Microbial leaching of heavy metal contaminated dredged materials using modified Starkey medium.
Dredged spoils abound in the Niger Delta owing to the continued oil and gas exploration activities in wetlands particularly mangrove areas. Results from various studies suggest that the entire mangrove swamp of the Gulf of Guinea contains pyrites, with a heavy presence in the Niger Delta region [1-5]. Disturbance of mangrove through dredging, canalization and aquaculture have variously led to acidification [6-8]. Acidification is often caused by the microbial and chemical oxidation of pyrite forming sulphuric acid . Once it occurs, the impact of acidification is heavy on the environment including destruction of habitat and associated fauna including fisheries, water and heavy metal pollution.
The oil industry typically carry out dredging by removing the soil and sediment along the proposed project route and place them adjacent to the dredged canals, and because of lack of better ways to handle the spoils, they are abandoned. The natural weathering of the exposed spoils causes acidification and pollution of the waterways. But the oil industries are at loss on how best to handle the situation . However, there exist large stockpiles of abandoned dredged spoils scattered all over the Niger Delta in close proximity to oil installations and causing environmental pollution through natural weathering . The major challenge is that the recovery of metals from dredged materials using the conventional physical and chemical extraction techniques is very expensive due to high energy and capital inputs required, low extraction efficiency and environmental impacts arising from the production of secondary contaminations and further hazardous emissions [11, 12]. As environmental standards continue to stiffen, so are the costs for environmental protection and remediation continue to rise. Biotechnology particularly bioleaching, is regarded as one of the most promising solution to these problems, because it has the potential of reducing the capital cost of intervention and offers the opportunity to reduce environmental pollution . The advantages of bioleaching are its relatively low cost, the mild conditions of the process and the subsequent low demand for energy or landfill space compared with conventional technologies . The use of microorganisms for heavy metals extraction has attracted increasing attention particularly due to their low cost and high efficiency compared to conventional methods .
Amongst various microorganisms, the iron and/or sulphur-oxidizing bacteria of the genus, Acidithiobacillus is considered to be the best candidates for heavy metal bioremediation since they are able to leach more than 50% of the metals from the contaminated matrices . Acidithiobacillus thiooxidans is a chemolithotrophic acidophilic, obligately autotrophic bacterium which derives its energy by oxidizing reduced or partially reduced sulfur compounds and obtains its carbon by fixing carbon dioxide from the atmosphere . Since it oxidises both elemental sulphur and sulphide to sulphuric acid, A. thiooxidans plays a significant role in bioleaching of metals from sulphide ores [14, 17, 18]. The metabolite, mainly sulphuric acid, which shows growth in the exponential phase, plays a major role in bioleaching . Factors affecting the rates of bioleaching of heavy metals include temperature, pH, the nature of the bacteria, sulphur content, cell concentration and the surface area of metals . A. thiooxidans is tolerant to extremely acidic environment even at pH < 1 . Hence, it is used in the mining industry for the extraction of metals especially from low grade ores, where physico-chemical techniques are uneconomic or impractical.
In this study, we consider the prospect of controlled microbial leaching of dredged spoil to prevent environmental pollution in a bioremediation perspective. One of the medium used for the cultivation of sulphur oxidizing bacteria and mineral leaching is Starkey medium. Acidithiobacillus species is a chemolithothroph, which utilizes sulphur as energy source. Since the mangrove soil and sediment of the Niger Delta contains pyrite and sulphur, we therefore consider leaching of metals from the spoils using Starkey's medium modified by not adding the energy source. The results obtained were compared to that of a previous leaching experiment using the standard Starkey's medium .
Materials and methods
This study was carried out in a dredged canal (5[degrees]31"N, 5[degrees]31"E) leading off a tributary of the Warri River in the mangrove swamp of the Niger Delta about 20km from Warri in Delta State, Southern Nigeria. The vegetation here is typical of mangrove swamp dominated by Rhizophora species. The area is characterized by high relative humidity (80-92%) and annual average rainfall exceeding 2800mm. Although, there are two seasons (wet and dry), measurable precipitation occurs in all the months of the year. Notwithstanding, the period of April to October is often regarded as raining season, while November--March is regarded as dry season. Atmospheric temperature ranged between 27[degrees]C to 29[degrees]C .
