, molybdopterin and thi-
amine, as well as Fe-S clusters in proteins and thionucleosides
in tRNA (Marquet 2001). Identifying the enzyme responsible for
cysteine degradation was beyond the scope of this study. How-
ever, based on a literature survey briey presented below, it
appears no cysteine-degrading enzyme has been shown to be
active extracellularly. For instance, cysteine desulfurase is an
intracellular enzyme that catalyzes the conversion of L-cysteine
to L-alanine and sulfane sulfur via the formation of a cysteine
persulde intermediate (Mihara and Esaki 2002). Cystathionine
β-synthase was also found to produce H2S upon reaction of cys-
teine in Bacillus anthracis (Devi et al. 2017). On the other hand,
cysteine desulfhydrase catalyzes the conversion of L-cysteine to
sulde, ammonia, and pyruvate (Takagi and Ohtsu 2016). From
this perspective, Bacillus sp. Abq seems to possess a cysteine
desulhydrase or a cystathionine β-synthase able to release sul-
de from cysteine. The formation of cysteine-Pb(II) complexes
was reported at high pH values (9.1–10.4) and at mole ratios vary-
ing from 2.1 to 10.0, with CPb(II)=0.01 and 0.1 M (Jalilehvand et al.
2015). The experimental conditions used in the current study do
not support the possibility to form such complexes.
According to the available results, it is reasonable to conclude
that cysteine is internalized by Bacillus sp. Abq, then enzymat-
ically degraded at the intracellular level and a portion of the
released sulde is exported to the extracellular milieu as H2S.
Extracellularly, H2S dissociates and sulde reacts with Pb(II),
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8FEMS Microbiology Ecology, 2020, Vol. 96, No. 9
Figure 6. TEM analysis of biogenic PbS produced by Bacillus sp. Abq under aerobic conditions, (A), HRTEM image of a cluster of randomly oriented nanocrystals and
their Fourier Transform (FT, inset), in which the spatial frequencies in the nanocrystals are represented by intensity maxima producing rings. The diameters of the
rings correspond to d-spacings as indicated by the green numbers, all of which are consistent with the structure of galena. (B), HRTEM image and its FT (inset) of
an individual galena nanocrystal, viewed with the electron beam parallel to the [1–10] crystallographic axis. (C), EDS spectrum of biogenic PbS particles produced by
Bacillus sp. Abq under aerobic conditions. The spectrum shows the presence of Pb and S in addition to C, N, O and minor P, K, and Ca. The Cu peak is an artifact from
the TEM support grid.
yielding PbS (Fig. 8). H2S has an acid dissociation constant, pKa
∼7.0 (Rumble 2018). The extracellular transfer of H2S to react
with Pb(II) is further supported by Fig. S4 (Supporting Infor-
mation) showing a brown halo surrounding bacterial colonies
grown aerobically on Pb and cysteine containing media.
The model of Pb(II) precipitation using cysteine by Bacillus sp.
Abq is supported by the following results: (i) Pb is removed as a
function of incubation time (Fig. 2A), (ii) Cysteine is the source
of sulde used for PbS precipitation (Fig. 4), (iii) Increasing cys-
teine concentration accelerates Pb removal rate, (iv) PbS parti-
cles form extracellularly (Fig. 5) and (v) A brown halo forms out-
side bacterial colonies, indicating the release of sulde and the
precipitation of PbS (Fig. S4, Supporting Information).
Because Pb is toxic for bacteria, sequestering it as a low sol-
ubility mineral offers the advantage of neutralizing its toxicity.
Cysteine is a critical amino acid for bacterial metabolism, being
an important stock molecule, and taken from the environment
or/and synthesized endogenously from serine (Takagi and Ohtsu
2016). The limited choice of Bacillus sp. Abq for cysteine as a
strategy for Pb precipitation might be explained in the nature of
this amino acid itself. Sulfur is present in cysteine as sulde, a
highly reactive valence state with high afnity for metals (Staicu
et al. 2019). Consequently, the possibility to readily obtain sulde
from cysteine, bypassing the time-consuming production of S2−
from high-valence states of sulfur (e.g. sulfate, sulte, thiosul-
fate), might help bacteria to counteract Pb toxicity in a fast and
efcient manner. It is noteworthy to mention that H2S has been
reported to act as a universal defense against antibiotics in bac-
teria (Shatalin et al. 2011) and is exported to the extracellular
milieu by diffusion (Mathai et al. 2009).
It is possible that initially Pb(II) enters the cell and triggers a
detoxication reaction by pumping out Pb(II) ions. However, this
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Staicu et al. 9
Figure 7. XRD of biogenic PbS produced by Bacillus sp. Abq under aerobic conditions.
Figure 8. Model of Pb detoxication in Bacillus sp. Abq using cysteine and forming low-solubility PbS. Figure not drawn to scale.
strategy is both energy intensive and not efcient on medium
and long term since Pb(II) can enter in the cell again. The use of
cysteine as a feedstock for sulde could be the second response
mechanism of bacteria, as forming an insoluble mineral out-
side the cell renders Pb non-bioavailable. Roane (1999) docu-
mented the formation of Pb precipitates intracellularly (Bacillus
megaterium) and extracellularly (Pseudomonas marginalis). How-
ever, the study did not investigate the mineralogical nature of
the two precipitates, while in the case of P. marginalis it was
hypothesized the involvement of an exopolymer in the seques-
