Nova Biotechnol Chim (2018) 17(2): 125-131  

                          DOI: 10.2478/nbec-2018-0013 
 

 

 Corresponding author: peter.pristas@upjs.sk 

Nova Biotechnologica et Chimica 

Genetic variability in Acidithiobacillus spp. – a working horse  

of environmental biotechnologies 

Peter Pristas1,, Jana Kiskova1, Ivana Timkova1, Lenka Malinicova1, Alena Luptakova2, 

Maria Kusnierova2 and Jana Sedlakova-Kadukova1 

1Department of Microbiology, Institute of Biology and Ecology, Faculty of Science, Pavol Jozsef Šafarik University  

in Košice, Šrobárova 2, Košice, 041 54, Slovakia 
2Institute of Geotechnics, Slovak Academy of Sciences, Watsonova 45, Košice, 043 53, Slovakia 
 

 

Article info 
 

Article history: 

Received: 16th October 2018 

Accepted: 21st December 2018 

 

Keywords: 

Acidithiobacillus 

Complex species 

Environmental biotechnology 

Genetic variability 

Phylogeography 

 
 

 

 

 

 

 

 

 

 
 

 

 

 

 

Abstract 
 

The genus Acidithiobacillus comprises 7 species of Gram-negative obligatory 

acidophilic chemolithotrophic bacteria that derive energy mainly from the oxidation 

of reduced sulphur compounds. Four of the species also catalyse the dissimilatory 

oxidation of ferrous iron while three (A. thiooxidans, A. albertensis, and A. caldus) 

do not. Bacteria from the genus Acidithiobacillus are often associated with mineral 

biotechnologies (biomining) and acid mine drainage. While acceleration of mineral 

solubilisation is a positive aspect in environmental biotechnologies, it is undesirable 

in acid mine drainage with strong negative ecological impact and there is profound 

interest in genetics and genomics of these bacteria. Representatives  

of Acidithiobacillus genus occur world-wide, however there are limited data  

on Acidithiobacillus spp. variability from Slovakia. In our work the variability  

of Acidithiobacillus spp., from Slovakia was analysed and the presence  

of A ferrooxidans was detected. In addition, for the first time we report here on the 

occurrence of A. albertensis as well. Comparative analyses confirmed pronounced 

genetic and genomic diversity within the genus, especially within A. ferrooxidans 

and A. thioxidans complexes. Based on data presented, several Acidithiobacillus 

species could be considered as a complex species and the description of several new 

species is very probable in the near future. 
 

 University of SS. Cyril and Methodius in Trnava
 
 
 

 
 
 

Introduction 
 

The genus Acidithiobacillus comprises a group  

of Gram-negative obligatory acidophilic 

chemolithotrophic bacteria that derive energy 

mainly from the oxidation of reduced sulphur 

compounds. The members of the genus are motile 

by one or more flagella and comprise both 

mesophiles and moderate thermophiles (Kelly and 

Wood 2000). Acidithiobacilli use various sulphur 

compounds as electron donor and therefore, are 

often found in metal sulphide deposits all over the 

world    (Karavaiko    et al.   2003),    fresh    waters  

associated with sulphide deposits (Gonzalez-Toril 

et al. 2003) and seawater (Kamimura et al. 2003). 

The final electron acceptor is usually oxygen 

(Kelly and Wood 2000) however, Fe3+ and S0 were 

reported to be alternative electron acceptors for 

some species (Ohmura et al. 2002). The ability  

to fix CO2 makes acidithiobacilli independent  

of an organic carbon source and allows them  

to thrive in mine waste ecosystems where the 

organic carbon concentration is extremely low 

(Dold et al. 2005).  

The Acidithiobacillus genus was formed when the 

former Thiobacillus genus was split into the genera 

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Nova Biotechnol Chim (2018) 17(2): 125-131 

126 

Acidithiobacillus, Halothiobacillus, and 

Thermithiobacillus (Kelly and Wood 2000).  

The type species of the genus, A. thiooxidans was 

described nearly 100 years ago as Thiobacillus 

thiooxydans (Waksmann and Joffe 1922) and  

up to now 7 species within the genus were validly 

described. All species are autotrophs capable  

of growth utilising inorganic sulphur compounds  

as sole energy substrate. Four of the species  

(A. ferriphilus, A. ferrivorans, A. ferrooxidans, and 

A. ferridurans) also catalyse the dissimilatory 

oxidation of ferrous iron while three  

(A. thiooxidans, A. albertensis, and A. caldus)  

do not. Representatives of Acidithiobacillus genus 

occur world-wide in a diverse range of natural (acid 

rock drainage, sulphur springs, etc.) and industrial 

settings (ore concentrates, leaching solutions of the 

mining industry, etc.), with varying 

physicochemical characteristics.  

