Nova Biotechnol Chim (2017) 16(1): 48-53 

                  DOI: 10.1515/nbec-2017-0007 
 

 

 Corresponding author: simona.kvasnova@umb.sk 

Nova Biotechnologica et Chimica 

Zinc bioaccumulation by microbial consortium isolated from nickel 

smelter sludge disposal site 

Simona Kvasnováa,, Ľudmila Hamarováb and Peter Pristašc 

a Department of Biology and Ecology, Matej Bel University, Tajovského 40, Banská Bystrica, SK-914 01, Slovak Republic  

b Institute of Biology and Ecology, Pavol Jozef Šafárik University in Košice, Šrobárová 2, Košice, SK- 040 01, Slovak Republic  

c Institute of Animal Physiology, Slovak Academy of Science, Šoltésovej4-6, Košice, SK- 040 01, Slovak Republic  

 

 
 

Article info 
 

Article history: 

Received: 20th February 2017 

Accepted: 11th May 2017 

 

Keywords: 

Bacteria 

Consortium 
Heavy metals 

Bioremediation 

Zinc bioaccumulation 
Arthrobacter 

 

 
 
 

 

 
 

 

 

 

Abstract 
 

Heavy metal pollution is one of the most important environmental issues of today. 

Bioremediation by microorganisms is one of technologies extensively used 

for pollution treatment. In this study, we investigated the heavy metal resistance 

and zinc bioaccumulation by microbial consortium isolated from nickel sludge 

disposal site near Sereď (Slovakia). The composition of consortium was analyzed 

based on MALDI-TOF MS of cultivable bacteria and we have shown that 

the consortium was dominated by bacteria of genus Arthrobacter. While consortium 

showed very good growth in the zinc presence, it was able to remove only 15 % 

of zinc from liquid media. Selected members of consortia have shown lower growth 

rates in the zinc presence but selected isolates have shown much higher 

bioaccumulation abilities compared to whole consortium (up to 90 % of zinc 

removal for NH1 strain). Bioremediation is frequently accelerated through injection 

of native microbiota into a contaminated area. Based on data obtained in this study, 

we can conclude that careful selection of native microbiota could lead  

to the identification of bacteria with increased bioaccumulation abilities. 
 

 University of SS. Cyril and Methodius in Trnava

 

 
 

Introduction 
 

Elevated concentrations of heavy metals are 

introduced into the environment through 

metalliferous mining, metal smelting, metallurgy 

industry activities, and waste disposal sites. 

This global environmental problem has developed 

over the past few decades due to the rapid increase 

in industrialization and urbanization (Dixit et al. 

2015). 

Some heavy metals are essential elements  

for the existence of living organisms because they 

perform several functions in biological systems. 

These  heavy metals  such as Ca, Co, Cr, Cu, Fe, K,  

Na, Ni, and Zn are essential nutrients; some 

of heavy metals are relatively harmless but can be 

toxic in larger amounts or certain forms. Essential 

metals function as catalysts for biochemical 

reactions are stabilizers of protein structures 

and bacterial cell walls and serve in maintaining 

osmotic balance. Many other metals have 

no biological role (e.g. Ag, Al, Cd, Au, Pb, and Hg) 

are non-essential and potentially toxic to living 

organisms, especially microorganisms (Hussein 

et al. 2005). Heavy metals toxicity leads to 

decreases in biodiversity and productivity 

and thereby, results changes in structure 

and function of ecosystems (Mayor et al. 2013). 

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Nova Biotechnol Chim (2017) 16(1): 48-53 

 

49 

Heavy metals polluted environments are however 

a unique source of poly-extremotolerant 

microorganisms for modern biotechnology 

and bioremediation applications (Pristas et al. 

2015). 

The available physicochemical methods of metal 

removal are not only expensive but also produce 

secondary sludge and are, therefore, not eco-

friendly. Hence, the need to remove hazardous 

trace metals with eco-friendly method is acute 

(Singh et al. 2012). 

The opposite of these physicochemical strategies is 

bioremediation. This process use microbes 

to transform pollutants in environment into less 

toxic substances or immobilization in order 

to reduce their bioavailability. Bioremediation 

gained more attention recently to clean up 

the polluted environment and ensures a safe 

and economical alternative to commonly used 

physicochemical methods (Guo et al. 2010). 

