Utility of Eucalyptus tereticornis (Smith) bark and Desulfotomaculum nigrificans for the remediation of acid mine drainage

Bioresource Technology 100 (2009) 615–621

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Utility of Eucalyptus tereticornis (Smith) bark and Desulfotomaculum nigrificans for the remediation of acid mine drainage Evvie Chockalingam, S. Subramanian * Department of Materials Engineering, Indian Institute of Science, CV Raman Road, Bangalore 560 012, Karnataka, India

a r t i c l e

i n f o

Article history: Received 1 August 2007 Received in revised form 30 June 2008 Accepted 2 July 2008 Available online 28 August 2008 Keywords: Acid mine drainage E. tereticornis (Sm) Bioremediation Dsm. nigrificans Sulphate reduction

a b s t r a c t The efficacy of the bark of Eucalyptus tereticornis (Smith) as an adsorbent for the removal of metal ions and sulphate from acid mine water was assessed. About 96% of Fe, 75% of Zn, 92% of Cu and 41% of sulphate removal was achieved from the acid mine water of pH 2.3 with a concomitant increase in pH value by about two units after interaction with the tree bark, under appropriate conditions. The adsorption isotherms adhered to Freundlich and Langmuir relationships and were exothermic in nature. The free energy of the adsorption process was found to be negative attesting to the feasibility of the reaction. The adsorption kinetics followed the first-order Lagergren rate equation. The filtrate obtained after treatment with E. tereticornis (Sm) bark was found to contain essential elements like potassium, magnesium, calcium, sodium and phosphate apart from carbon which served as a successful growth medium for the sulphate reducing bacteria (SRB) namely Desulfotomaculum nigrificans. Bacterial growth studies showed that about 57% and 72% of sulphate reduction could be achieved at initial pH values of 4.1 and 5.5 respectively of the acid mine water. Pretreatment of the acid mine water with tree bark followed by bioremoval using Dsm. nigrificans resulted in about 75% and 84% respectively of sulphate reduction at pH 4.1 and 5.5, cumulatively by biosorption and bioreduction. The mechanisms of metal ion removal using tree bark and sulphate reduction using Dsm. nigrificans are discussed. Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction One of the major environmental problems facing the mining industries is acid mine drainage (AMD). Sulphide minerals, mainly pyrite and pyrrhotite, which are often present in mine wastes, generate acidity when they are exposed to atmospheric oxygen and water in the presence of acidophilic bacteria such as Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Leptospirillum ferrooxidans. The resulting acid mine waters typically contain high concentrations of dissolved heavy metals and sulphate, possess a high turbidity and have pH values as low as 2. Untreated acid mine water may thus have a detrimental effect on terrestrial and aquatic ecosystems. Treatment of AMD can be categorized into two types namely, active and passive treatment systems (Johnson and Hallberg, 2005a). The active treatment method, which in its basic concept is a low technology approach for AMD remediation, involves the addition of chemical neutralizing agents like lime, calcium carbonate, caustic soda etc. This results in the production of voluminous sludge and again the sludge disposal becomes a further environmental problem. In the passive treatment method, constructed wetlands have emerged as a viable option for the treatment of * Corresponding author. Tel.: +91 80 22932261; fax: +91 80 23600472. E-mail address: [email protected] (S. Subramanian). 0960-8524/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2008.07.004

AMD and hold promise as a more cost-effective treatment (Johnson and Hallberg, 2005a,b). A scrutiny of the literature reveals the utility of various low cost organic adsorbents in the constructed wetland for the treatment of AMD. These organic substrates showed a high metal sorption capacity, which had been attributed to the presence of humic substances containing carboxyl, amino, hydroxyl and other functional groups. These functional groups are responsible for surface adsorption, ion exchange and chelation with the metal ions in the AMD. The removal of metals from AMD by biosorption using grape stalks and cork powder proved to be an effective strategy (Santos et al., 2004). The chemistry of conventional and alternative treatment systems for the neutralization of acid mine drainage has been extensively reviewed by Kalin and co-workers (2006). The use of microbes for cost reduction of metal removal from the mining industry waste streams has been summarized by Cohen (2006). The ability of poly (lactic acid) to serve as a long-term source of lactic acid for bacterial sulphate reduction activity in zinc smelter tailings and the benefits of such amendments for passive treatment of mine drainage have been investigated by Edenborn (2004). The effect of molybdate on methanogenic and sulfidogenic activity of biomass has been reported (Patidar and Tare, 2005). About 85–95% lead removal through biological sulphate reduction has been achieved by Hoa et al. (2007). The utility of lignocellulose as a carbon source for sulphate reduction in chemically and

