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For Review OnlyPHYSICO-CHEMICAL PROPERTIES, HEAVY METALS AND METAL-TOLERANT BACTERIA PROFILES OF ABANDONED GOLD MINE TAILINGS IN KRUGERSDORP, SOUTH AFRICA

Journal: Canadian Journal of Soil Science

Manuscript ID CJSS-2018-0161.R2

Manuscript Type: Article

Date Submitted by the Author: 03-Feb-2020

Complete List of Authors: Fashola, Muibat; Lagos State University, Ojo Lagos, Department of MicrobiologyNgole-Jeme , Veronica ; College of Agriculture and Environmental Sciences, UNISA, Florida, Environmental SciencesBabalola, Olubukola; North West University, Department of Biological Sciences

Keywords: Soil properties, Metal tolerant bacteria, Mine tailings, Tudor shaft

Is the invited manuscript for consideration in a Special

Issue?:Not applicable (regular submission)

https://mc.manuscriptcentral.com/cjss-pubs

Canadian Journal of Soil Science

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CJSS-2018-0161

TITLE PAGE

PHYSICO-CHEMICAL PROPERTIES, HEAVY METALS AND METAL-TOLERANT

BACTERIA PROFILES OF ABANDONED GOLD MINE TAILINGS IN KRUGERSDORP,

SOUTH AFRICA

Muibat Omotola Fashola 1, 2, Veronica Mpode Ngole-Jeme3 and Olubukola Oluranti Babalola 1

1. Food Security and Safety Niche Area, Faculty of Agriculture, Science and Technology, North-West

University, Private Bag X2046, Mmabatho 2735, South Africa; [email protected]

(O.O.B.)

2. Department of Microbiology, Faculty of Science, Lagos State University, Ojo, Lagos, Nigeria

[email protected] (M.O.F.)

3. Department of Environmental Sciences, College of Agriculture and Environmental Sciences,

UNISA, Florida, Private Bag X6 Florida, Roodepoort 1710, South Africa;

[email protected]

Correspondence: [email protected]; Tel.: +27183892568

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ABSTRACT

Mine tailings are a potential source of heavy metals that can be toxic to microbes, plants and animals in

aquatic and terrestrial ecosystems. Bacteria have evolved several mechanisms to tolerate the uptake of

heavy metal ions. This study aimed to assess the physicochemical properties, concentrations of selected

heavy metals and metalloids (As, Ni, Pb, Zn, Cd, and Co) and isolate potential metal tolerant bacteria

present at three abandoned gold mining sites with a view of understanding how tailings characteristics

vary and the implications on microbial activities in tailings dumps. Heavy metal tolerant bacteria were

isolated from the samples using minimum inhibitory and maximum tolerable concentrations of the Ni,

Pb, Zn, Cd, and Co. The substrates of the studied sites were acidic and deficient in nutrients. High

metals and metalloid concentrations in the order Zn > Ni > Co >As > Pb > Cd were recorded in some of

the studied sites and its adjacent soil which exceeded South African recommended values for soil and

sediments. Heavy metal tolerant bacteria that showed multiple tolerances to Ni, Pb and Zn were

isolated and putatively identified using biochemical tests as belonging to the phylum Proteobacteria,

Actinobacteria, and Firmicute. Gold mine tailings enriched the soil with heavy metals and also affects

soil physicochemical properties. Proper management of mine wastes must be ensured to prevent their

adverse effects on the diversity, composition and activity of soil microorganisms that help in

maintenance of the ecosystem.

Keywords: Soil properties, Metal tolerant bacteria, Mine tailings, Tudor shaft

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INTRODUCTION

Heavy metal (HM) and metalloid contamination associated with gold mining is one of the foremost

environmental concerns in areas where such mines are located. Heavy metals are naturally occurring

metals having an atomic number greater than 20 with a density greater than 6 g cm-3 and a molecular

weight greater than 53 (Howlett and Avery, 1997; Ali and Khan, 2017). Metalloids on the other hand

are chemical elements which in their standard state, have (a) the electronic band structure of a

semiconductor or a semimetal, (b) an intermediate first ionization potential (750−1,000 kJ/mol), and (c)

an intermediate electronegativity (1.9−2.2) (Vernon, 2013). The metalloid arsenic (As) and heavy

metals cadmium (Cd), cobalt (Co), copper (Cu), manganese (Mn), mercury (Hg), nickel (Ni), uranium

(U), and zinc (Zn) have been reported in soils around gold mines in different parts of the globe (Lee et

al., 2005; Jian-Min et al.; 2007; Abdul-Wahab and Marikar, 2012; Armah et al., 2014). These elements

persist for long periods in both aquatic and terrestrial ecosystems and adverse effects can span across

trophic webs from soil through plants to animals including humans (Chary et al., 2008). Elevated levels

of HM in soils may lead to their uptake by plants which can reduce crop productivity as a result of their

inhibitory effects on multiple physiological processes in plants (Singh and Kalamdhad, 2011).

Contamination of aquatic environments with HM has also been shown to alter food webs and reduce

diversity in aquatic ecosystems (Förstner and Wittmann, 2012). Human health effects associated with

HM ingestion (primarily via contaminated aquatic biota) include complications of the nervous, skeletal

endocrine and digestive systems (Fashola et al., 2016).

In addition to enriching the soil with HM, mining activities and mine waste generation also can disrupt

nutrient dynamics in soils as a result of dynamic and interaction alterations in physical, chemical and

microbiological processes (Adewole and Adesina, 2011). Numerous studies have emphasized the

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significance of soil properties including organic matter (OM), particle size distribution including clay

content, redox potential, electrical conductivity (EC), moisture content, cation exchange capacity

(CEC), and pH on the behavior of HM in soils (Sourkova et al., 2005; Keskin and Makineci, 2009).

Metal mobility has been shown to be lower in fine compared to coarse textured soils especially if the

mineralogical assemblage of the clayey soil is dominated by 2:1 tetrahedral: octahedral silicate clay

minerals (e.g illite or vermiculite with high cation exchange capacities). A high content of OM also can

enhance metal adsorption, thereby reducing mobility in the environment whereas acidic conditions

decrease soil exchange capacities of metal cations and increases metal solubility in the soil environment

making them more mobile (Sheoran and Choudhary, 2010 ; Ayangbenro and Babalola, 2017).

Acid mine drainage originating from abandoned gold mines has resulted in acidification of nearby soils

with consequences for the mobility of HM and microbial diversity in the soil (Fashola et al., 2016).

