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Aug 12, 2012 - Gavrilescu 2004; Volesky 2001). Biosorption ...... accumulation by rotating biological contactor (RBC) biofilms. J .... ron Sci Biotechnol 2:9–33.

Appl Microbiol Biotechnol (2013) 97:5113–5123 DOI 10.1007/s00253-012-4316-5

ENVIRONMENTAL BIOTECHNOLOGY

Biosorption of heavy metals in a photo-rotating biological contactor—a batch process study Sanaz Orandi & David M. Lewis

Received: 12 June 2012 / Revised: 13 July 2012 / Accepted: 16 July 2012 / Published online: 12 August 2012 # Springer-Verlag 2012

Abstract Metal removal potential of indigenous mining microorganisms from acid mine drainage (AMD) has been well recognised in situ at mine sites. However, their removal capacity requires to be investigated for AMD treatment. In the reported study, the capacity of an indigenous AMD microbial consortium dominated with Klebsormidium sp., immobilised in a photo-rotating biological contactor (PRBC), was investigated for removing various elements from a multi-ion synthetic AMD. The synthetic AMD was composed of major (Cu, Mn, Mg, Zn, Ca, Na, Ni) and trace elements (Fe, Al, Cr, Co, Se, Ag, Mo) at initial concentrations of 2 to 100 mg/L and 0.005 to 1 mg/L, respectively. The PRBC was operated for two 7-day batch periods under pH conditions of 3 and 5. The maximum removal was observed after 3 and 6 days at pH 3 and 5, respectively. Daily water analysis data demonstrated the ability of the algal–microbial biofilm to remove an overall average of 25–40 % of the major elements at pH 3 in the order of Na > Cu > Ca > Mg > Mn > Ni > Zn, whereas a higher removal (35–50 %) was observed at pH 5 in the order of Cu>Mn>Mg>Ca>Ni>Zn>Na. The removal efficiency of the system for trace elements varied extensively between 3 and 80 % at the both pH conditions. The batch data results demonstrated the ability for indigenous AMD algal–microbial biofilm for removing a variety of elements from AMD in a PRBC. The work presents the potential for further development and scale-up to use PBRC inoculated with AMD microorganisms at mine sites for first or secondary AMD treatment. Keywords Acid mine drainage . Indigenous AMD microorganisms . Micro-algae . Photo-rotating biological contactor . Biosorption S. Orandi (*) : D. M. Lewis Micro-algae Engineering Research Group, School of Chemical Engineering, University of Adelaide, North Terrace Campus, Adelaide 5005, SA, Australia e-mail: [email protected]

Introduction Mining activities contribute significantly to the production of acid mine drainage (AMD), which consists of a variety of heavy metals (Fe, Cu, Pb, Zn, Cd, Co, Cr, Ni, Hg), metalloids (As, Sb) and other elements (Al, Mn, Si, Ca, Na, K, Mg, Ba) (Nganje et al. 2010; Lottermoser 2010; Das et al. 2009b). Physical and chemical methods have been developed for metal removal from contaminated effluents. However, these methods are commercially impractical or inefficient for AMD treatment. For example, precipitation, which is a commonly used method in mine sites, is not effective for removal of many heavy metals as its efficiency is adversely affected by low pH and the presence of other salts or ions (Ahluwalia and Goyal 2007; Gavrilescu 2004). Evaporation, as another remedy commonly used in mine sites, produces potentially hazardous sludge and wastes valuable resources, e.g. water. Other methods such as ion exchange, membrane techniques and reverse osmosis are extremely expensive for AMD treatment (Das et al. 2008; Wang and Chen 2006; Gavrilescu 2004; Volesky 2001). Biosorption, particularly by live cells, has received a lot of attention and is considered a potentially efficient, cost-effective and sustainable method for removing metals from AMD (Gadd 2004, 2010; Das et al. 2008; Sheoran and Bhandari 2005; Ahalya et al. 2003). However, due to the environmental stress of AMD to aquatic life by low pH and dissolved metals, strains of extremophilic microorganisms are required for any biotreatment remedy (Das et al. 2009a; De la Pena and Barreiro 2009). Indigenous AMD microorganisms, including micro-algae, fungi and bacteria, are extremophiles that have adapted to survive under the hostile conditions of AMD (Lottermoser 2010; Aguilera 2006a; Malik 2004; Johnson and Hallberg 2003). These microbes thrive in extensive biofilms along acidic drainages, attached to substrates (Aguilera et al. 2008). Coexistence and synergistic relationships among the multi-species microbial community of the biofilm protect