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biologically generated sulphidic environments has been recently demonstrated by Roman et al. (2008). The enhancement of bioremediation by augmentation of indigenous microorganisms including SRB with Ralstonia sp. in sediment polluted by cadmium and zinc has been reported by Park et al. (2008). The growth of sulphate reducing bacteria under acidic conditions in an up flow anaerobic bioreactor as a treatment system for acid mine drainage has been investigated by Elliott et al. (1998). The potential for bioremediating highly acidic, sulphate contaminated waste waters using SRB in a bioreactor has been shown by Jong and Parry (2006). The kinetics of copper precipitation from acid mine water using SRB has been studied (Luptakova and Kusnierova, 2005). Almost complete removal of heavy metals with an attendant increase in pH from 3 to 6.9 was achieved in a pilot plant study for the treatment of acid lignite mine flooding water by microbial sulphate reduction (Glombitza, 2001). The chemical characterization of natural organic substrates for biological mitigation of acid mine drainage has been carried out by Gibert et al. (2004). The significant role played by sulphur-reducing bacteria in a successive alkalinity producing system treating acid mine drainage has been established based on a case study (Riefler et al., 2008). The precipitation of mixed metal residues from wastewater utilizing biogenic sulphide has been carried out by Bhagat et al. (2004). The Pacques technique for the treatment of AMD uses the sulfidogenic bioreactor treatment systems which are designed to optimize wetland treatment systems without the presence of wetland plants. This technique strictly depends on bacterial activities which involve the in situ generation of hydrogen sulphide for metal sulphide precipitation. This technique was first implemented at the Budelco zinc refinery in the Netherlands for treating zinc contaminated ground water using ethanol as the nutrient medium for the bacteria (Johnson and Hallberg, 2005a). This is a novel biological process, which has been successfully commercialized for the safe and cost-effective production of sulphide from elemental sulphur, sulphuric acid or sulphate present in effluents, wherein gaseous or dissolved H2S is produced on-site and on-demand in an engineered, high rate bioreactor (Huisman et al., 2006). The biological treatment of AMD involves the choice of appropriate organic substrates which can also serve as electron donors for SRB. The electron donors employed for biological sulphate reduction has been well reviewed (Liamleam and Annachhatre, 2007). In this context, a number of complex organic substrates have been considered which can serve as energy sources for biological sulphate reduction. These include oak chips (Chang et al., 2000), compost, sheep manure, poultry manure and oak leaf (Gibert et al., 2004) and leaves such as pine needles and eucalyptus (Frank, 2000) etc. The utility of rice husk was also assessed as an organic substrate in the treatment of AMD (Evvie et al., 2005). The use of tannery effluent (Boshoff et al., 2004a) and micro-algal biomass (Boshoff et al., 2004b) as carbon source for biological sulphate reduction has also been reported. Although the removal of heavy metal ions from synthetic solutions using various tree barks have been previously investigated (Seki et al., 1997), no systematic studies have been undertaken using tree bark for the mitigation of AMD. Motivated by this, the present study was taken up with a two-fold objective of not only using the bark of Eucalyptus tereticornis (Smith) as a low cost biosorbent for the removal of metal ions from acid mine water and for lowering of its acidity, but also to assess the utility of the bark filtrate as a growth medium for Desulfotomaculum nigrificans, a typical sulphate reducing bacteria (SRB). To fulfill these objectives, detailed characterization of the tree bark substrate as well as the filtrate obtained by interaction of the tree bark with Milli-Q water was carried out with respect to the organic and inorganic constituents. The filtrate obtained after the treatment of acid mine water with tree bark was then inoculated with Dsm. nigrificans to assess