Two different composite dredged spoil samples were collected during sampling namely spoil 1 (matured spoil i.e. about 5 years of abandonment) and spoil 2 (recent spoil i.e. < 4 months of abandonment). The samples were air-dried at ambient conditions. They were pounded and sieved through 2mm mesh and consequently preserved for further analysis. Result of laboratory analysis  show that the spoils are contaminated by heavy metals (Table 1).
In order to isolate the mesophilic, chemolithotrophic, acidophilic bacteria of the genus Acidithiobacillus, 1 g of spoil sample was suspended in 100ml each of Starkey medium in Erlenmeyer (shake) flask. The composition of the medium used was according to Starkey  containing per litre: 0.3g [[N[H.sub.4]].sub.2]S[O.sub.4] 3.5g K[H.sub.2]P[O.sub.4], 0.5g MgS[O.sub.4] x 7[H.sub.2]O, 0.25g Ca[Cl.sub.2] x 2[H.sub.2]O, 0.01g [Fe.sub.2][[S[O.sub.4]].sub.3] x 9[H.sub.2]O and 10g elemental sulphur and was adjusted to pH 3.5 using concentrated sulphuric acid. Starkey medium is a specialized medium for the isolation of sulphur oxidizing bacteria Acidithiobacillus thioxidans . Each shake flask was plugged using cotton wool and incubated at 28 + 2 [degrees]C for 4 weeks. The cultures were shaken for 1 hour each day at 150 rpm (semi-static conditions). Growth was evident by increase in turbidity, colour change of the media and microbial population increase. Cultures (10 ml) were transferred to fresh medium containing sterile spoils. This transfer continued until the population of active growing Acidithiobacilli was in the order of 106 MPN/100 ml.
Ten grams each of spoils 1 and 2 were weighed into each of a 250 ml conical flask containing 100 ml of modified Starkey's medium (i.e. without the addition of the energy source, sulphur to the growth medium). The samples were sterilized at 121[degrees]C for 30 minutes. Each spoil was subjected to leaching processes using Acidithiobacilli inocula. A control set of samples was prepared without the addition of the bacteria. Eight pairs of media were prepared for the two spoil samples corresponding to Day 0, 7, 14, 21, 28, 35, 42 and 49. The cultures were incubated at 28[degrees] [+ or -] 2[degrees]C in an incubator. Samples were collected weekly at each sampling day (0, 7, 14, 21, 28, 35, 42 and 49). Day 0 samples were collected after 30 minutes of incubation. Samples were analysed for microbial species, redox potential, pH, turbidity, optical density at 430nm, ferrous iron, sulphate TDS and conductivity. The remaining samples were digested using HN[O.sub.3]/HCl[O.sub.4]/[H.sub.2]S[O.sub.4] for heavy metal analysis. Heavy metals were analysed using Buck Scientific 200A Atomic Absorption Spectrophotometer (AAS) for copper, cadmium, chromium, nickel, manganese and zinc . Redox potential and pH were analysed using a combination platinum/reference (Ag/Agcl) electrode (ATI Russel,) pH/Eh meter, turbidity was determined using Hach turbidimeter 2100P, TDS and conductivity with conductivity/TDS meter (Hach CO. 150), sulphate using turbidimetric method (Hach turbidimeter 2100P) and optical density (X = 430nm) using Hach 4000 Spectrophotometer. A correlation study was carried out among the parameters using SPSS version 11 (Lead Technologies Inc, 2001) and the significance of the correlation was determined at 0.05 and 0.01 probability levels (two tailed).