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them from environmental stresses and enable them to survive under the extreme conditions of AMD (Aguilera et al. 2007, 2010; Levings et al. 2005). The significant role of these microorganisms in AMD is their natural cleansing ability for removing heavy metals through different functions including bioaccumulation, biosorption and biomineralisation (Das et al. 2009b; Singh et al. 2006; Malkoc and Nuhoglu 2003; Niyogi et al. 2002; Brake et al. 2001). Aguilera et al. (2010, 2007, 2006b) studied eukaryotic biofilms in extremely acidic river, Rio Tinto, in Spain. The biofilm was mainly formed by the autotrophic species of flagellated or filamentous green microalgae (60 %), chemo-lithotroph bacteria and heterotrophic microbes such as bacteria, fungi, amoebae, small flagellates and ciliates. Micro-algae have an important role in AMD biofilms due to their phototrophic nature which is the basis of the food chain production, and micro-algae provide organic carbon for heterotrophic microorganisms (Amaral Zettler et al. 2002). Metal bioremediation by the application of a judicious consortium of live metal-resistant cells can ensure better removal through a combination of bioprecipitation, biosorption and continous metabolic metal uptake after physical adsorption (Malik 2004). The biotreatment potential and the strength of the binding affinity of biofilms depend largely on biosorption conditions such as pH, temperature, contact time, competing ions and ion size, initial concentration of metals and the composition of extra polymeric substances (EPS) (Das et al. 2009a, b; Mack et al. 2007; Wang and Chen 2006; Mathure and Patwardhan 2005; Van Hullebusch et al. 2003). Aguilera et al. (2008) analysed the composition of EPS extracted from 12 biofilms, isolated from Rio Tinto. The results showed the heavy metal content of the EPS which closely resembled the water composition. The indigenous mining microorganisms in the extremophilic acidic biofilms could be the most efficient candidates for AMD treatment via bioremediation technologies. In the reported study, an algal–microbial consortium, dominated with filamentous green micro-algae, was collected from AMD at Sarcheshmeh copper mine in Iran to be used as the biosorbent for AMD biotreatment. The pH of the mentioned AMD was recorded between 3 and 5, and the water was contaminated with heavy metals (Orandi et al. 2007). The in situ microbial analysis revealed the potential of these microorganisms to accumulate heavy metals (Orandi et al. 2007). A photo-rotating biological contactor (PRBC) was used in this study to immobilise the isolated microbial consortium and to develop an algal–microbial biofilm for heavy metal removals from a multi-ion synthetic AMD. Rotating biological contactors (RBCs) can facilitate the immobilisation of microorganisms as attached biofilm on the support media (Rodgers and Zhan 2003; Patwardhan 2003). High interfacial areas generated by the discs, simple and feasible design and operation, low land occupancy, low energy consumption, low cost of operation and maintenance, high

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reaction rate and treatment efficiency are the principal advantages of this type of reactor (Cortez et al. 2008; Mathure and Patwardhan 2005). A number of studies reported the metal removal capacity of immobilised live microorganisms in a batch or continous systems using different type of reactors included rotary systems such as RBCs, with only one to three cations in solution (Akhtar et al. 2008; Bayramoglu et al. 2006; Travieso et al. 2002; Costley and Wallis 1999, 2000, 2001a, b; Gyure et al. 1987). A previous review (Malik 2004) stated that these biotreatment studies that demonstrated high efficiency for metal removal did not perform well in field trials. It is apparent that these investigations used synthesised wastewater with only a few selected metals that did not truly represent actual industrial effluents such as AMD. The reported study is a novel approach to exploit indigenous AMD microorganisms as an effective and environmentally friendly biosorbents for removing a variety of elements, including heavy metals, from a multi-ion synthetic AMD. This paper presents the efficiency of immobilised resistant algal–bacterial biofilm and PRBCs to be used for the first or secondary treatment of AMD at mining sites under acidic conditions.

