Bioresource Technology 218 (2016) 659–666

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

The bioleaching potential of a bacterial consortium

Mauricio Latorre a,b,c, María Paz Cortés a,b, Dante Travisany a,b, Alex Di Genova a,b, Marko Budinich a,b,
Angélica Reyes-Jara d, Christian Hödar b,c, Mauricio González b,c, Pilar Parada e,
Roberto A. Bobadilla-Fazzini e, Verónica Cambiazo b,c, Alejandro Maass a,b,f,⇑
a
Mathomics, Center for Mathematical Modeling, Universidad de Chile, Beauchef 851, 7th Floor, Santiago, Chile
b
Center for Genome Regulation (Fondap 15090007), Universidad de Chile, Blanco Encalada 2085, Santiago, Chile
c
Laboratorio de Bioinformática y Expresión Génica, INTA, Universidad de Chile, El Líbano 5524, Macul, Santiago, Chile
d
Laboratorio de Microbiología y Probióticos, INTA, Universidad de Chile, El Líbano 5524, Macul, Santiago, Chile
e
BioSigma S.A., Loteo Los Libertadores, Lote 106, Colina, Chile
f
Department of Mathematical Engineering, Universidad de Chile, Beauchef 851, 5th Floor, Santiago, Chile

h i g h l i g h t s

 A consortium of five bacteria involved in copper bioleaching was sequenced.

 A model of metabolic pathways representing the bioleaching was constructed.
 The consortium showed a high capacity to resist heavy metals.
 This is the first operational industrial bioleaching consortium described to date.

a r t i c l e i n f o a b s t r a c t

Article history: This work presents the molecular foundation of a consortium of five efficient bacteria strains isolated
Received 11 May 2016 from copper mines currently used in state of the art industrial-scale biotechnology. The strains
Received in revised form 2 July 2016 Acidithiobacillus thiooxidans Licanantay, Acidiphilium multivorum Yenapatur, Leptospirillum ferriphilum
Accepted 4 July 2016
Pañiwe, Acidithiobacillus ferrooxidans Wenelen and Sulfobacillus thermosulfidooxidans Cutipay were
Available online 6 July 2016
selected for genome sequencing based on metal tolerance, oxidation activity and bioleaching of copper
efficiency. An integrated model of metabolic pathways representing the bioleaching capability of this
Keywords:
consortium was generated. Results revealed that greater efficiency in copper recovery may be explained
Bioleaching
Metabolic pathways
by the higher functional potential of L. ferriphilum Pañiwe and At. thiooxidans Licanantay to oxidize iron
Metal resistance and reduced inorganic sulfur compounds. The consortium had a greater capacity to resist copper, arsenic
Bacterial consortium and chloride ion compared to previously described biomining strains. Specialization and particular com-
ponents in these bacteria provided the consortium a greater ability to bioleach copper sulfide ores.
Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction iron and reducing inorganic sulfur compounds used as energy

sources for different metabolic processes. These properties have
Research related to microorganisms which inhabit extreme being used for economic reasons, where metal recovery from min-
environments has focused on the study of archaic life forms as erals ores cannot be processed by conventional methods and
signs of the origin of life or for applied interests, such as industrial require other procedures for their exploitation.
and biotechnological operations (Cavicchioli et al., 2011). Mining One of the primary natural processes studied in extremophile
environments are characterized by the presence of microbial organisms is copper bioleaching. This process is defined as the
organisms able to survive despite high concentrations of metals, use of microorganisms to solubilize copper from low-grade ores
elevated temperatures, and high levels of acidity (Dopson et al., (Rawlings and Johnson, 2007). It is widely known that copper
2014). Many of these extremophile species are capable of oxidizing bioleaching involves a complex community of microorganisms,
where numerous ferrous iron and sulfur oxidizing bacteria produce
the chemical precursors required to generate the scenario for
⇑ Corresponding author at: Beauchef 851, Piso 7, Santiago, Chile. leaching reactions to occur (Mosier et al., 2013).
E-mail address: amaass@dim.uchile.cl (A. Maass).

http://dx.doi.org/10.1016/j.biortech.2016.07.012
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
660 M. Latorre et al. / Bioresource Technology 218 (2016) 659–666