its utility as a growth medium for SRB and to bring about sulphate reduction. The possible mechanisms of adsorption of metal ions and sulphate reduction are discussed. 2. Methods 2.1. Substrate The bark of E. tereticornis (Sm) was used as the substrate and was collected as and when it was shed. The bark was washed thoroughly with distilled water to remove sand and other debris materials. The washed bark was then dried in an oven at 100 °C, ground and sieved using BSS 44 mesh (355 lm). It was then stored in an airtight container to prevent moisture adsorption and used without any physical or chemical treatment. 2.2. Bacterial strain A pure strain of sulphate reducing bacteria Dsm. nigrificans (NCIM 2834) was obtained from the National Collection of Industrial Microorganisms (NCIM), National Chemical Laboratory (NCL), Pune, India. 2.3. Acid mine water The acid mine water was collected in the month of January 2003 from an abandoned pyrite mine pit near the Ingaldahl copper mines, located in the northern Chitradurga district of Karnataka, India. The pH of the acid mine water was 2.3 and its redox potential (ESCE) was 451 mV. The constituents of the acid mine water were (mg L1): Fe2+ – 127; Fe3+ – 172.3; Na – 77; K – 6.4; Ca – 378; Cu – 9; Zn – 29 and SO2 4 – 2200. 2.4. Reagents Ferrous sulphate, zinc sulphate and copper sulphate used for the adsorption isotherm studies were of analytical grade and obtained from Merck. Deionised water obtained from a Milli-Q system (Millipore, USA) was used for all the experiments. The resistivity of the water was below 20 MX cm. 2.5. Characterization of the E. tereticornis (Sm) substrate The size analysis of the ground E. tereticornis (Sm) bark was performed using a Mastersizer particle size analyzer, manufactured by Malvern Instruments, UK and the BET nitrogen surface area using Quantasorb analyzer, USA. The total carbon, hydrogen and nitrogen contents of the bark were determined using ThermoFinnigan Flash EA 1112 CHNS – O Analyser. The functional groups present in the bark, before and after interaction with acid mine water were characterized using JASCO model 410 FTIR Spectrometer, adopting the KBr pellet technique. The spectrum was recorded in the range of 4000–400 cm1. 2.6. Characterization of tree bark E. tereticornis (Sm) filtrate The filtrate was prepared by interacting 10 g of the tree bark with 100 ml of deionised, Milli-Q water for 1 h at 200 rpm and 30 °C. After interaction, the suspension was filtered through a Whatman 1 filter paper. The cations and anions present in the filtrate were analysed using a VG Elemental Model PQ3 ICP-MS. The filtrate was then lyophilised using a Virtis Sentry freezemobile (3 + SL) Lyophilizer, and analysed for total carbon, hydrogen and nitrogen contents using a ThermoFinnigan Flash EA 1112 CHNS Analyser. The amount of tannin present in the lyophilised sample

E. Chockalingam, S. Subramanian / Bioresource Technology 100 (2009) 615–621

was determined using a Shimadzu UV-260 UV–visible spectrophotometer. The functional groups present in the lyophilised sample were ascertained using a JASCO model 410 FTIR Spectrometer adopting the KBr pellet technique. 2.7. Test procedure for initial treatment of acid mine water with E. tereticornis (Sm) bark In each experiment, 100 ml of the acid mine water was taken in a 250 ml Erlenmeyer flask. To it, 10 g of E. tereticornis (Sm) bark was added and agitated at 200 rpm, at 30 °C in an Orbitek orbital shaker for different intervals of time. In another set of experiments, the quantity of the E. tereticornis (Sm) was varied keeping the time of interaction constant at 1 h at 200 rpm and 30 °C. After interaction, the solution was filtered through a Whatman 1 filter paper. The pH and the redox potential (ESCE) of the filtrate was measured and the concentrations of the various metal ions and sulphate were determined. 2.8. Adsorption isotherm Initially, different concentrations of metal ions such as ferrous, zinc and copper were prepared and the pH adjusted to 2.3 corresponding to that of the acid mine water. In each experiment, 5 g of E. tereticornis (Sm) bark was added to 100 ml of the desired metal ion of specified concentration in an Erlenmeyer flask and equilibrated for 1 h at 200 rpm in an Orbitek orbital shaker at 30 °C and 50 °C ± 0.5 °C. The final pH after interaction with E. tereticornis (Sm) bark was found to be 3. The contents were filtered through a Whatman 1 filter paper and the residual concentrations of the chosen metal ions were analysed. 2.9. Adsorption kinetics For the kinetic studies, a series of Erlenmeyer flasks of 250 mL capacity containing 5 g of the tree bark substrate and 50 mL of a known concentration of ferrous or zinc or copper was equilibrated in a Orbitek orbital shaker at 30 °C ± 0.5 °C and 200 rpm. At given time intervals, the suspension was filtered through a Whatman 1 filter paper and the supernatant solution was analysed for the chosen metal ions. 2.10. Procedure for the growth of Dsm. nigrificans Dsm. nigrificans was initially cultured in sealed serum glass bottles under anaerobic conditions using modified Baars’ medium, which has the following composition: K2HPO4 – 0.5 g L1; NH4Cl – 1 g L1; CaSO4 – 1 g L1; MgSO4  7H2O – 2 g L1; sodium lactate – 40 ml L1; yeast extract – 1 g L1; FeSO4(NH4)2SO4  6H2O – 0.5 g L1 and sodium thioglycollate – 0.02 g L1. After the culture reached the stationary growth phase, it was first filtered using a Whatman 1 filter paper of 11 lm pore size to remove the iron sulphide precipitate. The filtrate containing the bacterial cells was then centrifuged using a Sorvall RC-5B refrigerated centrifuge for 20 min at 15,000 rpm. The supernatant solution was decanted and the cell pellet was washed twice with double distilled, de-ionized water and centrifuged again. The supernatant was discarded and the bacterial cell pellet thus obtained was used for the sulphate reduction studies. The entire preparation of the cell pellet was carried out under anaerobic conditions. 2.11. Sulphate reduction test procedure using Dsm. nigrificans For the sulphate reduction tests, two samples each of 100 ml of the acid mine water of pH 2.3, were interacted for 2 h with 20 g of