Zinc solubility in soil has been reported to increase 100-fold for every unit decrease in soil pH

(Mortvedt et al., 1991). Chuan et al. (1996), also reported high solubility of Pb, Cd and Zn in

contaminated soils as the pH decreased from 5.0 to 3.3. Changes in soil pH have been known to disrupt

certain microbial metabolic pathways via inhibiting activities of pH-dependent enzymes or altering the

availability of key nutrients and HM, the latter which can be toxic to soil bacteria (Kapoor et al., 2015;

Ndeddy Aka and Babalola, 2017). Changes in soil microbial community structure and activities as a

result of mining related changes in soil physico-chemistry could affect such key ecosystem processes as

soil organic matter turn over leading to a decline in overall ecosystem functioning and may also lead to

indirect cascading impacts on metal mobility.

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Witwatersrand in South Africa is home to the largest known gold reserves in the world, making the

country one of the world leaders in gold mining (Hart, 2014). However, gold mining has also resulted

in the generation of thousands of tailings dumps along the gold mining corridor from Nigel to

Randfontein (Bobbins, 2015). Gold mine tailings present great risk to soils, plants, surface and

groundwater because of the dissemination of particles containing potentially toxic metals and

metalloids through wind action and/or by runoff from the tailings to streams that drain these tailings

(Naicker et al., 2003; Ayangbenro and Babalola, 2018). In many residential areas near abandoned

mines in Krugersdorp, dust from tailings has been a long-term environmental hazard (Bobbins, 2015).

Multiple studies have reported high concentrations of toxic metals and radioactive elements in different

gold mine tailing dumps and adjacent water resources in this area (McCarthy, 2011; Kamunda et al.,

2016; Olobatoke and Mathuthu, 2016; Ngole-Jeme and Fantke, 2017) as well as in many other gold

mines tailings and surrounding soils in other areas in South Africa (Mitileni et al., 2011; Matshusa et

al., 2012) and beyond (Rafiei et al., 2010; Bempah et al., 2013).

An understanding of the soil/substrate physicochemical characteristics and microorganisms around gold

mines may be important to inform remediation strategies aimed at reducing HM concentrations or

bioavailability (Fashola et al., 2016). In the present study, we investigated physicochemical properties

and six HM (Pb, Ni, Zn, Cr, Cd, Co) in abandoned gold mine tailings dams and adjacent soils in

Krugersdorp, South Africa. Additionally, we enriched for and isolated HM tolerance bacteria on

modified L-B medium from these substrates to help determine how the selected physicochemical

properties may impact on the activities of microorganisms in the tailings and the implications of these

impacts on potential bioremediation strategies.

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DESCRIPTION OF STUDY AREA

The study area was Krugersdorp (260 61S and 270 461E), a mining town in Gauteng province, South

Africa. This province houses the Witwatersrand basin which covers an area of 1600 km2 and has some

400 km2 of mine tailings dams and 6 billion tons of pyrite tailings containing 430,000 tons low-grade

uranium (CSIR., 2009).The soils are dominated by plinthic, duplex and hydromorphic soils, which all

carry land and soil limitations for agricultural crop production (Bredenkamp, 2002). The vegetation

around the study area is made up of grassland and savanna biomes comprising 71% and 29%

respectively of Gauteng area.

MATERIALS AND METHODS

Sample collection and preparation

Tailings and soil samples were collected from three abandoned gold mine sites in Krugersdorp at

depths of 0-15 cm with a Dormer steel soil auger. The three sites as indicated in Figure 1 were MA

(27.80764 E, -26.14265 S), MB (27.81576, -26.12771) and TS (27.80362, -26.13191). Samples

collection was done once every two months between February and September, spanning over a time

period of eight (8) months to accommodate the different seasons in South Africa. From each of the

three sites, three tailings and three soil samples were collected a few meters apart and combined to

obtain a composite sample representative of the site. During each sampling period, samples were

collected at different sites of the three tailings dumps. The samples were air-dried, crushed, and sieved

through a 2 mm sieve and the sieved soils used for the analyses of selected physical and chemical

parameters.

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Samples characterization

Physicochemical characterization of samples.

The moisture content of samples was determined using the gravimetric method described by Odu et al.

(1986) whereas particle size distribution of the < 2 mm fraction of the samples was determined using

the Bouyoucos hydrometer method (Gee et al., 1986; Filgueira et al., 2006) after dispersing the

particles with sodium hexametaphosphate, (Na6(PO3)6). A Jenway 3520 - pH meter was used to

determine sample pH, in a 1:2.5 (m: v) soil-water suspension (Van Reewijk, 1992). Sample EC was

determined in the same suspension using a Hanna multi range HI8733 EC meter (Thomas, 1996). The

CEC and OM content of the soil samples were determined using the ammonium acetate extraction

technique and the modified Walkley-Black methods respectively (Abollino et al., 2002).

Chemical characterization of samples.

Sample chemical properties determined included sulphate, carbon, nitrogen, sulphur and HM contents.

To determine sulphate content, samples were extracted with a solution comprising 39 g NH4OAC in 1

liter of 0.25 M acetic acid on a shaker at 200 rpm for 30 mins after which 0.25 g of activated charcoal

was added. The mixture was again shaken for an additional 3 mins and filtered through a sulphate free

filter paper (Whatman no 42) which had been washed with the extracting solution. Ten milliliters of the

filtrate were then pipetted into a 50 ml Erlenmeyer flask and 1 ml of acid seed (6 M HCl + 20 mg of

K2SO4 and 50 ml of 40 mg standard solution plus 50 ml of concentrated HCl) solution was added. The

solution was swirled, 0.5 g of Bacl2.H20 crystals added, and allowed to rest for another minute with

frequent swirling to dissolve the crystals. Sulphate content in the resulting solution was determined

using a UV spectrophotometer at a wavelength of 420 nm (Singh et al., 1995). Nitrate content of the

samples was determined using the equilibrium extraction method described by Willis and Gentry

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(1987) using a spectrophotometer automated flow analyzer. The content of total C, N, and S in each

sample was determined using a LECO CNS Trumac Analyzer calibrated with a LECO soil reference

standard (CNS LECO part no 502-309). The instruments settings and operations conditions were done

in accordance with the manufacturer’s specifications. Conventional aqua regia (3:1 HCl: HNO3)

digestion technique was performed according to the method of Chen and Ma (2001), and Ngole and

Ekosse (2012) to extract metalloid and HM including As, Pb, Ni, Zn, Cr, Co from the soil samples.

Heavy metal and metalloid concentrations in the sample extracts were determined using a contraAA

300 Atomic Absorption Spectrometer.

Isolation and identification of heavy metal Tolerant bacterial strains.

Metal tolerant bacteria were isolated from the samples using the spread plate method as follows. One

gram of duplicate composite tailings and soil samples was suspended in 9 mL of saline solution (8.5 g/

L of NaCl) in distilled water and vortexed for 1-2 mins at room temperature. Each suspension was

serially diluted (10-1 to 10-7) and aliquots of 0.1 ml dilution from 10-4, 10-5 and 10-6 spread with a glass

rod over triplicate Luria–Bertani (LB) medium supplemented with 1 mM of ZnS04, NiCl2·H20, Pb

(N03)2, CrCl2, CdCl2·2H20 and 0.5 mM CoCl2·6H20 (Van Nostrand et al., 2007). To minimize metal

ion complexation, the medium was adjusted to pH 7 using 1.0 N NaOH or 1.0 N HCl as was necessary.