The activity of microorganisms may lead to 

adsorption and precipitation of potentially toxic 

elements by biosorption or bioaccumulation, but 

also to increase their solubility or formation 

of volatile compounds with mechanisms such as 

biovolatilization or bioleaching (Boriová et al. 

2015). 

It is well known that certain species, such as 

Pseudomonas sp., Escherichia sp., Bacillus sp., 

Acinetobacter sp., and Enterobacter sp. can 

effectively remove metals in pure cultures, but 

multispecies consortia can better withstand extreme 

conditions often encountered with industrial waste 

like spikes of pH or high metal concentration. 

They provide a microenvironment, which could be 

very beneficial for metal precipitation. The positive 

interaction between constituent species may also 

facilitate the survival of sensitive strains.  

In view of these facts, the applicability of consortia 

in bioremediation processes, offer promising 

solution for combating heavy-metal pollution in the 

environment (Carpio et al. 2016; Malik, 2004). 

This work investigates influence of heavy metals 

on bacterial growth, metabolic activity 

and compares zinc bioaccumulation  

by pure cultures and microbial consortium  

isolated from heavy metals contaminated  

nickel sludge disposal site. 

Experimental 

 
Sample collection, isolation, growth, 

and identification of bacteria 

 

Sludge samples were obtained from sludge waste 

disposal site of Nickel smelter near Sereď 

(Slovakia). Samples from the surface and 10 cm 

depth at three different locations were aseptically 

collected, transferred to the laboratory  

and stored in the cold (4 °C) prior to 

microbiological analysis. 

To the 0.5 g of solid sample 10 mL of sterile PBS 

solution was added and after 20 min of intensive 

mixing 0.5 mL aliquot was transferred to the liquid 

diluted LB medium (LB medium (Becton 

Dickinson, USA) diluted 1 : 3 with distilled water) 

and cultivated aerobically at room temperature. 

After 24 h of cultivation the bacterial culture  

was transferred to the fresh diluted LB medium 

and after another 24 h of cultivation  

used in subsequent analyses. This mixed  

bacterial culture is referred as consortium 

in further. 

To analyse the composition of consortium, aliquots 

of consortium was spread on non-selective agar 

medium (TSA – Tryptone Soya Agar, Oxoid, USA) 

and cultivated under aerobic conditions 

at laboratory temperature for 48 h. The individual 

colonies were selected on the basis of cell 

and colony morphology and used for further 

analyses. After repeated cultivation and control 

for the purity, the isolates were typed and identified 

by the MALDI TOF MS approach (Kopcakova 

et al. 2014). 

 
The impact of heavy metals on metabolic activity 

of consortium 

 
For evaluation of heavy metal influence 

on metabolic activity of consortium we have used 

BIOLOG EcoPlates™, which contain 31 different 

carbon sources and a blank in triplicate (Insam, 

1997). Each well of the BIOLOG EcoPlate™ was 

inoculated with 100 μL of inoculum (overnight 

consortium culture diluted 1 : 50 in phosphate 

buffered saline, Thermo Fisher Scientific, USA) 

and incubated at 25 °C. Filter sterilized NiCl2 

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Nova Biotechnol Chim (2017) 16(1): 48-53 

 

50 

and CoCl2 were added at appropriate 

concentrations. Absorbance readings were taken 

periodically (after 24, 48, and 144 h) at 590 nm 

with a microplate reader (Synergy 2, BioTek, 

USA). 

The metabolic response (MR) of consortium was 

expressed as average well optical density of all 

carbon sources (MR=Σ ODi/31; Gryta et al. 2014).  

 
Bioaccumulation of zinc 

 
The bioaccumulation of zinc was tested in liquid 

diluted LB medium. Fresh overnight culture 

of consortium or selected isolates was inoculated 

(1 : 100) into diluted LB medium supplemented 

with 10 mM ZnCl2 and cultivated for 18 h at room 

temperature. Following cultivation, the cell 

biomass was removed by centrifugation at 3 000 g, 

the wet weight of biomass was measured 

and residual zinc concentration in the spent liquid 

medium was determined spectrophotometrically 

using dithizone method (Ahmed and Alam, 2003). 

All experiments were done at least in duplicates. 