In order to better understand the molecular mechanisms logs shared among all the considered bacteria were kept. Then,
involved in this mining process, several genomes of different bac- multiple alignments for each ortholog group were carried out
teria isolated from natural (acid drainage) and industrial bioleach- using MUSCLE v3.8.31. Ortholog groups that included genes with
ing sites (mines) have been sequenced (Mi et al., 2011; Travisany gaps more than 30% in the group alignment were discarded. Also,
et al., 2014, 2012; Valdes et al., 2008). The c-proteobacteria for simplicity, orthologs groups with more than one gene per gen-
Acidithiobacillus ferrooxidans is one of the most characterized ome were not considered in order to have a unique sequence for
model of acidophilic bacteria, with studies focused on descriptions each bacterium. Finally, using GBlocks 0.91b, poorly aligned
of particular metabolic and genomic features (Hodar et al., 2012; regions from each ortholog group were removed and all the align-
Latorre et al., 2016; Orell et al., 2010). These works have con- ments were concatenated in a single large multiple alignment. This
tributed to the identification of important mechanisms involved concatenated sequence alignment was used to construct a
in bioleaching and adaptation to extreme environmental condi- Neighbor Joining tree with 1000 bootstrap replications using
tions, principally related to metal resistance, iron and sulfur oxida- PHYLYP. The tree was plotted using the ape R package.
tion. However, the function of the bacterial consortium and the
specific participation of each microorganism during copper 2.3. Genome annotation, manual curation and metabolic pathway
bioleaching are less understood. identification
In this context, the aim of this work was to identify the molec-
ular basis of a consortium of five efficient copper bioleaching bac- A pipeline for prediction and annotation of coding sequences
teria, all of them frequently found in a mining environment. Using (CDS) was used for the genomes of the five biomining bacteria fol-
complete sequencing and functional annotation, common and par- lowing protocols described previously (Travisany et al., 2014,
ticular genomic features of this extremophile bacteria consortium 2012). Briefly, an ab initio CDS and tRNAs prediction was per-
were identified. An integrative analysis of genomic data allowed formed using REGANOR which implements a combination of
the generation of a metabolic consortium model able to indicate GLIMMER and CRITICA tools for gene finding and tRNAscan-SE
the potential of each bacterium and the metabolites required dur- for tRNA gene identification. All predicted CDS were functionally
ing the bioleach process. The results add experimental data to annotated using Hidden Markov Models tools for prediction of
demonstrate the high capacity of the consortium bacteria to bio- transmembrane helices TMHMM and the presence of signal pep-
leach copper in relation with other previously sequenced mining tide cleavage sites with SignalP. The BLASTP against the NCBI
strains. Finally, the bacterial consortium was composed of the Non Redundant, SWISSPROT, TCDB, OMNIOME, the Kyoto Encyclo-
major components of a stable, effective and continuously grown pedia of Genes and Genomes (KEGG), the Cluster of Orthologous
bioleaching consortium operating in a contemporary, large-scale, Groups database (COG) and InterproScan using PROSITE, Pfam, Pro-
and fully-operational biotechnology system. Dom and SMART. The consensus annotation was performed by
METANOR and InterPRO hits were used to assign Gene Ontology
(GO) terms to genes. Manual curation of automated annotations
2. Material and methods
was performed using the GenDB platform. An enrichment analysis
of COG categories was carried out for the gene sets from each of the
2.1. Genome sequencing and assembly
five biomining bacteria using Fisher’s exact test with Benjamini–
Hochberg multiple testing correction. Categories with corrected
Genomic DNA extracted from Sulfobacillus thermosulfidooxidans
p-value <0.05 were considered enriched.
strain Cutipay, Acidithiobacillus thiooxidans strain Licanantay (DSM
Based on the gene names of this protein list, a set of correspond-
17318), Acidiphilum multivorum strain Yenapatur, Leptospirillum
ing amino acid sequences was retrieved from the SWISSPROT.
ferriphilum strain Pañiwue and Acidithiobacillus ferrooxidans strain
These sequences were the input for a BLASTP against the eggNOG
Wenelen (DSM 16786) were used for whole genome shotgun
database to extend the list to other organisms using an e-value
sequencing. All sequencing reads were obtained using both Ion
of 1e 10 and sequence identity of 70%. The COG numbers of the
Torrent and 454 GS Junior platforms. Sequencing corresponding
high quality proteins were then obtained and clusters of these
to Sb. thermosulfidooxidans Cutipay and At. thiooxidans Licanantay
sequences were retrieved in order to build a database of resistant
have been previously reported (Travisany et al., 2014, 2012). A
proteins. This database was used to search for genes related to pro-
trimming process was executed, removing low complexity reads
tein resistance in the Chilean biomining bacteria draft genomes
and adaptors. Only reads with per base phred quality value greater
using BLASTP, considering a cutoff e-value of 0.01 and identity of
than 20 were maintained. These high quality reads were used for
35%. The matching sequences from the Chilean biomining bacteria
the assembly process. Assembly of each of the five bacterial gen-
obtained were further expanded to include all sequences from
omes was performed using wgs-assembler (Celera Assembler) ver-
their respective ortholog groups. Finally, each of these potential
sion 7.0. A summary of the results of genome assembly and
resistant protein sequences was manually curated through multi-
annotation of the five biomining bacteria under study can be found
ple sequence alignments to a reference resistant protein sequence
in Supplementary Table S1. All genome data are available at http://
and specific motive search when available (Banci et al., 2006; Nies,
biominingdb.cmm.uchile.cl/genomes/.
2003). For the identification of metabolic pathways, annotated
genomes from each bacteria were used to create pathway/genome
2.2. Phylogenomic analysis databases (PGDB) using the Pathway Tools software v 13.0. From
those databases, pathways of interest were selected (Supplemen-
All bacteria were isolated from a mining environment and their tary Table S3) and curated following the same protocol used in
corresponding strains from the same species. To construct the tree, the characterization of heavy metal resistance proteins.
was used the publicly available genomes of 19 biomining bacteria Comparative genomics strategy was used first to determine
(Supplementary Table S2) retrieved from the National Center for ortholog groups that shared a unique set of genes between each
Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov) of the five native bacteria genomes and a previously released gen-
and the DOE Joint Genome Institute (JGI; http://www.jgi.doe.gov) ome from the same species. The following were compared: At. fer-
databases. Conserved core genes were obtained as follows: first, rooxidans strain Wenelen versus strain ATCC 23270; At. thiooxidans
orthologs genes between all biomining bacteria were determined strain Licanantay versus strain ATCC19377; A. multivorum strain
using ORTHOMCL v1.4 with an e-value cutoff of 1e 5. Only ortho- Yenapatur versus strain DSM 11245; L. ferriphilum strain Pañiwe
 M. Latorre et al. / Bioresource Technology 218 (2016) 659–666 661