Control plates were also prepared with the same medium but with no HM supplement added. Both the

control and experimental plates were incubated at 37°C and 25°C for 24 - 48 h. Growth of bacteria was

observed and morphologically distinct colonies were selected and purified in the same medium and

growth conditions (Raja and Selvam, 2009). All cultures were stored at -80°C in LB broth with 20%

glycerol for further studies. To identify the isolates, the following tests were performed;

Gram staining was done to classify the bacteria into Gram positive and Gram-negative bacteria based

on their cell wall composition. Biochemical tests were performed to determine the metabolic diversity

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of the bacteria present. Indole, Methyl red, Voges-Proskauer, and citrate utilization test were done to

identify the coliform group that produces carbinol from glucose fermentation. Catalase, oxidase, nitrate

reduction, hydrogen sulphide production, starch hydrolysis was done to determine other intracellular

enzymes of the isolates while the sugar (glucose, lactose, sucrose and maltose etc) utilization tests were

performed to show the ability of the bacteria to ferment carbohydrates with the release of acid and gas.

The various tests were carried out as described by Cowan and Steel (Barrow and Feltham, 2004) and

the bacterial isolates were identified accordingly using this manual.

Determination of multiple metal tolerances levels of isolates.

The Maximum tolerable concentration (MTC) of Pb, Cr, Zn, Ni, Cd and Co by the identified bacteria

was determined by agar dilution method (Kannan and Krishnamoorthy, 2006). A log-phase culture of

the isolates was spot inoculated onto LB agar plates supplemented with increasing concentration of

metals. The concentrations in mM of the metal species were as indicated below:

Pb2+: 1, 2, 3, 4, 5, 6, 7, 8, 9

Ni2+: 1, 2, 3, 4, 5

Zn2+ :1,2, 3, 4, 5, 6, 7, 8, 9

Co2+ : 0.5, 1, 2

Cd2+ :1, 2, 3

Cr2+: : 1, 2, 3, 4, 5, 6, 7, 8, 9, 10

All metal salts used were added to the LB agar after autoclaving and cooling to 50°C from filter-

sterilized stock solutions. The plates were incubated at 37°C and 25°C for 24 - 48 h and observed for

bacterial growth. The highest concentration of the metal at which no bacteria growth was seen was

designated as the MTC.

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Quality control

All equipment used were calibrated with reference standards. Glass wares used for HM analyses were

rinsed in dilute HNO3 before used. Heavy metals used in bacteria isolation were filtered and sterilized

using membrane filters. All reagents and HM metal standards used were of analytical grade. Analyses

were carried out in duplicate to ensure reproducibility of the data obtained.

Statistical analysis

ANOVA was used to determine differences in the properties of the tailings and adjacent soil at the

different sites whereas correlation coefficient and regression analyses were used to understand any

relationship that existed between heavy metals and the physicochemical properties in the tailings and

adjacent soils and to determine the influence of soil physicochemical properties on HM behavior and

bacterial diversity. All tests were performed at a significance level of 5% using the SPSS statistical

package programme (version 21).

RESULTS AND DISCUSSION

Physicochemical properties of tailings and adjacent soils

Tailing moisture contents ranged between 7.23% - 12.07% with a mean of 6.61% whereas the moisture

content of adjacent soils ranged between 3.39% - 6.56% with a mean of 4.22%. Moisture content was

therefore higher in the tailings compared to the adjacent soils at all sites (Table 1). Moisture content in

mine soil is however not a stable parameter as it is affected by sampling period, organic carbon content,

height of dump, stone content, soil texture and thickness of litter layers on the dump surface (Maiti,

2006).The pattern observed may therefore change depending on the prevailing condition. The particle

size distribution of the samples ranged between 16-28% for clay, 12-34% for silt and 28-64% for sand.

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Clay contents in the adjacent soil samples were higher than that in the tailings (Table 1). Both the

tailings and adjacent soil samples had significant amount of clay-sized particles.The textural

classifications of the samples were sandy loam, silt loam, and sandy clay loam (Table 1). The host

rocks from which the Au is extracted is usually crushed to a fine texture to increase the surface area of

the ore body exposed for chemical extraction. This could explain the finer texture for the tailings

compared to the soil. Most of the tailings and soil samples analyzed over the eight-month period were

acidic to moderately acidic (Table 1) with the pH values ranging from 2.17-6.34 and mean of 4.48. All

tailings samples had lower pH values compared to the adjacent soils. The acidic values recorded in the

tailing soils may be due to oxidation of sulphide bearing minerals contained in the tailings, which

resulted in low pH (Dold, 2014).Generally,redox level varied between 194 - 380 mV, with highest level

observed in tailings samples. Soils around the tailings from all three sites had lower redox values than

the tailings (Table 1). Values for EC at the three sites varied with values ranging from 5.33 mS/m to

11.52 mS/m in the tailing’s samples and 0.49 mS/m to 3.39 mS/m in the adjacent soil samples (Table

1). The cation exchange capacities of the tailings varied from one site to the other (Table 1) with the

adjacent soils having higher CEC values compared to the tailing’s samples. The CEC values were

generally low reflecting the dominance of 1:1 clay mineralogy and further confirming that the fineness

of the tailings was not as a result of geogenic processes which would have resulted in the accumulation

of clay minerals that contribute significantly to soil CEC, but the physical process of grinding. Organic

matter also affects soil CEC with CEC generally increasing proportionately with OM content (Ngole-

Jeme, 2016), but the results in this study indicated low OM content (Table 1) as reflected by the mean

OM content of 0.89% in the tailings and 1.13 % in the adjacent soils also partially explaining the low

CEC. Soil organic matter in mine tailings has been reported to be low due to low vegetation cover and

bacterial diversity which play a major role in organic matter accumulation in soils and sediments

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(Sadhu et al., 2012). Our results are consistent with the findings of Rösner and Van Schalkwyk (2000);

Yang et al. (2003) and Ashraf et al. (2012) who all reported that mine associated soils generally were

low in organic matter.