Materials and methods Microbial inoculum A microbial consortium, dominated with filamentous green micro-algae, Klebsormidium sp., was collected from an AMD at Sarcheshmeh copper mine in Iran and used as inoculum for biofilm development. A previous study (Orandi et al. 2007) identified filamentous green micro-algae, Ulothrix gigas; the fungi, Geotrichum sp. and Aspergillus sp.; and the bacteria, Pseudomonas sp. and Thiobacillus sp. in the collected microbial sample. The microbial inoculum was collected in the plastic tubes with some of the AMD, leaving some air. Sampling and preservation were carried out according to Standard Methods (APHA/AWWA 1988). Photo-rotating biological contactor A single-stage laboratory-scale PRBC was constructed and operated as described previously (Orandi et al. 2012). Sixteen PVC discs, made of polyvinyl chloride (PVC), were mounted on a horizontal rotary shaft with a spacing of 2 cm (Fig. 1). Each disc’s surface was roughened with 5-grit grade sandpaper. The 25-cm-diameter discs were mounted on a horizontal shaft with a spacing of 2 cm. The shaft was mounted in a Plexiglas® trough so that the discs were submerged to a level of 40 % of disc surface area. The PRBC trough was covered with a plexiglas® lid. The volume of 15 L wastewater was provided. The dimensions are presented in Table 1.

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The disc shaft was coupled to a motor (REMX, EF series three-phase induction motor) operated with a speed controller (TECO SPEECON 7300 CV Series Inverters), which was used to maintain the rotational speed at 0.21 rad/s (2 rpm) during biofilm establishment and at 0.52 rad/s (5 rpm) during the treatment period (Orandi et al. 2012). To provide enough light for growth of algal–microbial biofilm in the PRBC, eight tubular cool white fluorescent lamps were installed inside an aluminium semi-cylindrical cover, as four pairs. The light intensity of each pair was189 μmol m−2 s−1, giving 756 μmol m−2 s−1 in total (Orandi et al. 2012). The intensity of sunlight illumination varries between less than 10 μmol m−2 s−1 and more than 2,000 μmol m−2 s−1 during a very overcast day to the direct sun light in a full day light, respectively (Hershey 1991), which is consistent with the settings used in this investigation. The light period was set to 12:12 h light–dark cycle. To reduce the heat effect of the lamps inside the cover, a fan was installed and operated during the 12 h of illumination. Synthesised AMD The PRBC was operated with a synthesised AMD (SynAMD) that was composed of various anions (Cl−, NO3−, NO2−, PO43+, SO42−, CO32−), major cations (Na, K, Ca, Mg) and trace metals (Cu, Mn, Zn, Ni, Co, Fe, Cr, Sb, Al, Ag, Pb, Se). The value of each parameter was calculated based on the natural AMD analysis from which the microbial consortium was collected. The amount of each reagent and Table 1 Dimensions of the PRBC (centimetres) Length of trough Width of trough Depth of trough Diameter of discs Thickness of discs Distance between discs Distance between outer edge of discs and wall of trough Distance between outer edge of discs and bottom of trough

41 30 33 25 0.3 2.0 2.5 2.0

process to make the Syn-AMD have been explained previously (Orandi and Lewis 2012). The Syn-AMD contained low concentrations of nitrate and phosphate, which are the major nutrients required for micro-algae growth. During the batch study, phosphate was provided in excess (5×) to sustain biofilm growth. The pH of the Syn-AMD was adjusted by adding sulfuric acid (0.2 M); 2 ml of sulfuric acid was added to 1 L of the Syn-AMD to decrease the pH from 5 to 3. The concentrations of the composed parameters are reported in Table 2 as milligrammes per litre. Table 2 Initial concentration of composed parameters in Syn-AMD (milligrammes per litre) at pH 3 and 5 Parameters

