Biosorption of Heavy Metals in Dumpsite Leachate by Metal-resistant Bacteria Isolated from Abule-egba Dumpsite, Lagos State, Nigeria

Authors designed the study, performed the statistical analysis and wrote the protocol. Author AOA wrote the first draft of the manuscript and managed literature searches. Authors HAS and AOA managed the analyses of the study. Both approved the final Aim: We studied the potentials of four metal-resistant bacterial strains to reduce the concentration of selected metals in metals ranged from 700-1500 µg/ml. The resistance to the metals was in the order: Pb ˃ Ni ˃ Cr ˃ Cd. Pseudomonas aeruginosa had the highest MIC to the metal combination (1300 µ g/ml) while the lowest was Proteus mirabilis (800 µ g/ml); Paenalcaligenes faecalis and Bordetella petrii had MIC values of 1000 µ g/ml and 1200 µ g/ml respectively. The biosorption set up showed that Paenalcaligenes hominis had a higher percentage reduction for Pb in the leachate with a reduction of 35.77%, while Bordetella petrii removed the highest concentration of Cd and Ni in the leachate with values of 32.81% and 34.91% respectively. However the highest percentage reduction for Cr (32.54%) was observed when the leachate was treated with a consortium of the four bacterial strains. Conclusion: This study revealed that these metal-resistant bacteria could be very useful in the biological treatment of metal-containing wastewater.


INTRODUCTION
Environmental pollution with toxic heavy metals is increasing worldwide as a result of rapid industrialization in the fields of metallurgy, agriculture, mining, petro-chemicals, electroplating, among others. Heavy metal pollution of soil and aquatic ecosystems is a significant environmental challenge [1,2]. Heavy metals are elements with specific gravity usually greater than 5.0 g/cm 3 , which is five times the density of water. Of the ninety naturally occurring elements, fifty-three (including arsenic) are classified as heavy metals. Heavy metals are transition elements which are characterized by the possession of incompletely filled d-orbitals, which provides them with the ability to form complexes. Thus, metal cations are vital as trace elements in sophisticated biochemical reactions [3].
Metals play an integral role in the life processes of living organisms. Some metals serve as microelements, components of various enzymes; and are essential in many redox-processes; while many others have no known biological roles and are non-essential. Virtually, all metals whether essential or non-essential can exhibit toxicity above a certain threshold, which for highly toxic metal species may be extremely low [4].
Various techniques have been employed for the treatment of metal-bearing wastewater, and they include adsorption, precipitation and electrochemical technologies; but these techniques are expensive, not environmentfriendly and usually dependent on the concentration of the waste especially in much diluted solutions. Therefore, the search for effective, cost effective and environmentallyfriendly remedies for wastewater treatment has been initiated. It was only in the 1990s that a new scientific area was developed to recover heavy metals and it was bioremediation, which is the use of biological agents; microorganisms (fungi, bacteria, algae), green plants or their enzymes to return the natural environment altered by contaminants to its original condition. Heavy metal bioremediation involves the removal of heavy metals from wastewater and soil through metabolically-mediated or physico-chemical pathways. Microorganisms have proved to have great potentials in metal removal from wastewaters [5].
The process of biosorption is mainly used to treat wastewater where more than one type of metal ions are present; especially in cases where the removal of one metal ion is not influenced by the presence of other metal ions. For example: the uptake of uranium by biomass of bacteria, fungi and yeasts was unaffected by the presence of other metals in the same solution as reported by Petrisor et al. [6]. The advantages of biological technologies for the removal of toxic pollutants are that they can be carried out in situ at the contaminated site, they are cost effective and usually environmentally benign (no secondary pollution). They have also been demonstrated to possess good potentials in replacing conventional methods for the removal of metal contaminants [7,8]. Therefore, this study is aimed at employing bacterial species in the removal/reduction of selected metals in leachate samples obtained from a refuse dumpsite in Lagos state, Nigeria.

Metal Resistance Assay
The resistance of each bacterial strain to metal ions was determined as the minimum inhibitory concentration (MIC) on nutrient agar (Oxoid, UK) plates supplemented with metals as described by Aleem et al. [10]. Graded concentrations of membrane filter-sterilized soluble salts of the respective heavy metals (NiSO 4 . 6H 2 O, K 2 Cr 2 O 7 , Pb(CH 3 COO) 2 and CdCl 2 ) was incorporated into the medium; with 100 µg/ml as the starting concentration for each metal. The concentration was thereafter increased by 50 µg/ml at a time. The plates were incubated at 35°C for 72 hours and observed for growth. The culture growing on one concentration was transferred to a higher concentration and the MIC was noted when the bacteria failed to grow on the metal-incorporated medium.