vs strain ML-04; and Sb. thermosulfidooxidans strain Cutipay vs BRL075 and DSM1467). For Sb. thermosulfidooxidans Cutipay strain
strain DSM 9293. The same strategy was applied to identify puta- and Sulfobacillus. acidophilus DSM 10332 strain the medium was
tive genes involved in chloride ion resistance. The complete set of incubated at 50 °C. The MIC was defined as the lowest concentra-
genes from each of the Chilean biomining bacteria and a counter- tion of the respective salt at which no growth was observed. MIC
part from the same genus or species with significantly lower chlo- determination assays were performed in triplicate and in three
ride minimum inhibitory concentration (MIC) were compared. In separate experiments.
each case, unique genes from each of the more resistant strains
listed in Supplementary Table S4 were selected and analyzed.
Genome structural analysis was performed by Mauve and Nucmer 3. Results and discussion
programs.
3.1. Consortium genome features

2.4. Experimental assays To determine the microbial diversity present in copper mining
bioleaching systems, the biodiversity of culturable bacteria and
Experimental bioleaching assays corresponding to copper achaea present in Chilean ore deposits was investigated. More than
recovery were obtained directly from patents US 2006/0094094 10,000 16S sequenced regions from DGGE experiments were col-
A1 and US 2007/0042482 A1 and Bobadilla-Fazzini et al. (2014). lected from native microbiota from different Chilean mining sites
The MIC was used to determine the effect of chloride (NaCl and (Fig. 1). The results confirm the low complexity (or low species
KCl), copper (CuSO4), and arsenic (As2O3) on growth of different richness) of these extreme environments (Tyson et al., 2004),
bacterial strains as described previously (Bobadilla-Fazzini et al., showing that the most abundant organisms were the gamma-
2014). Briefly, 9 K medium at pH 1.6 was inoculated with 100e7 - proteobacterium species, along with nitrospirae and clostridia.
cells/mL for each strain separately which was supplemented with Similar composition and bacterial diversity has been described in
different concentrations of salts. The medium was incubated at acid mine drainage (AMD)(Baker and Banfield, 2003) and suggests
20 °C and 30 °C for 5 days for At. ferrooxidans strains (Wenelen, a high potential to oxidize sulfur. Inside this group, five different
ATCC 23270 and ATCC 53993), A. multivorum strains (Yenapatur native Chilean bacteria were isolated according to their higher
and DSM 11245), At. thiooxidans Licanantay strain, Acidiphilium capacity to extract solubilized copper in comparison to other
cryptum DSM2389 strain and L. ferriphilum strains (Yagan, public bioleaching strains (Fig. 2): Acidithiobacillus thiooxidans