Concentration at pH 5

Concentration at pH 3

K Na Ca Mg Al Fe Ag Se Cu Sb Ni Co Mo

3 18 22 85 0.05 0.4 0.02 0.02 78 0.005 1.9 0.3 0.02

4 21 25 100 0.07 0.7 0.03 0.03 84 0.009 3.1 0.5 0.01

Mn Pb Zn Cr Si Cl NO3 +NO2

40 0.02 18 0.08 12 13

46 0.03 20 1 15 15

SO4 HCO3 +CO3 PO4

18 990 12 0.8

20 1,250 15 1

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Biofilm establishment and development As demonstrated previously (Orandi et al. 2012), the PRBC was inoculated with the microbial inoculum (10 % of the RBC solution volume). Since the inoculum was obtained from a dense microbial mat, it was necessary to homogenise the mat to obtain individual inoculums for the biofilm establishment and allow the various microorganisms to re-establish on a new substrate, i.e. the PBRC. The volume of the inoculum was 1.5 L with 0.1 g/L dry weight (microbial consortium) which was added to the PRBC solution. The PRBC was operated in batch mode for 12 weeks to establish and develop the algal–microbial biofilm. PRBC operation for heavy metal removal After biofilm establishment and development, the PRBC was drained and washed with distilled water for 2 h. Distilled water was used for washing the PRBC and the biofilm that was immobilized on the discs to ensure the removal of any traces of previously used Syn-AMD before each subsequent treatment period. The PRBC was refilled with the Syn-AMD, adjusted pH at 3, for a 7-day batch period. The Syn-AMD was analysed after preparation and then on a daily basis during the batch period, using Inductively Coupled Plasma–Mass Spectroscopy to evaluate the potential of the system to remove elements from the Syn-AMD. Sampling, preservation and analytical methods were carried out according to Standard Methods (APHA/AWWA 1988). The PRBC was operated with the Syn-AMD, at pH 5, for another 7-day batch period to estimate the efficiency of the system at the higher pH. Prior to the pH 5 batch experiment, the PRBC was drained and washed with distilled water for 1 day. To confirm the validity of the results, the process was replicated at both pH conditions. During the experiments, the physical properties of the Syn-AMD including pH, temperature and electrical conductivity (EC) were recorded on a daily basis at the same time of water sampling with a water quality logger (TPS-90 FL).

Results In the reported research, after inoculation, 60 g of biofilm developed on the disc surfaces of the PRBC after 12 weeks of operation (Orandi et al. 2012). The biosorption study was based on the elemental removal capacity of developed algal–microbial biofilm in the PRBC. The results for major and trace elements removal are presented below.

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Results from major elements removal under pH conditions of 3 and 5 The daily-recorded data from the concentration of the major elements (Cu, Mg, Mn, Zn, Ni, Ca, Na) in the Syn-AMD over the 7-day batch operation of the PRBC were compared with the initial concentration of each element. The results are reported as the daily removal percentages and presented as graphs in the Fig. 2, at both pH conditions 3 and 5. The results demonstrate the comparable removal patterns for the major elements at each pH. However, the daily removal percentage varied for each element. After the first day of the 7-day batch experiment at pH 3, the removal percentage for the major elements was in the order of Ca (38.1 %) > Na (29.3 %) > Zn (23.1 %) > Cu (13.7 %)>Mn (9.7 %)>Mg (5.6 %)>Ni (3.1 %). The removal percentages of these elements at the same retention time of the batch experiment under pH 5 were different in the order of Ca (48.1 %)>Cu (45.2 %)>Mn (43.9 %)>Na (42.2 %)>Mg (40.7 %)>Zn (40.8 %)>Ni (36.9 %). These data showed the lower removal potential of the system at the commencement of the operation (lower than 25 %) under the lower pH (3) for most of the elements, except Ca, Na and Zn, after a 24-h retention time, compared with the higher pH (5). The removal percentage for the major elements under pH 3 increased after the second and third day as one of the maximum removals during the 7-day batch period was observed after day 3 in the order of Na (61.8 %) > Zn (59.1 %)>Ni (59.1 %)>Cu (57.8 %)>Ca (57.8 %)>Mg (57.0 %)>Mn (56.5 %), shown in Fig. 2. The increasing potential of removal for these elements was similarly observed under pH 5, after the second and third day, as most of the elements were removed up to 60–70 % by the end of the third day. Na was the only element that achieved maximum removal after day 3 to the level of 61.8 and 67.4 % at pH 3 and 5, respectively. After the fourth day of the batch experiment, the efficiency of the system for removing most of the major elements decreased considerably at both pH conditions. The decrease in removal at pH 3 continued to the end of day 5 in the order of Cu (25.2 %)>Na (24.5 %)>Ca (12.1 %)>Mg (10.9 %)> Mn (10.1 %)>Ni (9.7 %)>Zn (5.2 %). The considerable decrease in removal was also observed by the end of the day 4 under the pH 5 condition, as Na, Ca, Zn and Ni were desorbed up to 10 % and a negative removal was recorded for these elements. Additionally, the removal efficiency for Cu, Mn and Mg decreased to 15.3, 10.9 and 3.4 %, respectively. The removal efficiency of the system returned to a high removal rate at pH 3 after 6 days as the elements were removed in the order of Na (49.4 %)>Cu (38.5 %)>Mg (28.0 %)>Ni (27.9 %)>Mn (25.6 %)>Ca (24.5 %)>Zn