tration of Pb.
Biomineralization process of PbS: Resource recovery
perspectives
Lead is used on large scale by various industrial applications,
generating waste materials (e.g. wastewater) charged with sig-
nicant amounts of Pb. If not properly treated, Pb will be released
to the environment leading to pollution and biodiversity loss
(Brink,Horstmann and Peens 2019). Such an industrial efuent
is spent lead-acid battery wastewater, a matrix containing high
levels of sulfate (grams L−1)andPb(<5mgL
−1)(Vuet al. 2019).
Most of the studies investigating this industrial wastewater
type only focused on its treatment, while the resource recovery
component was not taken into consideration. Importantly,
PbS (galena) is currently the main source of lead production
using pyrometallurgical processes (Rumble 2018). Because Pb
is present in spent lead-acid battery wastewater in its soluble
form, Pb(II), PbS formation offers the advantage of efcient
recovery in the form of a solid product. Moreover, recovering
PbS from a secondary resource would contribute to reducing
the pressure on natural resources in the framework of circular
economy (Cordoba and Staicu 2018; Kisser et al. 2020). The purity
of the recovered biogenic PbS is in contrast with the mined
minerals which are mixed with other minerals and gangue.
Pyrometallurgical processes for Pb production are energy
intensive and require numerous production phases, entailing
additional costs and environmental degradation. The increased
demand for metals using nite resources makes recycling of
secondary resources critical in the near future (Vidal et al. 2017).
CONCLUSION
This study documented a novel precipitation strategy used by
Bacillus sp. Abq, a novel strain isolated from an aquifer in New
Mexico, USA and belonging to Bacillus cereus sensu lato, against
lead (Pb) by means of degrading cysteine and extracellularly pre-
cipitating PbS (galena). The experimental dataset supports the
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10 FEMS Microbiology Ecology, 2020, Vol. 96, No. 9
model of cysteine import from the growth media, active degra-
dation of cysteine at the intracellular level and the export of
sulde, moiety released from this amino acid, followed by the
precipitation of Pb as highly insoluble and non-bioavailable PbS.
One signicant nding is the narrow size of PbS, <10 nm, and its
exclusive extracellular biomineralization. Its extracellular dis-
tribution is indicative of an efcient system that prevents the
reentering of toxic Pb in the cell. Biogenic PbS is crystalline
and shows a high degree of purity when compared with ref-
erence materials. Because PbS is currently the main source of
Pb industrial production, this study might open the possibility
to biologically recover galena from Pb-laden wastewaters and
use it as feedstock instead, replacing raw and limiting materials.
From a microbial ecology perspective, identifying new pathways
used by bacteria to precipitate Pb(II) would serve to decontam-
inate industrially-polluted soils that are a legacy of past heavy
industrial activity using a non-invasive, eco-friendly treatment
approach. Additionally, understating the microbial metabolism
of Pb is crucial in man-made infrastructures such as the aging
water supply systems made of lead conduits (e.g. the lead poi-
soning event in Flint, Michigan). In a follow up study, we plan
to investigate the potential of this bacterial isolate to precipitate
other toxic metals (e.g. Cd) using cysteine, therefore exploring
its full potential to form non-toxic and chemically stable metal
suldes.
SUPPLEMENTARY DATA
Supplementary data are available at FEMSEC online.
ACKNOWLEDGMENTS
LS and PJW acknowledge the National Science Centre (NCN),
Poland, Grant number 2017/26/D/NZ1/00408, for nancial sup-
port. LS acknowledges Dr Alina Maria Holban (University of
Bucharest, Romania), Prof Hisaaki Mihara (Ritsumeikan Univer-
sity, Japan) and Dr Rob van Houdt (Belgian Nuclear Research
Centre, Belgium) for productive discussions. FLJ was supported
by a Patricia Robert-Harris fellowship and the research was
supported, in part, by a grant to LLB from the US Depart-
ment of Energy through the Waste-Management Education and
Research Consortium and the Sandia National Laboratories
under Contract DE-AC04–90AL8500. The authors acknowledge
Tomasz Bajda (AGH-KGHM, Krakow) for help with XRD analy-
sis and Bartosz Rewerski (UW) for Pb analysis. The authors rec-
ognize Belinda Ramirez and Nada Kherbik for technical assis-
tance in this project. TEM studies were performed at the Nanolab
of the University of Pannonia, using Grants no. GINOP-2.3.3–15-
2016–0009 and GINOP-2.3.2–15-2016–00017 from the European
Structural and Investments Funds and the Hungarian Govern-
ment.
Conicts of interests. None declared.
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Supplementary Material
PbS biomineralization using cysteine: Bacillus cereus and the sulfur rush
Lucian C. Staicu1,*, Paulina J. Wojtowicz1, Mihály Pósfai2, Péter Pekker3, Adrian Gorecki1, Fiona
L. Jordan4, and Larry L. Barton4
1Faculty of Biology, University of Warsaw, Miecznikowa 1, 02-096 Warsaw, Poland
2Department of Earth and Environmental Sciences, University of Pannonia, Egyetem u. 10, H-8200,
Veszprém, Hungary
3Research Institute of Biomolecular and Chemical Engineering, University of Pannonia, Egyetem u. 10, H-
8200, Veszprém, Hungary
4Department of Biology, University of New Mexico, MSCO3 2020, Albuquerque NM 87131, USA