A. B.

Genus Number Percent

Acidithiobacillus 3900 40.30%

Ferroplasma 1657 17.10%

Unknown 995 10.30%

Leptospirillum 994 10.30%

Unknown bacteria 659 6.80%

Sulfobacillus 423 4.40%

Alicyclobacillus 313 3.20%

Acidiphilium 107 1.10%

Stenotrophomonas 94 1.00%

Thermoplasma 76 0.80%

Anoxybacillus 58 0.60%

Sulfolobus 44 0.50%
Others under 0.5% 347 3.60%

Total 9667 100.00%

Fig. 1. Biodiversity of cultivabe bacteria in Chilean copper mines. (A) Number of DGGE sequences assigned to each genus. (B) Phylogenetic concatenated tree generated with
the core of conserved genes present in the Chilean consortium species and reported bioleaching bacterial strains (Supplementary Table S2).

At. ferrooxidans At. thiooxidans Sulfobacillus

40 40 50
Abiotic Abiotic Abiotic
35 35 45
Cu extraction (%)

Cu extraction (%)

Licanantay
Cu extraction (%)

Wenelen 40 Cutipay
30 30 DSM 504 DSM 10332
ATCC23270 35
25 25 30
20 20 25
15 15 20
15
10 10
10
5 5 5
0 0 0
0 10 20 30 40 50 0 10 20 30 40 50 60 0 10 20 30 40

Time (days) Time (days) Time (days)

Fig. 2. Copper extraction efficiency between the native Chilean and ATCC strains. Experimental details patents Pub. No.: US 2006/0094094 A1 (Wenelen), Pub. No.: US 2007/
0042482 A1 (Licanantay) and Bobadilla-Fazzini et al., 2014 (Cutipay)(Bobadilla-Fazzini et al., 2014).
662 M. Latorre et al. / Bioresource Technology 218 (2016) 659–666

Licanantay (DSM17318), Acidiphilium sp. Yenapatur (DSM27270)

(here classified as Acidiphilium multivorum), Leptospirillum fer-
riphilum Pañiwe, Acidithiobacillus ferrooxidans Wenelen (DSM
16786) and Sulfobacillus thermosulfidooxidans Cutipay (DSM
27601).
These strains represent the four dominant bacterial taxonomic
groups involved in copper mines (Yin et al., 2008), covering more
than the 50% of the total bacterial genera present in the ecosystems
of Chilean mines. Finally, these five biomining bacteria were
selected to grow in a novel bioreactor designed to produce leaching
biomass (Patent Registration No. CL 48319), representing the major
components of a stable, effective and continuously grown
bioleaching consortium operating in a large-scale and fully
operational biotechnology system at CODELCO, Radomiro Tomic
Division (Antofagasta, Chile).
In order to analyze the molecular features related to the
bioleaching of copper, the complete genome sequences of these
five biomining bacteria were determined and analyzed. The result-
ing assemblies indicated that main features of sequenced chromo-
somes share the same general statistics (contig number, number of
annotated genes and assembly size) as average public biomining
bacteria genome sequences, discarding the presence of bias during
the assembly of the chromosomes (Fig. 3 and Supplementary
Table S1).
Fig. 3. Circular representation of COG categories of consortium bacteria. The Whole genome alignments showed notorious structural varia-
diagram shows the distribution of COG categories in each genome. COG families are
tions (insertions, deletions, inversions) between the five native
grouped as follows: Cellular Processes and Signaling (D, M, N, O, T, U, V, orange);
Information Storage and Processing (J, K, L, light blue); Basal Metabolism (C, E, F, G, bacteria and their corresponding ATCC reference strains
H, I, P, Q, green); Poorly Characterized (R, S, light grey) and No COG annotation (X, (Supplementary Fig. S1). Among them, an elevated set of unique
dark grey). Each concentric circles represents a consortium bacteria. From the outer genes that encode unknown proteins and putative transposases
to the inner circle: At. thiooxidans Licanantay (yellow): A. multivorum Yenapatur
were present in the consortium. At. thiooxidans Licanantay showed
(green), L. ferriphilum Pañiwe (light blue) At. ferrooxidans Wenelen (red), Sb.
thermosulfidooxidans Cutipay (pink). Bars within each circle represent the number
a total assembly 29% higher than that of public At. thiooxidans ATCC
of genes with different COG numbers in each category (details see Supplementary 19377, a genome structure variation which may impact the
Table S5). (For interpretation of the references to color in this figure legend, the bioleaching process (Darling et al., 2008). The five bacteria share
reader is referred to the web version of this article.) a conserved core of 478 genes, 47 of them unique to this