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(23.5 %). This reabsorbing process occurred immediately after day 4 under pH 5, and a higher removal was recorded on day 5 which continued to a maximum level on day 6 in the order of Ni (77.7 %)>Ca (77.3 %)>Cu (75.2 %)>Mg (74.8 %)>Mn (73.1 %)>Zn (67.5 %)>Na (59.1 %). In the reported research, the average of the major element removal at pH 3 was in the order of Na (38.2 %)>Cu (32.6 %)>Ca (30.8 %)>Zn (26.5 %)>Mg (25.5 %)>Mn (25.3 %)>Ni (25.0 %), whereas a different ranking was observed at pH 5 in the order of Cu (52.7 %) > Mn

(50.7 %)>Mg (46.3 %)>Ca (45.7 %)>Zn (42.5 %)>Ni (42.3 %)>Na (36.7 %). Trace element removal under the pH conditions of 3 and 5 The concentration of the trace metals (Al, Se, Ag, Fe, Mo, Co, Cr, Pb) were recorded daily over the 7-day batch operations of the PRBC and compared with the initial concentration of each element. The results are reported as the daily removal percentage and presented as graphs for both pH conditions in the Fig. 3.

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Over the batch experiment, Ag, Pb and Fe were the only trace elements which showed significant potential for biosorption. The graphs shown in Fig. 3 illustrate the higher removal of Ag under the lower pH as it was removed between 70 and 90 % at pH 3, whereas 50 to 80 % removal was reported for Ag at pH 5. The removal efficiency for Pb increased from 35 % up to50 % and from 25 % up to 60 %, under pH 3 and 5, respectively. The removal percentage of Fe increased gradually from 15 % after the first day of the experiment, up to 50 % by the end of the batch experiment at both pH. In the reported study, Al and Cr were removed with an increasing trend under pH 5, whereas they presented an increasing trend in desorption under pH 3. The removal efficiency for Al increased from 30 to 50 % over

the experiment at the higher pH of 5. But this element was mostly desorbed at pH 3. Al was removed from 5 to 20 % only after the first and third day of batch period under pH 3. However, the removal efficiency for Al decreased considerably between −5 and −30 % during the other days. The removal efficiency of the algal–microbial biofilm for Cr was comparable with Al at pH 3, as 25 % removal was observed after the first day, followed by a continous desorption in the range of −1 to −10 % during subsequent days. Cr removal was between 50 to 70 % at pH 5, over the 7-day experiment. Mo, Se and Co were the only trace metals removed more at the lower pH, 3. Mo was removed up to 20 % after the first