617

E. tereticornis (Sm) bark. After interaction, the suspensions were filtered and the residue discarded. The final pH of the filtrate was found to be 4.1. The pH of one of the filtrate samples was adjusted to 5.5 with 1 N KOH. The two samples of initial pH 4.1 and 5.5 were first sterilized and de-aerated with flowing nitrogen gas and then inoculated with 108 cells mL1 of Dsm. nigrificans in glass serum bottles fitted with tight fitting rubber stoppers. After inoculation, nitrogen gas was again passed through a syringe into the bottles to maintain anaerobic conditions and sealed using aluminium crimp. The parameters such as pH, bacterial cell count, and sulphate concentration were monitored periodically. 2.12. Analytical techniques The concentration of iron was determined by the orthophenanthroline method using a Shimadzu UV-260 UV–visible spectrophotometer (Vogel, 1989). Zinc and copper concentrations were measured using a Thermo Jarrell Ash Video 11E atomic absorption spectrophotometer. Sulphate concentration was measured by turbidimetric method using barium chloride at 420 nm in a Shimadzu UV-260 UV–visible spectrophotometer (APHA, 1989). Tannin concentration was determined by ammonium molybdate method at 380 nm in a Shimadzu UV-260 UV–visible spectrophotometer (Snell et al., 1961). 3. Results and discussion 3.1. Characterization studies on E. tereticornis (Sm) substrate The particle size analysis of the E. tereticornis (Sm) bark showed the average size (d50) corresponding to 126.5 lm and the surface area was found to be 0.74 m2 g1. It has been reported that finesized materials with the necessary reactive surface area facilitate the metal removal and pH neutralization (Drever, 1997). The CHNS analysis of the E. tereticornis (Sm) bark indicated the presence of total carbon: 45%, H: 5.5% and O: 49.5%. The FTIR spectra of E. tereticornis (Sm) bark, before and after interaction with acid mine water was recorded (Spectra not shown). The spectrum before interaction with acid mine water showed a strong band at 3432 cm1, indicating the presence of hydroxyl groups on the bark surface. The weak stretching frequency at 2928 cm1 was observed which was due to the hydroxyl groups bound to –CH3 groups. A peak at 2918 cm1 due to the C–H stretching frequency and weak bands at 1462 and 1505 cm1 attributing to aromatic C@C were also seen. A sharp peak at 1041 cm1 attributing to C–O stretching frequency and two sharp peaks at 1734 and 1622 cm1 characteristic of carbonyl group stretching were also observed. All the bands present in the spectra were mainly due to the tannin present in the E. tereticornis (Sm) bark. The spectrum after interaction with acid mine water showed a decrease in the intensity of the peaks at 3432, 1734,1622 and 1041 cm1, presumably due to chelation of the metal ions with carboxyl, carbonyl and hydroxyl groups present in the tree bark. 3.2. Characterization of E. tereticornis (Sm) filtrate The E. tereticornis (Sm) filtrate contained about 505 mg L1 of potassium, 57 mg L1 of sodium, 39 mg L1 of magnesium, 13 mg L1 of calcium, 19.3 mg L1 of phosphate, 17 mg L1 of sulphate and trace amounts of manganese, iron, silicon, zinc, phosphorous, aluminium, nitrate and fluoride. The solid product obtained after lyophilization of the filtrate contained carbon: 35%, H: 4% and O: 61%. The FTIR spectrum of the lyophilised filtrate was also recorded (spectrum not shown). All the bands present in the spectrum were characteristic of tannin present in the filtrate.