Metal recovery/leaching efficiency were calculated using the following equation :
Metal recovery, % = Metal content in leachate X 100/ Initial metal content in dredged spoil
The heavy metal leaching efficiencies of the modified Starkey's medium was compared to that of the standard Starkey's medium obtained in a similar study .
Results and discussion
The results from a previous study are presented in Table 1, which show that both spoils are contaminated with heavy metals. The population of actively growing Acidithiobacillus species increased from Day 0 to Day 49 (Fig. 1). Visual observations during the leaching indicate that the increased microbial growth is also associated with sulphur transformation. For instance, the colour of the leaching media changed from slight brown at the start of the experiment through yellow to deep yellow, indicating the oxidation of pyrite to elemental sulphur (Table 2). Also, turbidity and microbial slime production increased. Physico-chemical parameters monitored (Fig. 2) during the study tend to agree with the visual observations. For instance, the measured turbidity increased along with the visually observed turbidity as the population of acidithiobacilli increased. Other parameters that increased include conductivity, TDS, sulphate, iron and optical density. While pH decreased during the period, redox potential did not show any discernable pattern. In the leaching experiment using spoil 1, acidithiobacilli population strongly correlated with turbidity, iron and optical density (Table 3) indicating that the growth of acidithiobacilli is linked to increased in turbidity, optical density and iron concentrations. pH correlated with sulphate, TDS, conductivity and turbidity. Sulphate similarly, correlated strongly with conductivity, TDS, turbidity and optical density. From these correlation studies, it appears that the pattern of bioleaching involves the microbial oxidation of pyrite leading to the production of sulphur and sulphate, which decreased the pH and increased other parameters including turbidity, slime production, colour intensity, optical density, iron, conductivity and TDS.
After the 49 day experiment, the leaching efficiencies obtained for dredged spoil 1 are copper (82%), cadmium (100%), chromium (50%), nickel (80%), manganese (63%) and zinc (93%). The leaching efficiencies obtained for dredged spoil 2 are copper (93%), cadmium (96%), chromium (50%), nickel (86%), manganese (46%) and zinc (96%). These leaching efficiencies were quite similar (P>0.05) to that obtained when the standard Starkey medium was used (Fig. 3), suggesting that the modified Starkey medium is adequate for the bioleaching of pyritic dredged spoils. In a similar study using Matin's medium under semi static conditions in laboratory shake flasks, and after seven weeks of bioleaching, the heavy metal recoveries from dredged spoils are copper (81%), cadmium (86%), chromium (34%), nickel (61%), manganese (63%) and zinc (82%) . Other authors have similarly recorded high leaching efficiencies during the microbial leaching of heavy metals from sediments, dredged spoils, mine spoils and sewage. Chen and Lin  reported the leaching of metals from contaminated sediments using sulphur oxidising bacteria; Mn (62-68%), Cu (97-99%), Zn (96-98%), Ni (73-87%) and Pb (31-50%). In another study, Chen and Lin  recorded 85-95% release of heavy metals (Cu Zn, Mn, Pb, Ni and Cr) using indigenous Acidithiobacillus sp. for bioleaching. Xiang et al.  reported the following metal leaching efficiencies obtained from the microbial leaching of sewage sludge: Zn 83%, Cr 55%, Cu 92%, Ni 54% and Pb 16%. Liu et al.  reported 97.54% Zn, 97.12% Cu and 44.34% Pb during the microbial catalyzed leaching of heavy metals from acid mine tailings.
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The study demonstrated that the modified Starkey medium is adequate for the bioleaching of heavy metals from pyritic dredged spoils. Leaching of heavy metals from contaminated dredged spoils prior to their abandonment will prevent environment pollution. The use of modified Starkey medium for leaching will reduce the overall cost of the treatment.
We wish to express our gratitude to Dr. (Mrs.) M.O. Benka-Coker for supervising the Ph.D. research of the first author.