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and second day. However, the removal efficiency of the system decreased for Mo to about 6 % by the end of the 7-day batch period under pH 5. The removal efficiency increased up to 35 % for Mo under pH 3. Co was removed between 10 and 20 % at pH 3, whereas the maximum removal for Co was recorded up to 6 % at pH 5. The potential of the system for removing Se varied from 30 % for the first 3 days of experiment to the level of 50 % for the rest of the batch experiment at pH 3. The removal potential for Se was variable over the 7-day experiment at pH 5, as adsorption and desorption values were recorded alternatively. A removal of 10–30 % was observed after days 2 and 4, whereas desorption of Se was recorded −5 to −20 % by the end of days 1, 3, 5 and 6. The average of the trace elements removal at pH 3 was recorded in the order of Ag (83.6 %)>Se (36.7 %)>Pb (35.5 %)>Fe (32.4 %)>Mo (30.9 %)>Co (15.0 %)>Cr (3.1 %)>Al (−6.3 %), whereas a different removal was observed at pH 5 in the order of Ag (71.8 %) > Cr (61.4 %)>Pb (48.4 %)>Al (38.5 %)>Fe (26.6 %)>Mo (12.7 %)>Se (0.5 %)>Co (3.3 %). The physical properties, pH and EC, were recorded daily during the experiments, and the results are shown in Figs. 4 and 5, respectively. pH increased during batch processes of metals (Fig. 4), from 3 to 4 and 5 to 6.5, respectively. EC presents the amount of charged ions in solution (Lottermoser 2010). Since metals are more soluble at acidic pH, the EC of the initial PRBC solution was obtained at the higher value of 2,000 μS/cm at pH 3 and compared with the lower initial value of EC of 1,700 μS/cm at pH 5. EC decreased significantly to less than 1,000 μS/cm on the fourth and seventh day of the experiment at pH 5 that was comparable to the time that most major metals were removed (Fig. 5). A similar decrease was observed for EC from 2,000 to 1,500 μS/cm on the fourth day of the batch experiment at pH 3, which was comparable with the metal removal. Biotreatment of metals that remove elements through biosorption or bioprecipitation contributes to the decrease of EC in AMD before discharge to

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the environment. The reported study was undertaken at ambient temperature which fluctuated between 18 and 20 °C over the experimental period, whereas the water temperature was between 20 and 23 °C.

Discussion The efficiency of various biosorption systems reported in the scientific literature for metal removal are highly variable and difficult to compare (Akhtar et al. 2008; Bayramoglu et al. 2006; Costley and Wallis 1999, 2000, 2001a, b; Dvorak et al. 1992). As it was mentioned previously, the mechanism of metal biosorption is a complicated process and depends on a variety of factors including the ambient and environmental conditions such as pH and temperature; the initial concentration of metal ions and biomass; and competition of ions for biding sites (Das et al. 2009a, b; Lottermoser et al. 1999; Mathure and Patwardhan 2005). In the reported study, pH as the most important factor affecting the selectivity of ions in biosorption process was implemented. A comparison between the graphs in Fig. 2 illustrates the greater removal efficiency of the algal–microbial biofilm for removing the major elements at the higher pH (5) compared with the lower pH (3). The acidity of a solution affects the chemistry of the metals, the activity of the functional groups in the biomass and the competition of the metallic ions (Aguilera et al. 2007; Das et al. 2008; Malik 2004). Low external pH reduces both surface binding and intracellular influx (Gyure et al. 1987; Tsezos and Volesky 1982) due to the presence of hydrogen ions which compete successfully with other cations for binding sites and hence occupy many potential metal binding sites, resulting in poor metal biosorption results (Mehta and Gaur 2005; Gadd 1988). The metal uptake of biofilms under an acidic condition is substantially less than at neutral conditions (Ferris et al. 1989). In general, release of ions from a bond state is result of a lower pH, while higher pHs tend to favour their chelation (Wilson et al. 2001; Warren and Haack 2001).

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Additionally, the presence of some competing ions such as Na and especially Ca in solution impedes the sorption of heavy metal ions to biomass or reduces the binding capacity in some extent (Wang et al. 2010; Dimitrova 2002). Light metal ions such as Na and Ca are commonly present in many industrial wastewaters. High Na concentrations generate high ionic strengths which adversely affect heavy metals bonding (Schiewer and Wong 2000). Na, as a typical “hard” ion (Stumm and Morgan 1996), does not participate in covalent bonding. Therefore, Na does not compete directly with the covalent binding of heavy metals by the biosorbents. Ionic strength of Na is resulting from the Na competition with the heavy metals for electrostatic binding to the biomass. The negatively charged carboxyl or sulphate groups in the microbial cell walls such as green algae attract any cation, electrostatically. However, the presence of Na in solution affects more on the uptake of weakly bond metals such as Zn or Ni and strongly bond metals such as Cu are less affected by the ionic strength (Wang and Chen 2006; Schiewer and Wong 2000). Schiewer and Volesky (1996) reported that the electrostatic attraction was only influenced by the binding of light metals on biomass. They tested heavy metal cation binding by the marine algae Sargassum sp. under the presence of Na ions. According to Schiewer and Volesky’s results, Na binding can be neglected unless present at high concentrations because it only binded through weak electrostatic attraction and did not compete significantly with the binding of protons and divalent metal ions (Wang et al. 2010). In the reported study, the concentration of Na is comparable with some of metal concentrations in the Syn-AMD, which attributed to the higher Na removal due to the reasons explained above. The first step of passive biosorption is metabolism independent and proceeds rapidly by one or a combination of the following metal binding mechanisms: coordination, complexation, ion exchange, physical adsorption (e.g. electrostatic) and inorganic micro-precipitation. The passive mechanism of biosorption is a dynamic equilibrium of reversible adsorption–desorption. Metal ions bond on the surface can be eluted by other ions, chelating agents or acids (Das et al. 2008; Ahluwalia and Goyal 2007). The desorption stage which was observed in the reported study was related to the above-mentioned equilibrium of reversible adsorption–desorption. Additionally, the desorption stage observed at both pH conditions indicates the self-refurbishing nature of these microorganisms to survive under extreme conditions. This is one of the significant advantages of using live cells as biofilm for biosorption, where cells are protected from toxicity (Van Hullebusch et al. 2003). However, a sustainable treatment system with considerable efficiency is required for mine sites. The application of sequential PRBCs could improve the efficiency of the system for a consistent removal and biosorption capacity.