Sample Preparation
The dumpsite leachate was filtered using Whatman filter paper No 4 and tyndallized in a water bath to eliminate the initial microbial flora present. The tyndallized leachate was cultured on both Nutrient agar and Potato Dextrose Agar to ascertain the sterility of the leachate, and no growth was observed.

Metal Biosorption Setup
The metal biosorption set up was done in 150 ml Erlenmeyer flasks containing 100 ml of the tyndallized leachate samples each and into which nutrient medium has been incorporated to support the growth of the organisms according to the modified method of Murthy et al. [11]. The set up was inoculated with bacterial cells (wet weight) separated from nutrient broth by centrifugation at 10000 rpm for 10 min. The individual strains were inoculated with 10±1 mg cells, while the consortium was combined in the ratio 1:1:1:1, making up 10 mg of cells. The set up was subsequently incubated inside a rotary shaker (G24 Environmental Incubator shaker) at 35°C for 14 days at a revolution of 200 rpm. The experiment was conducted in triplicates. After the experimental duration, the set up was centrifuged at 10000 rpm for 15 min at 4°C to remove the bacterial cells and this was done using a Hitachi High Speed Refrigerated Centrifuge (HIMAC CR21GII).

Determination of the metal composition of the leachate
The metal composition in the treated and control leachate samples was determined using an Atomic Absorption Spectrometer (Perkin Elmer AAnalyst 200 Atomic Absorption Spectrometer), after the sample has been digested using concentrated acids (HCl and HNO 3 in the ratio 3:1). Standards of chromium, cadmium, lead and nickel solutions, e.g., 0.2, 0.4, 0.6, 0.8 and 1.0 mg/l were made from 1000 mg/l stock solutions analytes of the respective heavy metals. The set of standard solutions were analyzed using the Atomic Absorption Spectrophotometer (AAS) (UNICAM 929, London Atomic Absorption Spectrophotometer powered by SOLAAR software) for calibration. The detection limit of the metals in the samples was 0.0001 mg/l. The cathode lamp of each metal was used for the analysis of the respective mineral oils in the standards and the metal concentration in the sample filtrates. Gas mixtures were used to generate the flame [12].

Metal Reduction in the Leachate
The ability of the bacterial strains to reduce the metal composition of the dumpsite leachate was calculated as shown below: where: F 0 : Metal concentration in the control sample F 1 : Metal concentration after treatment

Statistical Analysis
The analysis of the metal concentration was done using the SPSS version 20, while the Duncan Multiple Range Test (DMRT) was for the mean separation.

Heavy Metal Resistance
The bacteria demonstrated the ability to tolerate the four selected metals used for this study. The MIC of all the isolates on Pb 2+ amended medium was considerably higher than the values for the other metals, and these values are slightly higher than the MIC value (1100 µg/ml) for Pb Acinetobacter calcoaceticus and Kocuria varians isolated from spent oil contaminated site and reported by Oriomah et al. [13]. The MIC values for Pb 2+ by the strains used in this study ranged from 1200-1500 µg/ml. The MIC for Cr ranged from 800-1000 µg/ml, and this is higher than the MIC for Cr 2+ (490 µg/ml) for some bacterial strains isolated from sewage as reported by Narasimhulu et al. [14]. The MIC values for the

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The analysis of the metal concentration was done using the SPSS version 20, while the Multiple Range Test (DMRT) was used

RESULTS AND DISCUSSION
The bacteria demonstrated the ability to tolerate the four selected metals used for this study. The amended medium was considerably higher than the values for the other metals, and these values are slightly higher than the MIC value (1100 µg/ml) for Pb 2+ by Kocuria varians isolated from spent oil contaminated site and reported by Oriomah et al. [13]. The MIC values by the strains used in this study ranged 1500 µg/ml. The MIC for Cr ranged 1000 µg/ml, and this is higher than the µg/ml) for some bacterial strains isolated from sewage as reported by Narasimhulu et al. [14]. The MIC values for the bacteria for Ni 2+ and Cd 2+ on the amended medium in this study ranged from 900 µg/ml and 700-900 µg/ml for the two metals respectively ( Table 1). The metal resistance pattern of the bacterial isolates on the metal combination is shown in Fig. 1, the MIC ranged from 800-1300 µg/ml with aeruginosa having the highest MIC value (1300 µg/ml) while the lowest MIC was observed for Proteus mirabilis (800 µg/ml). Paenalcaligenes faecalis and Bordetella petrii had MIC values of 1000 µg/ml and 1200 µg/ml on the metal combination respectively.