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Elijah I. Ohimain *, Emmanuel N. Ogamba and Lovet T. Kigigha
Biological Sciences Department, Niger Delta University, Wilberforce Island, Amassoma, Bayelsa State, Nigeria
Corresponding Author E-mail: email@example.com
Table 1: Heavy metal composition of abandoned dredged spoils. Metal (mg/kg) Spoil 1 Spoil 2 Copper 93.3 [+ or -] 0.26 121.1 [+ or -] 0.30 Cadmium 122 [+ or -] 2.00 130.8 [+ or -] 0.53 Chromium 95.2 [+ or -] 0.20 143 [+ or -] 1.00 Nickel 93 [+ or -] 0.20 138.5 [+ or -] 0.36 Manganese 251.4 [+ or -] 0.80 296 [+ or -] 2.00 Zinc 118.1 [+ or -] 0.78 131.5 [+ or -] 0.50 Source: Ohimain  Table 2: Visual observations during leaching of dredged spoil using modified Starkey medium inoculated with culture of sulphur oxidizing bacteria. Sampling 0 7 14 21 Day Observation SPOI SPOI SPOI SPOI SPOI SPOI SPOI SPOI L L L L L L L L 1 2 1 2 1 2 1 2 Turbidity + + + + ++ ++ ++ ++ Microbial - - - - + + ++ ++ slime * Colour of SB SB Y Y Y Y YY YY Sampling 28 35 42 49 Day Observation SPOI SPOI SPOI SPOI SPOI SPOI SPOI SPOI L L L L L L L L 1 2 1 2 1 2 1 2 Turbidity ++ ++ ++ ++ +++ +++ +++ +++ Microbial ++ ++ ++ ++ ++ +++ ++ +++ slime * Colour of YY YYY YY YYY YY YYY YY YYY the leaching medium ** * Increasing turbidity and microbial slime production: -none/clear; +low; ++moderate; +++high; ++++very high ** Increasing intensity: SB--Slightly brownish; Y- Yellow of increasing intensity (Y-low; YY-moderate; YYY-high; YYYY-very high) Table 3: Correlation coefficients of monitoring parameters. Spoil 1 Acidithio- pH Sulphate Redox Iron bacillus Acidithio- 1 bacillus pH 0.584 1 Sulphate 0.631 0.967 ** 1 Redox -0.151 0.028 -0.074 1 Iron 0.950 ** 0.696 0.719 * 0.035 1 TDS 0.730 * 0.950 ** 0.970 ** -0.116 0.785 * Conductivity 0.684 0.957 ** 0.974 ** -0.061 0.750 * Turbidity 0.857 ** 0.751 * 0.760 * 0.160 0.974 ** Optical 0.994 ** 0.627 0.676 -0.200 0.932 ** Density Spoil 1 TDS Conduc- Turbi- Optical tivity dity Density Acidithio- bacillus pH Sulphate Redox Iron TDS 1 Conductivity 0.996 ** 1 Turbidity 0.793 * 0.770 * 1 Optical 0.772 * 0.727 * 0.834 * 1 Density Spoil 2 Acidithio- pH Sulphate Redox Iron bacillus Acidithio- 1 bacillus pH 0.416 1 Sulphate 0.771 * 0.836 ** 1 Redox -0.725 * -0.110 -0.375 1 Iron -0.189 0.374 0.236 0.402 1 TDS 0.257 0.951 ** 0.700 -0.118 0.330 Conductivity 0.249 0.951 ** 0.694 -0.106 0.340 Turbidity 0.786 * 0.717 * 0.949 ** -0.249 0.148 Optical 0.703 * 0.887 ** 0.988 ** -0.277 0.226 Density Spoil 2 TDS Conduc- Turbi- Optical tivity dity Density Acidithio- bacillus pH Sulphate Redox Iron TDS 1 Conductivity 1.000 1 Turbidity 0.541 0.536 1 Optical 0.755 * 0.749 * 0.945 ** 1 Density * Correlation is significant at the 0.05 level (2-tailed). ** Correlation is significant at the 0.01 level (2-tailed).