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The concentration of tannin was found to be 1464 mg L1 by treating 10 g of tree bark with 100 mL of deionised Milli-Q water. 3.3. Removal of metal ions present in acid mine water using E. tereticornis (Sm) bark 3.3.1. Kinetics The removal of iron, zinc and copper ions using E. tereticornis (Sm) bark was studied as a function of time and the results are portrayed in Fig. 1a. The percentage removal of the chosen metal ions increased steeply in the first thirty minutes and thereafter only a marginal removal was observed. For example, 52% of iron, 33% of zinc and 60% of copper was removed from the acid mine water in thirty minutes. The percentage removal of the chosen metal ions increased to about 66% for iron, 45% for zinc and 70% for copper in about 180 min. Additionally, about 23% sulphate reduction was achieved in 60 min which slightly increased to 27% in 600 min. The corresponding changes in the pH and ESCE (mV) are depicted in Fig. 1b. The pH increased from 2.3 to 3 in 30 min and to about 3.1 by 180 min and remained at that value up to 600 min. The low pH of the acid mine water may be due to the mineral acidity related to the dissolved metal ions. After treatment with the tree bark, the pH of the acid mine water increased, which may be attributed to the removal of mineral acidity by the adsorption of metal ions. Further, the presence of phosphate and carbonate groups contributes to the removal of acidity (Gazea et al., 1996). From Fig. 1a and b, it is evident that E. tereticornis (Sm) bark is a good adsorbent for the metal ions present in the acid mine water and is also able to reduce its acidity. The values of ESCE (mV) were also reduced from

3.2

an initial value of 451 mV to 200 mV after 180 min and remained at that value up to 600 min. Therefore, for all further experiments, the equilibration time was fixed at 180 min. 3.3.2. Effect of E. tereticornis (Sm) bark loading The effect of the amount of E. tereticornis (Sm) bark added on the uptake of the metal ions was also investigated (Fig. 2a). In these experiments, the initial pH was 2.3 and the equilibration time was fixed at 180 min. The addition of 10 g of E. tereticornis (Sm) bark significantly reduces the concentrations of iron and copper by 72% and 74% respectively and zinc by 57%, from the acid mine water. A further increase in the E. tereticornis (Sm) bark loading to 20 g increases the removal efficiency to 96% for iron, 92% for copper and 75% for zinc. The sulphate concentration could also be appreciably reduced to 41% with 20 g of the substrate loading. The changes in the pH and ESCE (mV) as a function of E. tereticornis (Sm) bark loading are shown in Fig. 2b. It is noteworthy that the pH steadily increases from 2.3 in the absence of E. tereticornis (Sm) bark to about 3.2 after the addition of 10 g of E. tereticornis (Sm) bark. The pH further increases to about 4.1 with the increase in the substrate loading to 20 g. Taking into consideration the fact that the pH of the acid mine water increases with increase in E. tereticornis (Sm) bark loading, it becomes pertinent to examine the stability of the chosen metal ions as a function of pH. The pH values for the precipitation of Fe2+, Zn2+ and Cu2+ are reported to be 7, 6 and 6 respectively from dilute solutions (0.01 M) (Venkatachalam, 1998). Consequently, the adsorption isotherm experiments were carried out at pH 3.2 to minimize or avoid the precipitation of the chosen metal ions.