2
Figure S1. Triple Sugar Agar (TSI) test. Left (control, uninoculated slant), right (slant inoculated with
Bacillus sp. Abq). The bottom of the tube turned yellow, indicative of glucose fermentation. The fact no
darkening appeared in the inoculated tube shows the incapacity of Bacillus sp. Abq to reduce sulfate and
thiosulfate to hydrogen sulfide, that would have reacted with the Fe(II) present in TSI, thus forming a black
iron sulfide mineral precipitate.

Figure S2. Analysis of the family of M-peaks produced by Pb in an EDS spectrum obtained from a cluster of nanocrystals. Both graphs show a raw
spectrum with a fitted background, a modeled curve fitted to the peaks, and a curve for the residual intensity (the remaining intensity after subtracting
the modeled value from the net intensity), as indicated in the graph. (a) Modeled fit with S present; (b) modeled fit with S absent; as seen from the
higher values of the residual curve in (b), modeling with S present produces a far better result, suggesting the particles contain both Pb and S.

Figure S3. Selected-area electron diffraction pattern of a cluster of Pb-bearing nanocrystals. The rings
indicate that the particles are crystalline and occur in random crystallographic orientations. The rings
correspond to distinct periodicities (d-spacings) in the crystals, as shown by the values (in Å) written on
them. All d-spacings and their relative intensities are consistent with the structure of galena, PbS.

Figure S4. Growth of Bacillus sp. Abq on agar-LB plates containing 1mM cysteine, 1 mM Pb(II), and 1 mM cysteine + 1 mM Pb(II). The brown
halo on the third plate supports the hypothesis that sulfide is exported outside bacterial colonies where it reacts with Pb(II) forming black PbS.

6
Table S1. Biochemical characterization of Bacillus sp. Abq (+, positive reaction; -, negative reaction).
API 20E
Incubation
time
API 20NE
Incubation
time
Acronym
Enzyme
24h
48h
Acronym
Enzyme
24h
48h
ONPG
ß-galactosidase
-
-
NO3
Nitrate reduction to NO2
+
+
ADH
Arginine dihydrolase
-
+
TRP
Indole production (TRyptoPhane)
-
-
LDC
Lysine decarboxylase
-
-
GLU
D-glucose fermentation
-
+
ODC
Ornithine decarboxylase
-
-
ADH
L-Arginine dihydrolase
+
+
CIT
Citrate utilization
-
+
URE
Urease
-
-
H2S
Hydrogen sulfide production
(from thiosulfate)
-
-
ESC
Hydrolysis (ß-glucosidase) (ESCulin)
+
+
URE
Urease
-
-
GEL
Hydrolysis (protease) (GELatin)
+
+
TDA
Tryptophan deaminase
+
+
PNPG
4-Nitrophenyl-β-D- glucopyranoside
-
-
IND
Indole production
-
-
GLU
Assimilation (GLUcose)
+
+
VP
Acetoin production
-
-
ARA
Assimilation (ARAbinose)
-
-
GEL
Gelatinase
+
+
MNE
Assimilation (ManNosE)
-
-
GLU
Glucose fermentation/oxidation
+
+
MAN
Assimilation (MANnitol)
-
-
MAN
D-Mannitol
fermentation/oxidation
-
-
NAG
Assimilation (N-Acetyl-Glucosamine)
+
+
INO
Inositol fermentation/oxidation
-
-
MAL
Assimilation (MALtose)
+
+

7
SOR
D-Sorbitol fermentation/oxidation
-
-
GNT
Assimilation (potassium GlucoNate)
+
+
RHA
L-Rhamnose
fermentation/oxidation
-
-
CAP
Assimilation (CAPric acid)
-
-
SAC
D-Saccharose
fermentation/oxidation
-
-
ADI
Assimilation (ADIpic acid)
-
-
MEL
D-Melibiose
fermentation/oxidation
-
-
MLT
Assimilation (MaLaTe)
+
+
AMY
Amygdaline
fermentation/oxidation
-
-
CIT
Assimilation (trisodium CITrate)
-
+
ARA
L-Arabinose
fermentation/oxidation
-
-
PAC
Assimilation (PhenylACetic acid)
-
-