Nitrate levels were mostly higher in the soils compared to the tailings samples (Table 1). Sulphate

content in samples from the tailings and adjacent soils followed the order TS > MA > MB >TSC >

MAC > MBC with values from site TS being above the permissible limit of 200 mg/ml stipulated by

WHO in soils. Sulphate content was higher in the tailings than in the adjacent soil samples. High

sulphate content in the tailings could be indicative of the presence of sulphur containing minerals such

as pyrite, and chalcopyrite in the Au host rock (Bosman, 2009). According to Rosner (2007), a total

sulphur content of approximately 0.1% within the first meter below the surface of tailings indicates that

the tailings have almost fully oxidized. The values obtained for this study indicate that the tailings have

not been fully oxidized and may still present a risk of AMD generation. Results in Figure 2 showed that

mean total carbon (0.48%) and nitrogen (0.13 %) content in the tailings samples were lower than in the

adjacent soils (1.74% for C and 0.15% for N). The samples therefore had a low C: N ratio of 1: 0.086:

1:0.027. Nitrogen is generally known to be deficient in mine dumps (Sheoran and Choudhary, 2010),

which was observed in both the tailings and adjacent soil samples. This is as a result of decrease in

microbial population and unfavorable conditions for maintenance of soil vegetation cover and

formation of soil humus in the mining environment. The nitrogen content recorded in this study also

corroborates the values reported by Saviour and Stalin (2012), who also noted that the inadequate

mineralizable organic nitrogen and reduced mineralization rates of mine dumps lower nitrogen

contents. The results of the physicochemical properties of the tailings indicate the variations that may

exist in gold mine tailings and adjacent soils. These variations may be influenced by the nature of the

host rock at the site as well as the gold mining process utilized at the mine. It also generally shows that

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tailings have a significant influence on the physico-chemical properties of adjacent soils as shown in

the pattern of values for the different parameters at the different sites.

Heavy metals concentrations in tailings and adjacent soils

The concentrations of As, Cd, Co, Ni, Pb and Zn recorded in the tailings and adjacent soils after acid

digestion are shown in Figure 3. Arsenic content in tailings MA and MB and the adjacent soils MAC

and MBC were below the detection limit of the equipment. All metal concentrations excluding Cd in

tailings from TS and its adjacent soil TSC exceeded the recommended values of different standard

bodies as shown in Table 2. Cadmium concentration in site TS had values which fell within the

permissible levels recommended by South Africa standards for soils and sediment quality guidelines

but above Dutch pollutant standard. Highest concentration of all the metals in tailings and adjacent soils

were recorded in site TS and TSC (Figure 3 and Table 2). The concentration of Cd, Co and Ni found in

tailings from sites MA and MB fall within the acceptable limits recommended by several countries

(Table 2). The maximum and mean values of Pb and Zn (Figure 3) recorded in MAC and MBC

exceeded the recommended values stipulated by South Africa standards for soils and sediment quality

guidelines, Dutch pollutant standard and Canadian soil quality guidelines for the protection of human

and environmental health (Table 2).

Among all the samples analyzed, samples from sites TS and TSC had the highest values for HM

concentrations, with Zn having the highest concentrations among the six metals studied (Figure 3).The

concentrations of the elements are typical of what has been reported in gold mine tailings in other

studies and also highlight the variability that exist in the properties of mine tailings soil. The

concentration values for most of the metals were higher in the adjacent soil compared with the tailings.

This could be attributed to the properties of the soils relative to the tailings. Lower CEC values for the

tailings imply the cations are likely to be leached into adjacent soil where they may accumulate because

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they have a higher OM and CEC. All the metals except Pb were highly positively correlated (P ≤ 0.01

and P ≤ 0.05) with r values of 0.432 for Cd and As, 0.534 for Co and As, 0.592 for Ni and As, 0.582 for

As and Zn, 0.861 for Co and Cd, 0.863 for Ni and Cd, 0.848 for Zn and Cd, 0.964 for Co and Ni, 0.974

for Co and Zn and 0.976 for Ni and Zn indicating a common source for these HM. Lack of correlation

with Pb could be an indication that the Pb contained in the samples could be from other anthropogenic

inputs aside from gold mining activities. Gold mining activities in the studied areas has resulted in

deterioration of the surrounding environment through contamination of HM and metalloids in the soil.

This observation agrees with several studies conducted on gold mine tailings dams as potential source

of HM contamination in adjoining soils and sediments in South Africa (Bempah et al., 2013; Olobatoke

and Mathuthu, 2016).

Metal tolerant bacterial strains identified in the tailings and adjacent soils

Metal tolerant bacteria are usually found in sites polluted by high concentrations of metal species (Wei

et al., 2009 ; Xie et al., 2016). A total of 117 metal tolerant bacteria species were isolated from both the

tailings and the adjacent soils studied using 1 mM concentrations each of Pb, Zn, Ni, Co, Cr and Cd

(Table 3). There seem to be more metal tolerant isolates in the tailings compared to the adjacent soils

for two of the three sites studied. After screening using colonial and morphological characteristics;

thirty-five isolates showed distinct colony characteristics and were selected and putatively identified

using biochemical tests (Table 4 and 5). The various colonial and cellular morphology of the bacterial

isolates are shown in Figure 3. Out of the 35 isolates selected, 31.43% were Gram negative while the

remaining 68.57% were Gram positive. The biochemical results of the selected isolates (Table 4)

showed the effects of the HM on the various biochemical properties determined. Over sixty-eight

(68.57%) of the isolates were urease negative while 57.14% were oxidase positive and 97.14% had

catalase enzyme. Most of the sugars were also utilized by the isolates. Similar observations were made

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by Oste et al.(2001),who reported a decrease in the activities of various soil enzymes such as urease due

to metal pollution. The ability of the bacterial strains to utilize the sugars signifies aerobic metabolic

pathways (Samanta et al., 2012). The sugars are converted into pyruvate that are decarboxylated and

oxidized to become acetyl Co A which condenses with oxaloacetate to enter the TCA cycle. Also, the

utilization of the different carbon sources by the identified bacteria indicated diversity of metabolic

patterns that could enhance their degradative ability. The results of the biochemical tests (Table 4)

putatively revealed that the bacterial isolates belong to three phyla: Actinobacteria, Proteobacteria and

firmicutes (Table 5).

Inability to isolate the acidophilic heterotrophic iron and sulphur oxidizing bacteria such as

Acidiphilium, Acidithiobacillus ferrooxidans, Leptospirillum ferrooxidans, and some other iron

oxidizing bacteria that are known to only grow and proliferate in acidic pH 2.4 -5.0 in mine tailings

might have been due to the neutral pH of the culture medium used in enriching and isolating the

bacteria.

Previous researches have shown the abundance of these phyla in HM and metalloid impacted

environments (Jamaluddin et al., 2012; Bajkic et al., 2013; Hookoom and Puchooa, 2013). Their ability

to survive in these environments has been attributed to the composition of their cell walls that are able

to interact and bind effectively with the metals (Shin et al., 2012) as well as various genetic

mechanisms that enable them to overcome the effects of the toxic metals (Rensing and Grass, 2003;

Abou-Shanab et al., 2007; Dupont et al., 2011). Ability of a native bacteria to tolerate HM stress is a

major determinant in the utilization of such bacteria as a bioremediating agent. There are various

reports on different bioremediation strategies employed by indigenous bacteria isolated from mine

tailings. Autochthonous Bacillus sp isolated from mine tailing in South Korea with the ability to

effectively biomineralize active Pb ions to inactive form has been reported by Govarthanan et al.