A. B.
At. ferrooxidans At. ferrooxidans At. thiooxidans
Wenelen ATCC 23270 Wenelen ATCC 19377 Licanantay

148 2719 675 505 2466 1432

Sb. thermosulfidooxidans
At. thiooxidans Cutipay
Licanantay

A. multivorum L. ferriphilum
DSM 11245 Yenapatur ML-04 Pañiwe

314 3061 635 548 1925 601

Sb. thermosulfidooxidans
DSM 9293 Cutipay

438 3100 500

L. ferriphilum
A. multivorum Pañiwe
Yenapatur

Fig. 4. Gene conservation. (A) Venn diagram showing the unique and shared genes between the five native Chilean bacteria. (B) Total gene distribution between the native
Chilean consortium and ATCC strains.
 M. Latorre et al. / Bioresource Technology 218 (2016) 659–666 663

100

90 Cellular process and signaling

Percentage of total genes (%)

80

70 Metabolism

60

50 Storage and processing

40

30 Poorly characterized

20

10
No COG
0

A.ferrooxidans Wenelen
At. ferrooxidans Wenelen
12 A.thiooxidans Licanantay
At. thiooxidans Licanantay
Acidiphilium multivorum
A. multivorum BRL109
Yenapatur
L. ferriphilum
L.ferriphilium Pañiwe
BRL111 *
10 Sb. thermosulfidooxidans
S.thermosulfidooxidans CutipayCutipay *
Percentage of total genes (%)

*
8 *
*
*
6 *
* *
*
4 * **
* *
**
2
*

0
C D E F G H I J K L M N O P Q R S T U V

COG categories

Fig. 5. Individual COG category coverage. (A) percentage of total genes accounted by species for each meta-COG class. Bacterial clustering was performed with total genes of
each COG category. (B) percentage of total genes accounted by species and individual COG category ([C] Energy production and conversion, [D] Cell cycle control, cell division,
chromosome partitioning, [E] Amino acid transport and metabolism, [F] Nucleotide transport and metabolism, [G] Carbohydrate transport and metabolism, [H] Coenzyme
transport and metabolism, [I] Lipid transport and metabolism, [J] Translation, ribosomal structure and biogenesis, [K] Transcription, [L] Replication, recombination and repair,
[M] Cell wall/membrane/envelope biogenesis, [N] Cell motility, [O] Posttranslational modification, protein turnover, chaperones, [P] Inorganic ion transport and metabolism,
[Q] Secondary metabolites biosynthesis, transport and catabolism, [R] General function prediction only, [S] Function unknown, [T] Signal transduction mechanisms, [U]
Intracellular trafficking, secretion, and vesicular transport, [V] Defense mechanisms). Percent of genes not assigned in COG category (NoCOG): At. ferrooxidans Wenelen 26%,
At. thiooxidans Licanantay 29%, A. multivorum Yenapatur 17%, L. ferriphilium Pañiwe 30%, Sb. thermosulfidooxidans Cutipay 23% and E. coli K-12 19%. Asterisk denotes significant
enrichment (P < 0.05) compared to the total proportion in the bacterial genome.