480 100 pH

3.0

Cu Fe

Zn 2SO4

420

2.6

300

pH

360

b

2.4 E

240

60 40 20

a

0

SCE

2.2 80

180 500 Cu

70 60

4.0 400

Fe

3.6

50

pH

40

pH

Zn

30

2.8

2-

20

300

3.2

SO

200

ESCE(mV)

% Removal

ESCE (mV)

2.8

% Removal

80

4

10

a

0 0

100

200

300

400

500

600

Time (min) Fig. 1. (a) Percentage removal of iron, zinc, copper and sulphate from acid mine water and (b) variation in the pH and redox potential of the acid mine water as a function of time after interaction with tree bark.

E SCE

2.4

0

2

4

6

8

10

12

14

16

b 18

100 20

Weight (g) Fig. 2. (a) Percentage removal of iron, zinc, copper and sulphate from acid mine water and (b) variation in the pH and redox potential of the acid mine water as a function of tree bark loading.

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3.4. Adsorption isotherms

Amount adsorbed (mg/m2)

The adsorption isotherms of ferrous, zinc and copper for E. tereticornis (Sm) bark at 30 °C and 50 °C are depicted in Fig. 3. All the isotherms show a continuous increase in the adsorption densities as a function of equilibrium concentration. It is noteworthy that the adsorption densities at 30 °C are higher than that at 50 °C for all the metal ions studied, attesting to the exothermic nature of the adsorption process. The results obtained for the adsorption of ferrous, zinc and copper onto E. tereticornis (Sm) bark were analysed by the two widely used isotherm models namely Freundlich and Langmuir isotherm (Sawyer et al., 2003). All related parameters of Freundlich and Langmuir constants obtained from the plots (figure not shown) are given in Tables 1 and 2. In the case of the Langmuir isotherm for all the metal ions studied, the constant Qo (maximum adsorption capacity) was found to decrease with increase in temperature confirming the exothermic type of reaction and b (energy of adsorption) was found to increase with increase of temperature. The values of k (adsorption capacity) and n (adsorption intensity) obtained using Freundlich isotherm can be arranged as

2.0

1.5

1.0

0.5

0

5

10

15

20

25

30

35

40

Equilibrium concentration (mg/L) o

Cu Cu

Temp : 30 C Zn o Temp : 50 C Zn

Fe Fe

Fig. 3. Adsorption isotherms of iron, zinc and copper for tree bark at 30 °C and 50 °C.

Table 1 Langmuir constants for iron, zinc and copper

Iron Zinc Copper

Iron Zinc Copper

ð1Þ

where b (L/mol) is the Langmuir constant and C0 (mg/L) is the initial concentration of metal ions. The parameter RL indicates the shape of the isotherm accordingly: RL > 1 RL = 1 0 < RL RL = 0

(Unfavourable). (Linear). < 1 (Favourable). (Irreversible).

The values of RL indicated that the adsorption of iron, zinc and copper onto E. tereticornis (Sm) bark were favourable (RL < 1) at 30 and 50 °C and at all concentrations studied. 3.4.1. Thermodynamics of adsorption The thermodynamic parameters such as standard Gibbs free energy (DG°), standard enthalpy (DH°) and standard entropy (DS°) (Sawyer et al., 2003) for the adsorption process were also calculated and the results are shown in Table 3. The negative free energy values indicate the feasibility of the reaction process and the spontaneous nature of adsorption without any induction period. Additionally, the free energy slightly decreases with increase in temperature attesting to its exothermic nature.

b  105 (L mol1)

R2

30 °C

50 °C

30 °C

50 °C

30 °C

50 °C

1.01 5.13 60.97

0.99 4.22 40.65

22.79 6.31 0.37

74.98 11.58 0.69

0.99 0.98 0.94

0.97 0.97 0.94

K

The kinetics of the adsorption of iron, zinc and copper onto E. tereticornis (Sm) bark was next investigated at pH 2.3. The percentage of adsorption was obtained by determining the amount of iron, zinc and copper that remained in the solution. In this experiment, it was verified that the adsorption equilibrium was attained near to 180 min (Fig. 1). It is well recognized that the characteristics of the adsorbent surface is a critical factor which affects the adsorption rate parameters and that the film resistance plays an important role in the overall transport of the solute. The Lagergren rate equation (Ho and McKay, 1999) was used to evaluate the adsorption of iron, zinc and copper on E. tereticornis (Sm) bark. The values of log (qe  qt) were calculated from the kinetic data, and when plotted against time, straight lines were obtained (Fig. 4) attesting to the first-order adsorption kinetics. The values of the rate constant kads for iron, zinc and copper was found to be 1.03  102, 1.01  102 and 1.86  102 per minute respectively. 3.6. Adsorption mechanisms