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(2013). This bacterium also increases the urease enzyme activity of the tailings as well as significantly

reduced the bioavailability of Pb in the environment. Furthermore, Ndeddy Aka and Babalola (2016),

described the enhancement of phytoextraction capacity of B.juncea by three indigenous HM resistant

bacteria; Pseudomonas aeruginosa, Alcaligenes faecalis and Bacillus subtilis isolated from mine

tailings in South Africa. It was observed that the inoculation of the bacteria with B. Juncea, an

hyperaccumulator plant grown in soil spiked with different concentrations of Ni, Cd and Cr resulted in

higher accumulation of the three metals compared with the uninoculated plant. This shows that

indigenous HM tolerant bacteria could bring about revegetation of mine tailings and surrounding

polluted sites.

Determination of multiple metal tolerances

The isolates were screened against six metals (Co2+, Ni2+ Pb2+ Cd2+ Zn2+ Cr2+) to examine the extent of

multiple tolerances. All the isolates showed the ability to tolerate multiple metals found in the tailings

and surrounding soils. All the isolates could not grow beyond 1 mM concentration of Co. The pattern of

toxicity of the metals to the bacterial isolates was Co > Cd > Ni > Pb > Zn > Cr, which shows that Co is

the most toxic to the bacterial isolates out of the six metal species as shown in Table 6. The isolates

showed a high level of tolerance for Cr with 51.42% of the isolates tolerating 9 mM and 17.14%

tolerating 10 mM of Cr. 25.71% of the bacterial isolates tolerated up to 7 mM of Zn, 11.42% up to 7

mM of Pb and 17.14% tolerated 5 mM of Ni (Table 6).

The high level and widespread tolerance shown by the bacterial isolates against multiple metals (Table

6) could be attributed to the elevated concentrations of the metals recorded in the different samples

where the bacteria were isolated from. Bacteria when exposed to high concentrations of HM devise

various physiological and genetic mechanisms needed for their adaptation and survival under such

conditions (Fashola et al., 2016). Fashola et al. (2019), have also highlighted that exposure of bacteria

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to high concentrations of heavy metals extends the lag phase of their life cycle resulting in increased

survival rate. In addition, the efflux system which enables bacteria to pump out metals from the

cytoplasm to the periplasmic space with the help of ATPase found in the internal membrane of the

bacteria has been reported in Gram-negative bacteria (Blair et al., 2014). Other mechanisms such as

complexation, precipitation, biotransformation, bioaccumulation, oxidation-reduction reactions and

biosorption have also been reported to be responsible for bacterial tolerance to metals (Wei et al., 2009;

Sahmoune and Louhab, 2010 ; Govarthanan et al., 2013).

The MTC values recorded for Ni, Pb and Zn by the bacterial isolates obtained in this study are however

much higher than those reported by Choudhary and Sar (2009) and Govarthanan et al. (2013) in mine

polluted tailings and soil. The Ni tolerance level recorded is lower than the values reported by Bajkic et

al. (2013), where Ni tolerance as high as 11 mM was attained by the bacteria strain MS108 identified as

Staphylococcus sp. from a copper mining and smelting complex in Serbia. The differences in metal

tolerance by the bacterial isolates could be attributed to many factors such as the strength of the

medium used in isolation, presence of negatively charged ions like chloride, organic constituents, and

nature of the medium which determines availability of the metals to the bacteria (Kannan and

Krishnamoorthy, 2006). The multiple metals tolerant patterns observed by the bacterial isolates could

be attributed to the fact that, mine tailings are usually contaminated with a cocktail of metals, and

hence, bacteria surviving in mine tailings are likely to possess the ability to withstand high

concentrations of different metals. For example, Staphylococcus sp isolated from copper mining soil

was able to tolerate Cr, Ni and Cd (Bajkic et al., 2013). Similarly, the bacterial strain CCNWRS33-2

isolated from Lespedeza cuneate in gold mine tailings also showed high tolerance to Cu, Cd, Pb and Zn

(Wei et al., 2009). The present study also shows high tolerance, as well as tolerance to multiple metals

by bacterial isolates.The multiple heavy metal tolerance exhibited by the isolates could be used to bio

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augment environments contaminated with a cocktail of metals to improve the rate of natural

attenuation.

Implications of the physicochemical properties of soils and tailings on HM behavior and effects on

the adjacent environment

Some very strong negative and positive correlations were observed between HM and analyzed

parameters including pH, EC, and nitrates in the adjacent soils (Table 7). These correlations were none

existent in the tailings samples (Table 8). In both the soil and tailings samples, there were significant

correlations among the heavy metals (Table 7 and 8). Soil properties and heavy metals concentrations

in uncontaminated soils display natural associations because of the influence of these properties on

heavy metal behavior in the soils. Lack of correlation between soil properties and the heavy metals in

the tailings is an indication that these have been affected by anthropogenic activities, which in this case

may be the process of mining. There is strong correlation between the heavy metals in the tailings

compared to the adjacent soil indicating that these are possibly from the same source. Worthy of note is

the lack of correlation between the heavy metals studied in the adjacent soils and Pb. This may indicate

that the origin of Pb in the soils is not the tailings but some other activity.

The fate and activities of HM in soils and sediments are influenced by the physicochemical properties

of the soils which dictate their mobility and bioavailability (Osakwe, 2010; Ngole-Jeme, 2016). Particle

size distribution, pH, CEC, redox conditions, Sulphur (sulphide and sulphate content) and organic

matter contents are known to be the major soil properties affecting HM behavior in mining

environment. The acidic pH of the tailings and soil samples indicates that the metals and metalloids in

the samples around the different sites will be labile as HM mobility increases under acidic conditions.

The mobility of these contaminants would also be enhanced by the low organic matter content recorded

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in both the tailings and adjacent soils, as organic matter contains various functional groups which

improve the capacity of soil to bind metal ions (Hernandez-Soriano and Jimenez-Lopez, 2012). The

fine texture of the exposed tailings will increase their erodibility by wind which would result in their

dispersal to surrounding environments. The fine texture of the tailings is therefore a contributory factor

to the spatial extent of HM contamination around mining environments. The presence of the sulphide of

most metals are precipitated over a wide pH range whereas sulphates increases acidity in soil which

mobilizes HM. The effect of sulphur on the behavior of HM may therefore depend on whether the

reduced form of sulphur (sulphide) or the oxidized form (sulphate) is present.