consortium (i.e. not detected in the ATCC reference strains) (Fig. 4). Additionally, they also illustrate the bacterial complementarity
In this group, were highlight proteins involved in energy produc- that occurred during the bioleaching process, supporting previous
tion and protein biosynthesis (two processes that confer efficiency experimental strategies used to increase the efficiency to recover
to extract copper), unique elements probably acquired by horizon- copper from ores (Feng et al., 2013; Wang et al., 2012, 2014).
tal gene transfer events inside the community (Fig. S2 and
Supplementary Table S3)(Hemme et al., 2016). 3.2. Integrative bioleaching pathway model
A COG class clustering inside the consortium allowed for classi-
fication of the five bacteria into two main clades according to the Through the analysis of genome annotations and metabolic
bacterial abilities to use different energy sources (Fig. 5 and pathway reconstructions, species-specific components of key
Supplementary Table S5). One clade is composed of the metabolic routes that influence the bioleaching process were
chemolithoautotrophs At. ferrooxidans Wenelen, At. thiooxidans revealed. An integrated model of metabolic pathways representing
Licanantay and L. ferriphilum Pañiwe, and the other of the chemo- the bioleaching capability of the bacterial consortium was assem-
mixotroph Sb. thermosulfidooxidans Cutipay and the heterotroph A. bled (Supplementary Fig. 3 and Supplementary Table S3).
multivorum Yenapatur. The main differences in functional cate-
gories between these groups relates to the proteins involved in 3.2.1. Iron oxidation
inorganic ion transport and biogenesis. Differences suggest a The most extensively characterized mechanism of iron
strong dependence between the bioleaching process and the bacte- oxidation has been predicted only in At. ferrooxidans strains (rus
rial ability of using inorganic compounds as sole energy sources. operon) (Carlos et al., 2008). In silico predictions indicated that
664 M. Latorre et al. / Bioresource Technology 218 (2016) 659–666