Concentration of Metals in the Leachate
The concentration of the selected metals in the treated and control dumpsite leachate is shown in on the amended medium in this study ranged from 900-1200 900 µg/ml for the two metals y ( Table 1). The metal resistance pattern of the bacterial isolates on the metal combination is shown in Fig. 1   wastes; Notable among them was Kumar et al. [15] who reported values range of 0.010-0.865 mg/l of Pb in wastes collected from landfills and wastewater from selected locations in India. They reported a range of 0.032-7.60 mg/l for Ni, 0.018-8.56 mg/l for Cr while the Cd concentration was 0.001-0.523 mg/l. This suggests that the leachate employed in this study is heavily laden with metal, which is of serious environmental and public health concern, as this can easily permeate into underground water table thus causing contamination of aquifers. For all the metals analysed, the values in the control, were significantly higher at p≤0.05 than for all the treated samples, which suggests various degree of metal reduction by the bacterial strains employed in this study.

Metal Removal by the Bacterial Strains
The bacteria were able to reduce the concentration of metals present in the leachate at different rate. The highest percentage reduction in the Pb concentration was 35.77% by Paenalcaligenes hominis followed by Proteus mirabilis at a value of 32.75%. These values were higher compared to 37.40% and 30.08% Pb uptake obtained by Petrisor et al. [6], using waste biomass of Aspergillus niger and Penicillium sp. respectively in the pre-screening of biosorbents for biosorption of metals in leachate samples generated from two Romanian mine waste disposal sites. This is not however in accordance with a study carried out by Kumar et al. [15], who reported a Pb reduction of 93% by heavy metal acclimated Staphylococcus sp. isolated from soil and sludge and 100% Pb removal by Staphylococcus saprophyticus from a study carried out by Ilhan et al. [16]. Worthy of note is the lowered percentage reduction in the concentration of Pb when the dumpsite leachate was treated with the consortium of all the bacterial strains (Fig. 2). This might be due to competition for nutrient and antagonistic activities among the strains when combined.
The consortium containing all the bacteria employed in this study showed a capacity of reducing Cr from the leachate sample used in this study (Fig. 3). A reduction of 32.54% was recorded by the consortium for Cr; this value is comparatively higher than the 7.01% and 6.68% Cr reduction in Bauxite from a mining site leachate by Thiobacillus sp. and Pseudomonas sp. respectively [17]. The highest percentage Cr reduction in this study was by Paenalcaligenes hominis (36.89%), although the value in this study is comparatively lower than the percentage reduction of Cr in magnesite reported by the same authors for Pseudomonas sp. (40.01%).
The highest percentage reduction for Ni was 34.91% by Bordetella petrii (Fig. 4), and this value was higher than the value recorded by Petrisor et al. [6], who reported a reduction of 8.2% and 3.0% for a mine leachate sample obtained from Rosia Poieni; Although it should be stressed that their study was carried out using immobilized bacterial cells consisting of a mixture of heterotrophic and acidophilic microorganisms. The percentage reduction of Ni in the leachate was not enhanced when the consortium of all the bacterial isolates was utilised in this biosorption study.
The lowest Cd removal was for the leachate treated with Proteus mirabilis (27.68%), while the highest reduction was recorded with Bordetella petrii (32.81%) as shown in Fig. 5. The reduction rate from this study was lower compared to the report of a study carried out by Kumar et al. [15], who reported a Cd reduction of 50% in a liquid waste by Aspergillus niger, a fungus isolated from the soil environment.

CONCLUSION
The bacterial strains employed in this present study possess the ability to remove metals from the leachate generated from the refuse disposal site. Hence will be useful candidates for the clean-up of environments contaminated with toxic heavy metals. However further studies need to be carried out on the genetics of heavy metal resistance by these bacterial strains.