The implication of the observed associations could be felt in various environmental receptors. Metals

associated with mines located in this area have been reported to travel hundreds of kilometres and

impact downstream ecosystems (Olobatoke and Mathuthu, 2016). Considering that these particles could

be laden with HM, they could be a source of human exposure to HM. The health risk associated with

incidental inhalation and ingestion of HM contaminated tailings particles have been highlighted by

Ngole-Jeme and Fantke (2017). High incidence of dust related diseases has been reported among the

residents living around the mine dumps and this problem continue to increase as a result of increased

re-mining of the tailing dams due to recent high price of gold and development of new extraction

technology (Cairncross,2013). The particle size of the tailings as determined in this study will pose a

significant health problem. A number of potentially hazardous substances are usually found in the dust

particles, some of which are more soluble in human physiological fluids and available for absorption by

the body. These particles when inhaled are usually deposited in the respiratory system which can results

in a series of respiratory disorders.

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High soil organic matter and clay content increase cation adsorption in the soil and consequently the

CEC of the soil. The tailings and the surrounding soils samples however had very low CEC which

would further enhance cation mobility in the soil environment. The various values recorded for redox in

the tailings and surrounding soils showed that all the samples examined had moderately reduced redox

conditions (Seo and DeLaune, 2010). This is as a result of oxygen consumption by lithotrophic iron

reducers present in the samples to enable them use the metals for their growth and metabolic activities.

This would have resulted in the lowering of the redox potential of the samples (Chuan et al., 1996;

Bohrerova, 2004).

Implications of physicochemical properties of the tailings, soils and HM contents on bacterial

activities and diversities

As a result of the low pH and high metal concentrations recorded in the tailings and adjacent soil

samples, the metallophilic and acidophilic sulphur and iron oxidizing bacteria such as the

Acidithiobacillus spp, Acidiphillum spp are the predominant bacteria that can survive in these tailings

and the adjacent soil samples. This has also been explained by Natarajan, (2008), and Fashola et al.,

(2015). Several other bacteria such as Bacillus spp, Arthrobacter spp, Pseudomonas spp,

Achromobacter spp, Streptomyces spp and many others with ability to thrive in the acidic and high

metals conditions have also been identified (Jamaluddin et al., 2012; El Baz et al., 2015; Ndeddy Aka

and Babalola, 2017). The low content of organic matter in both the tailings and adjacent soils will result

in reduction of bacterial biomass and extractable carbon as well as bacterial community structure and

biodiversity. Similar observations have been made by Šourková et al., (2005), and Laudicina et al.,

(2015). But the low C:N ratio will help the bacteria present to synthesize their proteins.Insufficient

moisture content will lower microbial activity as microbes require adequate moisture for their growth

and metabolic activities. Redox potential is also an important soil property that affects all biochemical

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reactions and enzymatic activity of the bacteria cells (Husson, 2013). It greatly affects availability of

substrate and energy transformation which play a vital role in regulating soil microbial abundance,

diversity and community structure (Song et al., 2008). The moderately reducing conditions observed in

the tailings and surrounding soils will limit the bacterial community to those that can survive the

moderately reduced redox conditions.

The characteristics of the tailings and adjacent soil present potential challenges for the establishment of

a healthy soil bacterial community. There are likely important dynamic feedbacks between the tailings

and soil bacteria and plant establishment and broader ecosystem restoration. Understanding what

stimulates soil-like microbial communities could help spur this restoration.

CONCLUSION

Gold mining represent one of the major anthropogenic activities that leads to the release of HM into the

broader environment. Physicochemical properties of the mine waste sites and the adjacent soils pose

substantial challenges to reclamation. However, HM resistant bacteria as we have isolated from the

mine wastes could be explored as a potential future in-situ inoculant to reduce the bioavailability and

mobility of HM and spur plant growth and shifts towards more healthy soil-like communities

dominated by heterotrophs. Next-steps in this regionally and globally important African mining region

could build on the current work using molecular methods to better characterize community structure

and network interactions by determining mechanisms of bacterial resistant to the HM, by determining

optimal environmental conditions needed for inoculate utilization in removing HM from these and

similar polluted environments.

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Acknowledgments: Fashola Muibat Omotola acknowledges the sponsorship of Tertiary Education

Trust fund (TETFUND) and the scholarship of North West University, Mafikeng. OOB also gratefully

acknowledges NRF for grant (UID81192) that supports work in her laboratory.

Author Contributions: Muibat Omotola Fashola, Veronica Mpode Ngole-Jeme and Olubukola

Oluranti were involved in samples collection, wet laboratory analyses, and drafting and finalization of

the manuscript for publication.

Conflict of interest: There is no competing interest in relation to this manuscript.

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Figure 1: Map showing the sampling locations

Figure was created using Arc Map version 10.2 and assembled from the following data sources: Data

points (from field trip excel sheet), Contour lines (WR2000), Shape files (WR2000). Base map from

WR2000, courtesy of WRC South Africa’’. The study area was Krugersdorp (26061S and 270 461E), a

mining town in Gauteng province, South Africa. The three sites as indicated in Figure 1 were MA

(27.80764 E, -26.14265 S), MB (27.81576, -26.12771) and TS (27.80362, -26.13191).

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MA MAC MB MBC TS TSC0

0.5

1

1.5

2

2.5

3

N

C

S

key

Sampling sites

% o

f nut

rient

s

Figure 2: Average concentration of total nitrogen (N), carbon (C) and Sulphur (S) across mine sites.

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Conc

entr

atio

n (m

g/kg

)Co

ncen

trat

ion

(mg/

kg)

Conc

entr

atio

n (m

g/kg

)Co

ncen

trat

ion

(mg/

kg)

Conc

entr

atio

n (m

g/kg

)Co

ncen

trat

ion

(mg/

kg)

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Figure 3: Total concentrations of As, Cd, Co, Ni, Pb, and Zn recorded during the 8 months sampling periods.

C L Y O B L S T C F I I S E F R R C P N0

10

20

30

40

50

60

70

80

90

100

Frequency %

Figure 4: Colonial and cellular morphology of the bacterial isolates

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Keys: R, L, Y, O and B = cream, light brown, yellow, orange and brown coloration, L, S and T = large, small and tiny sizes, C, F and I = circular,

filamentous and irregular shape, I, S and E = irregular, serrated and entire margin, F, R = flat and raised elevation, R and C = rod and cocci cell

shape, P and N = Gram positive and Gram negative Gram reaction.