At. ferrooxidans Wenelen, L. ferriphilum Pañiwe and Sb. thermosulfi- the ATP-driven proton pumping activity of the F0F1-ATP synthase,
dooxidans Cutipay also contain components of the rus operon. It used as an energy source during oxidation of reduced copper ores.
has been demonstrated that leptospirilli species have a greater Moreover, glutathione precursors such as NAD(P) were also
affinity for ferrous iron and a lower sensitivity to inhibition by fer- detected as one of the most synthesized molecule by Acidithioba-
ric iron (Rawlings et al., 1999). The principal component rusti- cilli, indicating that these molecules have a key putative role in
cyanin was not detected in L. ferriphilum Pañiwe and Sb. optimizing the bioleaching process.
thermosulfidooxidans Cutipay genomes. This suggests that an The bacterial attach capacity to the mineral and the biofilm for-
uncharacterized functional homolog of At. ferrooxidans rusticyanin mation plays a crucial role during the bioleaching process (Yang
would be present and that molecular mechanisms of iron oxidation et al., 2014). All studied species present classical proteins involved
in these bacteria evolved independently (Orengo and Thornton, in the production of extracellular polymeric substances and
2005). In addition, through the bc1-NDH complex, the molecules biofilm-associated proteins, highlighting LuxR and VpsS-R tran-
NAD(P) can receive electrons from the rus system. One of the sec- scriptional factor family members as putative regulators of these
ondary metabolites produced during the synthesis pathway I for processes. Previous work indicates that elevated production of
NAD(P) is L-glutamate, an amino acid required during biosynthesis the second messenger c-di-GMP is directly correlated with biofilm
of HEM molecules and the principal co-factor of cytochromes formation (Ruiz et al., 2012). The high number of ydeH copies (at
(Quatrini et al., 2009). While these components are present in least five genes per bacterium) involved in the synthesis pathway
other species of the consortium, L. ferriphilum Pañiwe showed a of c-di-GMP suggests that the consortium possess an elevated
higher number of these proteins. This findings support the idea potential to generate biofilm.
that this species is the dominant iron-oxidizing bacterium inside
the mining consortium; thus, increased participation in the
4. Environmental resistance mechanisms
bioleaching process might be expected (Rawlings, 2005).
Biomining bacteria resist high levels of metals via different sys-
3.2.2. Reduced inorganic sulfur compounds (RISC)
tems such as active efflux or trapping of metal ions (Navarro et al.,
Classical sulfur dioxygenase and sulfite acceptor oxidoreductase
2013; Orell et al., 2010). During the bioleaching process, the
were predicted in the genome of At. ferrooxidans Wenelen, At.
increase of soluble oxidized metals can induce the formation of
thiooxidans Licanantay and Sb. thermosulfidooxidans Cutipay
reactive oxygen species (ROS). Although all bacteria contain the
strains, indicating the presence of an active RISC mechanism inside
classic ROS response genes (SOD, catalases, peroxidases, reduc-
the consortium. At. thiooxidans Licanantay contains an elevated
tases, and thioredoxines), the number of these components does
number of HDR gene copies (one of the principal protein complex
not differ from other microorganisms (Imlay, 2013). Thus, high
involved in RISC) (Bobadilla Fazzini et al., 2013), two copies for the
environmental resistance may relate to the ability to handle high
sulfur oxidizing gene pathway (sox) and one archaeal type sulfur
ionic strength, osmotic pressure and concentrations of metal and
oxygenase reductase gene system (sor). Unlike At. thiooxidans
other inhibitory ions and avoiding intracellular toxic effects
strains previously reported (Quatrini et al., 2009), At. thiooxidans
(Fig. 6). The analysis indicated that all five bacteria contain a high
Licanantay contains the complete cys operon involved in sulfate
amount of metal resistant determinants, principally ATPases type P
assimilation and cysteine biosynthesis. The presence of this system
and chaperons, classical from copper tolerant organism. First, the
provides a higher possibility to generate cysteines. Together, gluta-
high number and duplication of copper efflux proteins is directly
mate and cysteines are the amino acid precursors of glutathione, a
correlated with the environment where these bacteria grow
metabolite that plays a catalytic role in elemental sulfur activation.
(copper sulfide ores). At. ferrooxidans Wenelen showed the highest
In addition, cysteine produced by the bacterium can be used to
tolerance to copper versus all the tested strains, which also corre-
generate Fe-S cluster. This cluster is the principal co-factor of the
sponded with the highest number of copper resistant elements
HDR complex, which utilizes the reduced glutathione to assimilate
encoded in its genome.
sulfate, completing the assimilation cycle. A. multivorum Yenap-
In the case of the gram-positive Sb. thermosulfidooxidans
atur, unlike the other species of the consortium, contains a higher
Cutipay, the lack of outer membrane prevents the existence of
number of unique proteins classified as components of carbohy-
RND metal proteins. To compensate for this absence, a high num-
drate transport and protein metabolism. Thus supporting the fact
ber of CopA (copper ATPase) are encoded in this bacterium.
that this bacterium plays a role degrading organic metabolites,
Secondly, arsenic-resistant mining strains show a more efficient
highly toxic for chemolithoautotrophs, which in turn oxidizes thio-
leach activity (Hallberg et al., 1996). At. thiooxidans Licanantay
sulfate and prevents damage against Acidiphilium. This process
exhibited the highest tolerance against arsenic (more than 10
establish a mutualistic relationship inside the consortium (Okabe
times compared with the others species), a property that might
et al., 2007; Okibe and Johnson, 2004).
be conferred by the unique presence of ArsM, an arsenic methyl-
transferase involved in metal resistance (Morgante et al., 2015).
3.2.3. Basal metabolism and biofilm formation
Finally, high concentrations of chloride ions released during the
Based on the integrated model of metabolic pathways in the
process impact yields in the biomining of copper (Chang-Li et al.,
consortium, were able to link basal metabolic routes with the
2012), attributed to the loss of the outer membrane integrity and
bioleaching process. Circular pathways in which the function and
the complete inhibition of biooxidation (Bobadilla-Fazzini et al.,
synthesis of the components directly depended on the capacity
2014). Most of the native strains showed an elevated tolerance to
of the bacteria to reduce inorganic sulfur compounds and oxidize
chloride ions (Table 1). A comparative genomics strategy identifies
iron were found. Glutathione appears to play a crucial role in
a particular set of ion transporters potentially involved in chloride
metabolic processes directly or indirectly related with iron and
resistance, which are only encoded in the resistant strains of the
RISC oxidation. It was recently demonstrated that At. thiooxidans
consortium (Supplementary Table S4).
Licanantay produces a high concentration of glutathione under dif-
ferent growth conditions (Martinez et al., 2013). The resulting acid-
ification of the cytoplasm can be controlled by glutamate, one of 5. Conclusions
the principal mechanisms of acid resistance in different bacteria
(Foster, 2001). The high concentration of hydrogen can also be The genome analysis of these five bacteria provides specific
used for consortium species to generate ATP. This occurs through information about the role of each species in the consortium,
 M. Latorre et al. / Bioresource Technology 218 (2016) 659–666 665