 
Results and Discussion 
 
Heavy metals pollution is one of the most serious 

environmental issues, nowadays. Heavy metals 

significantly impact soil bacterial community due 

to their toxic effect. To transform pollutants 

into less toxic substances or immobilization 

in order to reduce their bioavailability, 

bioremediation by microorganisms is frequently 

used (Guo et al. 2010). In several countries 

e.g. in USA, 12 % of the in situ projects and 11 % 

of the ex situ projects have used bioremediation 

(US EPA, 2010). The activity of microorganisms 

led to adsorption and precipitation of potentially 

toxic elements by mechanism biosorption 

or bioaccumulation. Bioremediation is frequently 

accelerated through injection of native 

microorganisms into a contaminated area. 

The aim of our study was to analyse the native 

microbiota of nickel sludge disposal site near 

Sereď. This landfill belongs to the most serious 

ecological burdens in Slovakia and is composed 

of nickel sludge with extremely high heavy metal 

content: Fe (50–80 %), Cr2O3 (2.5–3.5 %), SiO2 

(6–8 %), Al2O3 (6–8 %), CaO (2.5–3.5 %), Ni 

(0.17 %), P2O3 (0.6 %) (Michaeli et al. 2012). 

To the best our knowledge of the microbiota 

of the sludge was never analyzed. Recently, 

Remenár et al. (2014) analysed cultivable bacteria 

from nickel and cobalt contaminated agricultural 

soils adjacent to the landfill. 

In our experiment, the bacterial consortium was 

isolated from the mixed sludge samples. 

The consortium showed relatively good growth 

in the presence of several heavy metals (data not 

shown). To test influence of nickel and cobalt 

on metabolic activity of obtained consortium 

BIOLOG EcoPlates™ were used. Both metals 

showed strong inhibition of metabolic activity 

of consortia (Fig. 1). At both concentrations tested, 

cobalt exerted much stronger inhibition effect 

on consortium compared to nickel. There are 

no general rules on the relative toxicity of nickel 

and cobalt because published data are often 

contradictory, even for similar type of microbial 

systems (Gikas 2008). Several authors however, 

reported stronger inhibitory effect of cobalt over 

nickel. Cobalt is more potent inhibitor than nickel 

of the growth of Escherichia coli and Pseudomonas 

putida (Wu et al. 1994). Schmidt and Schlegel 

(1989) who studied the effects of Ni(II) and Co(II) 

on several bacteria isolated from metal 

contaminated soils, have also reported that bacterial 

resistance to Co(II) is lower compared to Ni(II) 

resistance. Arthrobacter butzleri has been reported 

to be more sensitive to Ni(II) than to Co(II) (Otth 

et al. 2005). 

 
Fig. 1. Effect of heavy metals to metabolic activity of isolated 

consortium – expressed as average well colour optical density 

in each microplate. 

 

Microbial consortium obtained was tested  

for the heavy metal bioaccumulation using zinc 

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Nova Biotechnol Chim (2017) 16(1): 48-53 

 

51 

as a model. Upon cultivation in liquid zinc 

supplemented medium, the consortium was able 

to remove of about 15 % of zinc 

and bioaccumulation of zinc reached 0.18 mg/g 

of wet biomass weight (Fig. 2). Observed 

bioaccumulation abilities of isolated consortium are 

not very high and several authors reported much 

higher accumulation rates. Singh et al. (2012) 

reported that microbial consortium isolated 

from coal could efficiently remove over 80 % 

of metals, such as Ni, Zn, Cd, Cu and Cr, while 

the removal of Pb was nearly 45 %. According 

to Carpio et al. (2016) microbial consortium 

isolated from the contaminated water and sediment 

could remove more than 50 % of zinc 

from medium. 

Bacterial consortia isolated from contaminated 

environments are usually genetically complex 

and consist of multiple bacterial taxa (Carpio et al. 

2016) of different physiological and bio-

accumulation properties. 

 
Fig. 2. Comparison of zinc removal from medium 

by bacterial strains and consortium. 