Bacteria from the genus Acidithiobacillus are often 

associated with mineral biotechnologies 

(biomining) and acid mine drainage. Biomining 

utilises these bacteria for recovery of metals from 

sulphidic low grade ores and concentrates. Acid 

mine drainage results in acidification and metal 

contamination of soil and water emanating from the 

dissolution of metal sulphides from deposits and 

mine waste storage (Johnson 2014). Acidophilic 

microorganisms play a central role in these 

processes by catalysing aerobic oxidation  

of sulphides. While acceleration of mineral 

solubilisation is a positive aspect in mineral 

biotechnologies, it is undesirable in acid mine 

drainage with strong negative ecological impact 

and there is profound interest in genetics and 

genomics of these bacteria. 

DNA based analyses have already revealed 

pronounced genetic diversity within 

Acidithiobacillus genus, especially within  

A. ferrooxidans complex. The diversity probably 

could be not attributed to the geographic 

distribution of acidithiobacilli as practically all 

known species were detected worldwide (Nunez  

et al. 2017). More than 50 % of all 

Acidithiobacillus isolates characterised at 16S 

sequence level can be mapped to Asia, followed  

by Europe (20 %), South (9 %), and North America 

(7 %), and Africa and Oceania (less than 2 % each). 

From the Slovakia territory two isolates were 

characterised, both identified as A. ferrooxidans 

(Nunez et al. 2017). The aim of our study was  

to understand the variability within acidithiobacilli 

in Slovakia and worldwide based on genetic and 

genomic analyses.  

 
Experimental 

 
Molecular identification of Acidithiobacillus spp. 

isolates from the Slovakia 

 
Molecular identification of Acidithiobacillus spp. 

isolated recently from Smolnik (East Slovakia) acid 

mine drainage water – LC isolate (Luptakova et al. 

2012) and A. albertensis TS isolate from Hodruša-

Hámre (Central Slovakia) gold mine (Pristas et al.; 

manuscript in preparation) were performed using 

widely accepted 16S based methodology (Yarza  

et al. 2014). The sequences obtained were 

deposited into GenBank database under accession 

numbers MH740926 and MH796351 for LC and 

TS isolate, respectively. The sequences were 

compared against database of 16S sequences  

of type strains of bacteria and archaea using 

BLASTN analysis (Altschul et al. 1990). 

Phylogenetic placement of isolates was confirmed 

by multiple sequence comparison against 528 type 

strains of Acidithiobacillus spp. downloaded from 

RDP database (available at 

https://rdp.cme.msu.edu/) using the Neighbor-

Joining method implemented in MEGA 7 software 

(Kumar et al. 2016) with 500 bootstrap 

replications.  

 
Analysis of genetic variability within 

Acidithiobacillus genus 

 

To analyse genetic variability in Acidithiobacillus 

genus 16S rRNA sequences of LC and TS isolates 

obtained in this study were added to the pre-aligned 

16S Acidithiobacillus spp. sequences and analysed 

as described above. For genome comparison, 

available genome sequences of Acidithiobacillus 

spp. were downloaded from the GenBank database 

(https://www.ncbi.nlm.nih.gov/genome). Final 

dataset was composed of 8 sequences - 

GCA_001931655 for A. albertensis, 

GCA_000214095.3 for A. ferrivorans, 

GCA_000175575.2 for A. caldus, 

GCA_000227215.2 for A. thiooxidans,

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https://rdp.cme.msu.edu/
https://www.ncbi.nlm.nih.gov/genome


Nova Biotechnol Chim (2018) 17(2): 125-131 

127 

GCA_000021485 for A. ferrooxidans, 

GCA_000179815.2 for Acidithiobacillus sp. GGI-

221, GCA_002341825.1 for Acidithiobacillus sp. 

UBA2486, and GCA_002733395.1 for 

Acidithiobacillus sp. NORP59. For each pair  

of sequences ANI value (Average Nucleotide 

Identity) was calculated using EzTaxon server 

(https://www.ezbiocloud.net/tools/ani; Yoon et al. 

2017). Based on ANI values obtained a similarity 

dendrogram was constructed using UPGMA 

algorithm implemented in MEGA 7. For functional 

genome comparisons the RAST server pipeline 

(Rapid Annotation by Subsystem Technology) 

available at https://rast.theseed.org/ was used 

(Overbeek et al. 2014). 