At. ferrooxidans At. thiooxidans A. multiborum L. ferriphillum Sb. thermosulfidooxidans

Wenelen Licanantay Yenapatur Pañiwe Cutipay

C
O
P
P
E
R 20 £C 30 £C 20 £C 30 £C 20 £C 30 £C 20 £C 30 £C 50 £C
Wenelen >10 >10 Licanantay 8 8 Yenapatur 2 2 Pañiwe 5 5 Cutipay 3
ATCC 23270 5 5 DSM 2389 0,5 0,5 DSM14647 n.d. <3 DSM 10332 1
ATCC 53993 10 8 DSM 11245 0,5 0,5 .

A
R
S
E
N
20 £C 30 £C 20 £C 30 £C 20 £C 30 £C 20 £C 30 £C 50 £C
I
Wenelen 0,2 0,2 Licanantay 3,5 3,5 Yenapatur 0,2 0,2 Pañiwe 0,05 0,05 Cutipay <0,03
C ATCC 23270 0,2 0,2 DSM 2389 0,2 0,1 DSM14647 n.d. <0,03 DSM 10332 1
ATCC 53993 0,3 0,4 DSM 11245 0,1 0,2

Fig. 6. Copper and arsenic gene resistant clusters and ion tolerance. Definition of colored arrows (COG super-class) are as follows: orange, cellular process and signaling; light
blue, metabolism; green, storage and processing; light grey, poorly characterized; dark grey, NoCOG. A red asterisk denotes a unique component. Tables indicate minimum
inhibitory concentration results. A red value indicates the highest resistance between the same genus species. A green value denotes the highest tolerance between all the
tested strains. Gene Id codes in Supplementary Table S3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this
article.)

Table 1
Minimum inhibitory concentration (MIC) to chloride ions.

Chloride ion (KCl) [g/L] Chloride ion (NaCl) [g/L]

20 °C 30 °C 20 °C 30 °C
At. ferrooxidans Wenelen 5 6 5 5
At. ferrooxidans ATCC 23270 7 >10 3 5
At. ferrooxidans ATCC 53993 8 8 7 7
A. multivorum Yenapatur >10 >10 >10 >10
A. cryptum DSM 2389 (JF5) 4 4 5 5
A. multivorum DSM 11245 (AIU301) 4 4 5 5
L. ferriphilum Pañiwe 2 5 1 3
L. ferriphilum DSM14647 (ML-04) n.d. 2 n.d. >1
At. thiooxidans Licanantay >10 >10 >10 >10
Sb. thermosulfidooxidans Cutipay** 10 5
Sb. acidophilus DSM 10332** 3 1
**
Experiment temperature 50 °C.

an important advance useful for designing of heap bioleaching Acknowledgements

plant (Panda et al., 2015). These bacteria represent the first
native mining consortium isolated directly from copper mines. This work was supported by BioSigma S.A., Fondap Grant
In addition, this information can be used to intervene directly 15090007, Center for Genome Regulation, Basal Grant of the Center
the composition of the consortium inside the bioreactor (Feng for Mathematical Modeling UMI2807 UCHILE-CNRS and CIRIC-
et al., 2015a,b). Finally, these five bacteria constitute the indus- INRIA Chile project. The authors thank BioSigma S.A. for authoriz-
trial bioleaching consortium best described to date and represent ing the submission of the manuscript for publication, Mr. Cristián
the culmination of a decade of research in genomics of mining Murillo for graphic design support and Ms. Estela Blanco for edit-
bacteria. ing. Acknowledge to the National Laboratory for High Performance
Computing at the Center for Mathematical Modeling (PIA ECM-02.-
CONICYT) and the sequencing center Omics Solutions.
Author contributions

M.L., R.B., P.P., V.C., M.G. and A.M. analyzed the results and Appendix A. Supplementary data
wrote the manuscript. M.P.C., D.T., A.D. and M.B. performed all
the bioinformatics analysis. M.L., A.R. and C.H. participated in pro- Supplementary data associated with this article can be found, in
tein manual annotation. gDNA bacterial extraction was done by R. the online version, at http://dx.doi.org/10.1016/j.biortech.2016.07.
B. 012.
666 M. Latorre et al. / Bioresource Technology 218 (2016) 659–666

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