Table 1: Physicochemical and chemical properties of soils and tailings samples

Properties MA MAC MB MBC TS TSC

Location Kagiso Kagiso Chamdor Chamdor Tudor shaft Tudor shaft

Clay (%) 16 24 24 28 26 28

Silt (%) 28 12 12 44 18 34

Sand (%) 56 64 64 28 56 38

Texture Sandy loam Sandy clay loam Sandy loam Silt loam Sandy clay loam Sandy clay loam

Moisture content (%) 3.88 1.53 2.46 1.91 6.61 4.22

Ph 2.69 5.09 3.37 5.34 4.05 6.34

Redox (mV) 301.00 162.50 209.75 126.50 167.75 104.25

EC( mS/m) 4.59 0.09 2.49 0.20 4.07 6.34

CEC (meq/100) 15.84 21.10 20.31 13.93 10.07 13.65

Organic matter (%) 0.37 1.13 0.89 0.59 0.15 0.45

Sulphate (mg/kg) 184.25 9.19 152.64 3.72 296.62 17.77

Nitrates (mg/kg) 0.91 2.95 0.82 3.58 34.87 33.16

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Table 2: Recommended standards of heavy metals in soils and sediments

Element MA MB TS

MA MAC MB MBC TS TSC

South

AfricaaCanada b Netherlandsc

As < 1 < 4

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Table 3: Total number of metal species tolerant bacteria isolated from the three sites

Sampling

Site

Total number

of isolates

Number of

selected isolates

Designates

MA 19 8 OM6 142, OMF 002, OMF 811, OMF 812, OMF 813, OMF 815,

OMF 816, OMF 810

MAC 23 4 OMF 817, OMF 818, OMF 814, OMF 809

MB 22 8 OMF 001, OM4 274, OMF 808, OMF 807, 0M9 107, OMF 806,

OMF 804, OMF 805

MBC 16 3 OMF 819, OMF 820, OMF 821

TS 24 7 OMF 532, OMF 008, OMF 132, OMF 003, OMF 802, OMF 801,

OMF 321

TSC 13 5 OMF 832, OMF 835, OMF 800, OMF 803, OMF 005

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Total number of isolates 117 35 35

Key: TS (Tudor shaft), TSC (Tudor shaft surrounding soil), MA (Mine tailings A), MAC (MA surrounding soil), MB (Mine tailings

B), MBC (MB surrounding soil).

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Table 4: Biochemical characteristics of the bacterial isolatesTESTS

ISOLATECODES

MO

T

CA

T

OX

I

NIT

IN VP

MR

CIT

UR

E

SH GL

H2S

MA

N

XY

L

GLU

AR

A

SUC

GA

L

FRU

MA

L

LAC

OMF 012 + + + + + ─ ─ + ─ + + ─ ─ + + + + + + + +OMF 811 ─ + ─ + ─ + ─ + + + ─ ─ ─ + + + + + + + +OMF 812 ─ + ─ + ─ + + + ─ + ─ + + + + + + + + + +OMF 813 + + + + + + + ─ ─ ─ ─ ─ ─ ─ + + + + ─ ─ +OMF 814 + + + + + ─ ─ + ─ + + ─ ─ + + + + + + + +OMF 815 ─ + + + ─ ─ ─ ─ ─ ─ ─ ─ ─ ─ + ─ ─ ─ ─ ─ ─OMF 816 + + + ─ ─ + + ─ ─ ─ + + + + + + + + + + +OM6 142 ─ + + + ─ + + ─ ─ + ─ ─ + + + + + + + + +OMF 810 + + ─ + ─ + + + ─ + + ─ + + + ─ + + + + +OM8 321 + + + ─ ─ + ─ ─ ─ ─ ─ ─ + + + + + + + + +OMF 001 ─ + + + ─ + + + ─ + + ─ + ─ + ─ + + ─ + ─OMF 809 + + ─ + ─ + ─ + ─ + ─ ─ + + + + + + + + +OMF 274 + + + ─ ─ + ─ + ─ + ─ + ─ + ─ ─ ─ + ─ + +OMF 808 ─ + ─ + + ─ ─ ─ ─ + ─ ─ + + + + + + + + +OMF 807 + + ─ + + + + ─ ─ + + ─ + ─ ─ + ─ ─ + + ─OMF 107 + + + ─ ─ + + ─ ─ ─ ─ ─ ─ ─ + ─ + + + + +OMF 806 ─ + ─ + + ─ ─ ─ ─ + ─ ─ + ─ + + + + + + +OMF 805 + + ─ + ─ + + + ─ + ─ + + + + + + ─ + + +OMF 804 ─ ─ + ─ ─ + ─ + + ─ + ─ + ─ ─ ─ ─ ─ ─ + ─OMF 532 ─ + ─ + ─ ─ + + ─ + ─ ─ + + + + + ─ + + +OMF 008 ─ + + + ─ + ─ ─ ─ + ─ + + + + ─ ─ + + + +OM7 132 ─ + ─ + ─ + ─ + + + ─ ─ ─ + + + + + + + +OMF 003 + + + ─ ─ ─ ─ + + ─ + ─ ─ ─ + + + ─ + + +OMF 803 ─ + + ─ ─ ─ ─ ─ + + + + + + + + + + + + +OMF 802 ─ + + ─ ─ ─ ─ + ─ + + ─ + ─ + + + + ─ + +OMF 801 ─ + ─ + + ─ ─ + + + + ─ + ─ + + + ─ + + +OMF 800 + + ─ + ─ + ─ + + + ─ ─ + + + + + + + + +OMF 005 ─ + + + + + + ─ ─ + ─ ─ + + + + + + + + +OMF 817 + + ─ + ─ + ─ + ─ + + ─ + ─ + ─ + + + + +OMF 818 + + ─ + + ─ + + + + ─ ─ ─ + + + + + + + +OMF 819 ─ + + + ─ ─ + ─ + + ─ + + + + + ─ ─ + + ─OMF 820 ─ + + + ─ ─ ─ + + + + ─ + + + ─ + + ─ + ─OMF 821 ─ + ─ ─ ─ ─ + + ─ ─ ─ ─ + ─ ─ + + + + + +OMF 832 + + + + + + ─ + ─ + + + + + + + + + + + ─OMF 835 + + + + ─ ─ ─ + + + ─ ─ + ─ + ─ ─ + + + ─Key : MOT (Motility), CAT (Catalase), OXI (Oxidase), NIT(Nitrate), IN (Indole), VP (Voges Proskaeur), MR (Methyl red), CIT (Citrate), URE (Urease), SH ( Starch

hydrolysis), GL (Gelatin liquefaction), MAN (Mannose), XYL (Xylose), GLU (Glucose), ARA (Arabinose), SUC (Sucrose), GAL (Galactose), FRU (Fructose), MAL (Maltose), LAC (Lactose).