 
The composition of isolated consortium 

was analysed using cultivation approach 

and subsequent identification of isolates 

by MALDI-TOF MS analysis. The analysis showed 

that consortium is composed by gram-positive 

bacteria only and it is dominated by actinobacteria 

of Arthrobacter genus (Fig. 3). Among 22 

randomly selected isolates at least 16 were 

affiliated to this genus. Carpio et al. (2016) 

reported predominance of proteobacteria 

in microbial consortium isolated from 

the contaminated water and sediment. Remenár 

et al. (2014), who analysed microbiota of soils near 

nickel sludge in Sereď, reported the dominance 

of actinobacteria in these soils. According their 

research in nickel-contaminated soils dominate 

genus Streptomyces (18.1 %) followed 

by Arthrobacter (15.2 %), Rubrobacter (5.7 %), 

and Glycomyces (4.8 %) genera. The occurrence 

of Arthrobacter genus in heavy metal contaminated 

soil is not surprising and it was reported  

by several authors (Alvarez et al. 2017; Henne 

et al. 2009).  

The phylum actinobacteria and genus Arthrobacter 

constitute some of the main and most diverse phyla 

within the domain Bacteria. These taxa exhibit 

diverse physiological and metabolic properties, 

such as production of extracellular enzymes  

and the formation of a wide variety of secondary 

metabolites. This versatility in secondary 

metabolite production makes them important tools 

for biotechnological applications such as 

bioremediation (Ludwig et al. 2012). 

The environmental prevalence of Arthrobacter 

may be due to its ability to survive long periods 

under stressful conditions, such as temperature 

shifts, oxygen radicals, and toxic chemicals, 

including heavy metals. Arthrobacter sp. is one 

of the most important genus in relation to tolerance 

to heavy metals and its potential use 

in bioremediation (Alvarez et al. 2017). Based 

on these data several isolates (NH3, NH5, NH8, 

and NH9) identified as Arthrobacter spp. 

By MALDI TOF analysis. No reliable 

identification was obtained for NH1 isolate 

nevertheless the isolate was identified 

as Arthrobacter spp. by 16S rRNA sequencing 

(data not shown). 

The obtained results showed that growth  

in the presence of zinc and bioaccumulation 

abilities vary greatly among isolates.  

All isolates showed lower growth rates compared 

to consortium (Fig. 2) and no clear correlation 

was observed between growth rates and zinc 

bioaccumulation. While for the most isolates 

bioaccumulation ability was lower compared 

to consortium, NH1 isolate was able to eliminate 

the most of zinc from medium, despite the slowest 

growth rate. NH1 isolate is able to take up to 95 % 

of zinc (0.5 mg of zinc per 1 g of wet cell biomass) 

and therefore it is the most appropriate isolate 

for bioremediation use (Fig. 2). 

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Nova Biotechnol Chim (2017) 16(1): 48-53 

 

52 

 
Fig. 3. MALDI TOF based similarity cluster analysis of isolates from indigenous microbiota of nickel sludge disposal site near 

Sered. The MALDI TOF based identification is shown next to the clades. 

 

 

It has been suggested that stimulating the growth 

of indigenous microorganisms with metal 

bioaccumulation capacity may be a useful strategy 

for immobilizing metals in soils and preventing 

contamination of groundwater (Valentine  

et al. 1996). Several studies focused on microbial 

metal bioaccumulation involving multi-  

or single-component systems of pure  

cultures. The advantages of the individual 

components lies in the providing information 

on metal binding characteristics of individual 

organisms, but cannot be directly applied to the 

behaviour of metals in heterogeneous systems. 

Studies focused on the behaviour  

of multi-component systems, like a consortium 

of microorganisms, represent an approach, even  

if still simplified, closer to the real situations 

in which bacteria are found in natural systems 

(Ledin, 2000; Alvarez et al. 2017).  

These advantages mainly consist of synergistic 

interactions among members of the consortium. 

Our data suggest that analysis of contaminated 

environments could lead to the identification 

of bacteria with increased bioaccumulation 

abilities. 

 

Conclusions 
 

Indigenous microbiota of nickel sludge disposal 

site near Sereď have been found to be dominated 

by Arthrobacter spp. The microbiota was analysed 

from the point of metal bioaccumulation 

capabilities and while the highest growth rates  

were observed for mixed bacterial consortium, 

the highest zinc bioaccumulation rates were 

observed for individual strain. Despite extreme 

conditions, heavy metals contaminated sites could 

be source of autochthonous microflora, which 

could be used to concentrate, remove and recover 

metals from contaminated sites and thereby 

enhance the efficiency of treatment processes. 

 

Acknowledgements 
 
This work was in part supported by VEGA 1/0229/17 

and VVGS-2016-275 grants. 

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