 

Results and Discussion  
 

Two recently isolated Acidithiobacillus spp. 

isolates were identified using 16S rRNA 

sequencing followed by BLASTN analysis  

of sequences obtained. The 16S rRNA sequence  

of LC isolate from Smolnik acid mine drainage 

water showed the highest similarity (99.7 %) to the 

16S rRNA sequences of type species  

of A. ferrooxidans strain ATCC 23270. The 16S 

rRNA sequence of TS isolate from Hodruša-Hámre 

gold mine has shown the highest similarity  

(99.7 %) to the A. albertensis DSM 14366 

sequence. Multiple sequence alignments confirmed 

identification of both isolates resulted from 

BLASTN analysis (Fig.1). The LC isolate sequence 

grouped with A. ferrooxidans sequence, while this 

of TS isolate grouped with A. albertensis sequence.

The tree indicates that the ability to obtain energy 

from the dissimilatory oxidation of ferrous iron  

is probably of old evolutionary origin as all four 

species able to utilise ferrous ions (A. ferriphilus, 

A. ferrivorans, A. ferrooxidans, and A. ferridurans) 

formed well supported group phylogenetically 

distant from the species unable to utilize ferrous 

ions. The analysis indicates that A. caldus is the 

most distant species of Acidithiobacillus genus, 

forming a separate branch non-related to the other 

members of the genus (Fig. 1). 

Up to now, up to 120 Acidithiobacillus spp. isolates 

or environmental 16S rRNA sequences were 

identified from the European territory, most  

of them identified as A. ferrooxidans and  

A. thiooxidans and the occurrence  

of A. ferrooxidans related bacteria have already 

been reported from Slovakia as well (Nunez et al. 

2017). However, no A. albertensis related 

sequences have been reported neither from the 

Slovak nor European territory yet. A. albertensis 

species was described in 1983 (Bryant et al. 1983) 

and it is not studied very frequently. A. albertensis 

related isolates were reported up to now mainly 

from China and Canada (Nunez et al. 2017), while 

our observation is the first report on A. albertensis 

from Europe. 

Biotechnology and environmental protection 

interest led to the characterization of multiple 

Acidithiobacillus spp. isolates mainly from 

industrial environments. Most of the isolates were 

identified as A. ferrooxidans or A. thiooxidans  

as for many years these two species were the only 

validly  described  species  of the  Acidithiobacillus 

.

Fig. 1. Unrooted phylogenetic tree 

documenting phylogenetic placement 

of Acidithiobacillus spp. from Slovakia 

(A. ferrooxidans LC and A. albertensis 

TS; indicated with asterisks) inferred 

using the Neighbor-Joining method. 

The bar represents genetic distance 

shown in number of base substitutions 

per site. Numbers at nodes are 

bootstrap values after 500 repetitions. 

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https://www.ezbiocloud.net/tools/ani
https://rast.theseed.org/


Nova Biotechnol Chim (2018) 17(2): 125-131 

128 

 

 

genus (Jones et al. 2016). In the recent years great 

diversity within the genus was recognised and 

several new species of acidithiobacilli were 

described (Hallberg et al. 2010; Hedrich and 

Johnson 2013; Falagan and Johnson 2016).  

To understand genetic variability  

in Acidithiobacillus genus all available 16S rRNA 

sequence of Acidithiobacillus-related bacteria were 

downloaded from RDP database (Cole et al. 2014) 

and compared using phylogenetic approach. The 

resulted phylogenetic tree (Fig. 2) clearly 

documents extraordinary variability within 

Acidithiobacillus genus. While the recently 

described species e.g. A. ferrivorans and  

A. ferridurans form monophyletic, well supported 

clades in the tree, other species are spread over the 

tree and could be found in multiple branches, 

indicating genetic variability within the genus.  

The diversity within Acidithiobacillus genus have 

already been reported (Karavaiko et al. 2003; Ni  

et al. 2008) and based on our data the existence  

of at least 20 species could be proposed solely  

on 16s rRNA analysis. 

Recent advances in genomics allowed us to better 

understand the common and distinctive features 

some of Acidithiobacillus species isolated from 

similar habitats and provided data to understand 

potential adaptation mechanisms of these bacteria 

to the harsh environments. Complete or draft 

genome sequences of several Acidithiobacillus 

species have become available in the recent few 

years (Cárdenas et al. 2016). Comparative genomic 

analyses indicate that genomes of acidithiobacilli 

are relatively similar. The genome sizes vary from 

2.78 to 3.78 Mbp, G + C content vary from 52.5  

to 67.5 %, and numbers of predicted genes vary 

from 2648 to 4007. In all these parameters  

A. caldus is the most distant species, showing the 

highest G + C content, the smallest genome size, 

and the smallest number of genes (data not shown). 