Qo  105 (mol g1)

Table 2 Freundlich constants for iron, zinc and copper Adsorbates

RL ¼ 1=1 þ bC 0

3.5. Adsorption kinetics

0.0

Adsorbates

Fe > Zn > Cu. Further, it is observed that there is a significant decrease in the values of k and n with increase in temperature attesting to an exothermic type of reaction. The essential features of the Langmuir isotherm can be expressed in terms of a dimensionless separation factor or equilibrium parameter (RL), which is defined as:

The thermodynamic data obtained for the adsorption of the various metal ions onto tree bark lend support to a chemical interaction mechanism. The metal ions will also be adsorbed by ion exchange or electrostatic attraction with the negatively charged reaction sites (Davis et al., 2003). The functional groups present Table 3 Thermodynamic parameters for the adsorption of iron, zinc and copper on tree bark

R2

n

Adsorbates

30 °C

50 °C

30 °C

50 °C

30 °C

50 °C

0.561 0.073 0.023

0.175 0.016 0.010

3.49 1.25 0.78

1.88 0.82 0.68

0.97 0.96 0.98

0.93 0.99 0.98

Iron Zinc Copper

DG (kJ/mol) 30 °C

50 °C

21.12 24.36 32.03

19.32 24.33 31.88

DH (kJ/mol)

DS (kJ/K/mol)

48.45 24.76 33.88

0.09 0.0013 0.0061

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1E10

0.0

-1

-0.8

No. of cells m L

log (qe - qt )

-0.4

-1.2 -1.6 -2.0

1E9

1E8

Initial pH 4.1 Initial pH 5.5 Modified Baars' medium

-2.4 0

40

80

120

Cu

Time (min) Zn

160

a

200

1E7 7.5

Fe

Fig. 4. Plot of Lagergren pseudo-first-order kinetics for adsorption of iron, zinc and copper onto tree bark.

6.0

pH

on the surface of tree bark such as carboxyl, carbonyl and hydroxyl as revealed by IR spectroscopy facilitate metal binding by chelation. These organic substrates have carboxyl and phenolic acid groups attached to a larger organic molecule, which will bind metal ions. Further, these organic substrates also serve as energy source for the growth of SRB (Evvie et al., 2005; Evvie and Subramanian, 2006). Adsorption of metal ions onto hydroxyl and carboxyl groups can be represented as follows:

4.5

b 3.0

ð2Þ

2RCOO þ M2þ ! ðRCOOÞ2 M

ð3Þ

where M = metal ion, ROH and RCOO = hydroxyl group and carboxylate ion arising from the polyphenol structure of tannin present in the tree bark. 3.7. Growth and sulphate reduction studies using Dsm. nigrificans The foregoing adsorption studies showed that tree bark was able to remove 96% of iron, 92% of copper, 75% of zinc and only about 41% of sulphate from acid mine water. It was therefore considered appropriate to determine whether the anaerobic, neutrophilic sulphate-reducer Dsm. nigrificans could bring about sulphate reduction. 3.7.1. Growth and sulphate reduction studies with acid mine water The growth curves of Dsm. nigrificans in acid mine water samples of pH values 4.1 and 5.5 are shown in Fig. 5a. With an initial pH of 4.1, the cell number increases gradually from 1.4  108 cells mL1 to 1.3  109 cells mL1 in 60 days and remains more or less unchanged at this value till 90 days. In the case of the sample at pH 5.5, the cell number increases from an initial value of 1.2  108 cells mL1 to 5.2  109 cells mL1 in 60 days and to about 6.4  109 cells mL1 in 90 days. It is noteworthy that the bacteria could be grown at initial pH values of 4.1 and 5.5, even in the absence of the standard growth medium. As highlighted earlier, the characterization of the tree bark and the filtrate revealed the presence of organic and inorganic compounds, which served as essential nutrients for the growth of SRB. For comparison, the growth curve of SRB in the modified Baars’ medium is also shown in the same figure. The cell number increases from an initial value of 3  107 cells mL1 to 4  108 cells mL1 after 10 days and thereafter the stationary phase is attained. It is pertinent to note that the bacterial cell count is an order of magnitude higher in the case of the samples grown in tree bark filtrate at pH 4.1 and 5.5 vis-à-vis that grown using the modified Baars’ medium.