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Table 5: Probable identity of the bacterial isolates

Isolates code Putative identity

OMF 012 B. amyloliquefaciensOMF 811 Bacillus sp.OMF 812 Enterobacter spOMF 813 B. methylotrophicusOMF 814 Bacillus spOMF 815 Micrococcus yunnanensisOMF 816 B. pumilusOM6 142 E. faecalisOMF 810 B. subtilisOM8 321 Bacillus spOMF 001 B. pumilusOMF 809 B. anthracisOMF 274 E. aerogenesOMF 808 B. cereusOMF 807 Arthrobacter oxydansOMF 107 B. thuringiensisOMF 806 Arthrobacter spOMF 805 Enterobacter spOMF 804 Rhodococcus spOMF 532 E. asburiaeOMF 008 Alcaligenes spOM7 132 Enterobacter spOMF 003 B. cereusOMF 803 Arthrobacter spOMF 802 A. nigatensisOMF 801 B. cereusOMF 800 B. psychoduransOMF 005 Enterococcus spOMF 817 Serratia marcescensOMF 818 Citrobacter spOMF 819 M. variansOMF 820 M. luteusOMF 821 Delftia acidovoransOMF 832 Aeromonas hydrophiliaOMF 835 Burkhoderia cepacia

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Table 6: Maximum tolerable concentration of the tested metals against the bacterial isolatesIsolate codes MTC (mM)

Co2+ Ni2+ Pb2+ Cd2+ Zn2+ Cr2+OMF-012 1 2 4 1 3 8OMF-811 1 2 5 1 3 9OMF-812 1 2 4 1 4 9OMF-813 1 3 5 2 5 7OMF-814 1 3 6 2 5 9OMF-815 1 2 6 2 6 8OMF-816 1 3 4 1 4 8OM6-142 1 5 6 2 7 9OMF-810 1 5 6 2 7 9OM8-321 1 4 6 2 7 9OMF-001 1 4 5 2 7 9OMF-809 1 4 6 3 7 9OM4-274 1 4 6 3 6 7OMF-808 1 2 3 1 3 9OMF-807 1 2 4 2 4 10OMF-107 1 4 7 2 9 9OMF-806 1 4 6 2 5 9OMF-805 1 4 6 2 4 8OMF-804 1 2 5 2 4 8OMF-532 1 5 7 3 9 10OMF-008 1 4 6 3 6 9OM7-132 1 4 6 2 7 9OMF-003 1 5 7 3 9 10OMF-803 1 4 5 2 5 9OMF-802 1 4 5 2 5 9OMF-801 1 4 5 1 5 9OMF-800 1 3 6 2 5 10OMF-005 1 5 7 2 7 9OMF 817 1 2 4 2 4 7OMF 818 1 3 6 1 3 8OMF 819 1 2 5 3 3 7OMF 820 1 3 3 2 5 9OMF 821 1 2 4 2 7 6OMF 832 1 2 4 1 7 7OMF 835 1 4 4 2 5 6

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Table 7 : Correlations of the Physico chemical parameters with heavy metals in adjacent soils

pH EC (m

S/cm

)

Redo

x

% M

oist

ure

Org

anic

m

atte

r

CEC

Nitr

ate

Sulp

hate

mg

/ L Tota

l N

itrog

en

Carb

on

Sulp

hur

As Cd Co Ni

Pb Zn

pH 1.00 EC (mS/cm) 0.71 1.00 Redox -0.39 -0.06 1.00 % Moisture 0.43 0.59 0.08 1.00 Organic matter 0.05 -0.33 0.25 -0.33 1.00 CEC -0.62 -0.65 -0.12 -0.68 0.02 1.00 Nitrate 0.20 0.66 0.62 0.58 -0.21 -0.48 1.00 Sulphate 0.39 0.33 -0.55 0.31 -0.24 0.18 -0.03 1.00 Tot-Nitrogen 0.29 -0.13 -0.11 -0.43 0.29 0.04 -0.32 -0.06 1.00 Carbon 0.00 0.08 -0.39 0.00 -0.35 0.35 -0.17 0.66 0.16 1.00 Sulphur 0.20 0.22 -0.46 0.07 -0.38 0.16 0.04 0.35 -0.49 0.01 1.00 As 0.70 0.85 -0.27 0.68 -0.41 -0.39 0.54 0.68 -0.22 0.26 0.44 1.00 Cd 0.49 0.87 0.26 0.68 -0.30 -0.54 0.88 0.25 -0.32 -0.04 0.23 0.83 1.00 Co 0.66 0.92 -0.06 0.72 -0.37 -0.53 0.70 0.45 -0.30 0.07 0.35 0.95 0.94 1.00 Ni 0.68 0.90 -0.14 0.72 -0.40 -0.47 0.66 0.57 -0.27 0.17 0.40 0.98 0.89 0.98 1.00 Pb 0.20 0.11 -0.32 0.17 -0.30 0.17 0.03 0.60 0.05 0.47 0.31 0.32 0.00 0.12 0.33 1.00 Zn 0.68 0.86 -0.21 0.69 -0.42 -0.41 0.59 0.63 -0.23 0.23 0.43 1.00 0.86 0.96 0.98 0.29 1.00

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Table 8 : Correlations of the Physico chemical parameters with heavy metals in the tailings samples

pH EC (m

S/cm

)

Redo

x

% M

oist

ure

OM

CEC

Nitr

ate

Sulp

hate

m

g / L

Tota

l N

itrog

en

Carb

on

Sulp

hur

As Cd Co Ni

Pb Zn

pH 1.00

EC (mS/cm) -0.35 1.00

Redox -0.65 -0.07 1.00

% Moisture 0.22 -0.15 -0.16 1.00

Organic matter 0.16 -0.09 -0.41 -0.40 1.00

CEC -0.26 0.12 0.18 -0.49 0.29 1.00

Nitrate 0.27 -0.17 -0.29 -0.01 -0.22 -0.39 1.00

Sulphate mg / L -0.23 0.38 -0.06 -0.10 -0.24 0.22 0.06 1.00

Total Nitrogen 0.41 -0.24 -0.06 0.44 -0.58 -0.21 0.40 0.37 1.00

Carbon -0.15 0.44 -0.21 -0.01 -0.24 -0.06 0.34 0.54 0.28 1.00

Sulphur 0.40 0.23 -0.51 0.43 -0.48 -0.42 0.41 0.38 0.66 0.38 1.00

As 0.28 -0.20 -0.26 -0.08 -0.23 -0.48 0.97 0.06 0.34 0.31 0.38 1.00

Cd 0.23 -0.17 -0.27 -0.06 -0.14 -0.37 0.99 -0.03 0.28 0.32 0.30 0.96 1.00

Co 0.26 -0.10 -0.32 0.06 -0.24 -0.38 0.99 0.05 0.42 0.40 0.47 0.94 0.98 1.00

Ni 0.36 -0.04 -0.43 0.18 -0.34 -0.49 0.93 0.11 0.53 0.44 0.69 0.90 0.89 0.96 1.00

Pb 0.33 -0.02 -0.50 0.43 -0.40 -0.26 0.69 0.39 0.69 0.46 0.81 0.60 0.60 0.75 0.84 1.00

Zn 0.31 -0.07 -0.39 0.16 -0.30 -0.40 0.96 0.09 0.51 0.43 0.60 0.90 0.93 0.99 0.99 0.83 1.00

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physico-chemical properties, heavy metals and metal … · 2020. 5. 6. · for review only 1...

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