Based on the functional comparative genomic 

analyses of Acidithiobacillus species, it was shown 

that different metabolic pathways might allow these 

microorganisms to acquire energy, carbon sources, 

and nitrogen sources from acidic environments, 

ensuring their survival and proliferation in these 

environments (Fig. 3). Using RAST server pipeline 

(Overbeek et al. 2014) very similar numbers  

of genes were found e.g. those of involved in RNA 

metabolism or cell wall and capsule subsystems  

in all Acidithiobacillus species analysed.  

The species, however, differ significantly 

in number of genes classified to the Nitrogen or  

Phosphorus metabolism, and Protein metabolism, 

as well as stress response (Fig. 3). The comparisons 

indicate that non-motile species of the genus  

(A. ferrooxidans and A. thioxidans) completely lack 

the genes for motility and chemotaxis.  

The phenotypic diversity observed within the genus  

is thus a consequence of genetic and genomic 

diversity. 

To evaluate the genomic diversity in more detail,

Fig. 2. Unrooted phylogenetic tree 

documenting phylogenetic diversity within  

584 available 16S rRNA sequences  

of Acidithiobacillus spp. The tree was inferred 

using the Neighbor-Joining method.  

The placement of validly described species  

is shown. The bar represents genetic distance 

shown in number of base substitutions per site. 

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Nova Biotechnol Chim (2018) 17(2): 125-131 

129 

 
Fig. 3. Comparative genomics of selected Acidithiobacillus species. Number of genes classified to the selected subsystems  

as identified by RAST server is shown. 

 

average nucleotide identity (ANI) values were 

calculated for every pair of genomic sequences and 

compared. ANI was proposed recently as a means 

to compare genetic relatedness among prokaryotic 

strains. The method compares nucleotide-level 

genomic similarity between the coding regions  

of two genomes and is considered the best measure 

of genetic relatedness among bacterial strains 

(Figueras et al. 2014; Jain et al. 2018). ANI values 

in range from 0.993 to 0.640 were observed among 

analysed set of sequences. Based on ANI values 

relatedness tree of Acidithiobacillus spp. was 

constructed (Fig. 4). The tree shows congruent 

topology with 16S rRNA based tree (Fig. 1). 

The ferrous iron-oxidising species form a separate 

group distant from the species that are unable  

to utilize ferrous ions, and (like in 16S rRNA based 

tree) A. caldus is the most divergent species  

of Acidithiobacillus genus related distantly only  

to any other species. The tree also documents 

diversity within the genus when the yet 

uncharacterised isolates UBA2486 and NORP59 

have produced significantly low ANI values and 

therefore could be assigned as a new species within 

Acidithiobacillus genus. Similarity values higher 

than 0.95 are necessary to place the isolates to the 

same species (Figueras et al. 2014).  

The first species of the Acidithiobacillus genus was 

 

Fig. 4. UPGMA (unweighted pair 

group method with arithmetic 

mean) generated dendrogram 

showing relatedness  

of Acidithiobacillus sp. based 

on ANI (average nucleotide 

identity) values. 

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Nova Biotechnol Chim (2018) 17(2): 125-131 

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described already 100 years ago (Waksmann and 

Joffe 1922) and up to now 7 species  

of acidithiobacilli were validly described, most  

of them in the recent 20 years. However, 

application of modern genetic and genomic 

methods indicates that variability within the genus 

is highly underestimated and several species, 

especially A. ferrooxidans and A. thioxidans, have 

to be considered a complex species and  

the description of multiple new species  

of acidithiobacilli is very probable in the near 

future. 

 

Conclusions 
 

Our data indicate that genetic variability  

of Acidithiobacillus genus is highly 

underestimated. Several species, especially  

A. ferrooxidans and A. thioxidans, have to be 

considered a complex species and the description 

of multiple new species of acidithiobacilli is very 

probable in the near future. From the Slovakia 

territory A. ferrooxidans species is reported 

predominantly, but for the first time we report here 

on the occurrence of A. albertensis as well. 

 

Acknowledgement 
 
The work was financially supported by Research Agency  

of the Ministry of Education, Science, Research and Sport  

of Slovak Republic under the VEGA Project 1/0229/17. 

 

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