1600

Sulphate (mgL-1)

M2þ þ 2ROH $ ðROÞ2 M þ 2Hþ

1200

800

c

400

0

20

40

60

80

100

Time (d) Fig. 5. Changes in (a) cell number (b) pH and (c) sulphate concentration as a function of time during the growth of Dsm. nigrificans in acid mine water pretreated with tree bark.

The corresponding changes in the pH during the growth of the bacteria under different conditions are shown in Fig. 5b. There is not much change in the pH values. The pH increases slightly from 4.1 to 4.3 and from 5.5 to 6.1 in about 90 days for the two cases studied. The pH value in the modified Baars’ medium increases from 6.2 to 7.5 after 20 days and remains unchanged thereafter. Fig. 5c shows the change in the sulphate concentration as a function of time for the different conditions studied. In the experiments conducted with acid mine water of initial pH values of 4.1 and 5.5, the sulphate concentration decreases gradually in the time period investigated. The sulphate concentration decreases from 1286 mg L1 to about 624 mg L1 in 60 days and then gradually to about 558 mg L1 in 90 days at pH 4.1. At pH 5.5, the sulphate concentration decreases from 1257 mg L1 to 564 mg L1 in about 60 days and further to about 350 mg L1 in about 90 days. At pH

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4.1 about 57% and at pH 5.5 about 72% of sulphate could be reduced. There is also decoloration of the tannin present in the medium indicating that the bacteria could also degrade the organic compounds present in the solution. With respect to the sulphate reduction in modified Baars’ medium, the sulphate concentration decreases to about 880 mg L1 from an initial value of 1720 mg L1 in about 12 days and further no reduction is observed up to 60 days of experimentation. This confirms that the bacteria could bring about 50% of sulphate reduction in the modified Baars’ medium. However, pretreatment of acid mine water with tree bark followed by bioremoval using tree bark filtrate in the presence of Dsm. nigrificans indicates that about 75% and 84% sulphate reduction could be achieved at pH 4.1 and 5.5 respectively. It is well documented that the SRB utilize sulphate as an electron acceptor in the oxidation of the energy substrate with the production of hydrogen sulphide, which will react with heavy metals to precipitate them out as metal sulphides (Boshoff et al., 2004a, b). The mechanism of sulphate reduction in the presence of organic carbon could be illustrated as: þ 2CH2 O þ SO2 4 þ 2H ! 2CO2 þ 2H2 O þ H2 S 2

þ

M þ H2 S ! MS # þ2H

ð4Þ ð5Þ

where, CH2O represents the organic matter and M represents the metal ion. It is pertinent to note that an essential prerequisite for sulphide precipitation to occur is the presence of sufficient organic matter to maintain an anaerobic environment. 4. Conclusions Based on the detailed investigations carried out, the efficacy of the bark of E. tereticornis (Sm) as a biosorbent for the removal of metal ions from acid mine water was established. A notable feature was an increase in pH after interaction of the acid mine water with the tree bark. The adsorption isotherms of the various metal ions with respect to the tree bark adhered to both the Langmuir and Freundlich relationships. The adsorption process was found to be spontaneous, exothermic in nature and followed the first-order kinetics. Additionally, the growth of Dsm. nigrificans and consequent sulphate reduction could be successfully achieved using the filtrate obtained after treating the acid mine water with the tree bark. Acknowledgements The authors thank the Indo-French Centre for Promotion of Advanced Research (CEFIPRA) and the Institute for Research and Development (IRD) France, for grant of research projects to carry out this investigation. The authors profusely thank Ms. K. Eaazhisai for the assistance rendered in using the lyophilization unit and Ms. K. Shivapriya in recording the FTIR spectra and for the CHN–O analysis. References APHA, 1989. Standard Methods for the Examination of Water and Wastewater, 17th ed. American Public Health Association, Washington, DC. Bhagat, M., Burgess, J.E., Antunes, A.P.M., Whiteley, C.G., Duncan, J.R., 2004. Precipitation of mixed metal residues from wastewater utilizing biogenic sulphide. Miner. Eng. 17, 925–932.

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