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The increased removal efficiency of the system after 3 days, followed by a higher removal after 6 days, was attributed to the increasing capacity of the algal–microbial biofilm to remove elements by the end of the batch operations along with the multi-layer metal adsorption on cell walls. The adapted nature of the indigenous AMD microorganisms to the high concentration of different metals, and the previously adsorbed metal ions, may have favoured multi-layered adsorption of metals which would account for the increased metal removal by the end of the reported study. A continuous progression of metal uptake occurs with multi-layered adsorption as binding sites begin to adsorb a second layer of ions (Costley and Wallis 2000). As mentioned previously, the initial metal concentration and ion competition for binding sites are of the important parameters affecting the metal selectivity of the biofilm from a multi-ion solution (Bayramoglu et al. 2006). In the reported research, the concentration of Cu and Mg was higher than the other heavy metals in the Syn-AMD. This is likely to be the reason for these metals to out-compete other ions for available adsorption sites after 6 days. However, the close removal range of the other major elements such as Ni, Mn and Zn demonstrated that the uptake of these elements did not appear to be affected significantly by the presence of other metal ions. Additionally, Cu, Zn, Mn, Co and Ni are of the essential micronutrients for growth and metabolism of all micro-algae (Andersen 2005; Malik 2004). In both acidic conditions, Cu was readily selected and removed by the system. Many studies reported that Cu was preferentially adsorbed from a multi-ion solution (Costley and Wallis 2001a, b). In the reported study, the indigenous algal–microbial consortium was derived in a copper mine and grew in AMD that contained >80 mg/L of copper. The establishment of this copper-selective population could favour multi-layered adsorption of Cu, and the removal efficiency was not adversely affected by the presence of other ions. Ni is a recalcitrant pollutant, and many microorganisms have a relatively low Ni-binding capacity (Williams et al. 1998). Mn is also a recalcitrant metal which cannot be removed by chemical treatment in traditional systems (Malik 2004). In the current research, the average removal of 25 % and 40–50 % for Ni and Mn at pH 3 and 5, respectively, highlights the effectiveness of using indigenous mining biosorbents that are adapted to the high concentration of these elements in its aquatic habitat. Among trace elements, Ag was most removed element under both pH conditions. Ag is one of the most toxic of heavy metals to freshwater micro-organisms (Gad and Griffith 1978) but was readily removed in this study and did not appear to be toxic to the biofilm. In the reported study, Al and Cr were the only trace elements with an opposite removal efficiency under

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different pH conditions. These results showed that the biosorption of these elements were more pH dependant. In a previous study, the treatment process was ineffective in removing Al and Mg from a mildly acidic and multi-metal (Cu, Zn, As, Ni, Fe, Mg, Al) contaminated wastewater by sulphate-reducing bacteria in a packed bed reactor (Jong and Parry 2003). The removal of Al was achieved due to hydrolysis forming insoluble Al(OH)3 at around pH 10.5 (Jong and Parry 2003; Dvorak et al. 1992). Mo, Se and Co were the only trace metals that were removed more at the lower pH, 3. Travieso et al. (2002) used a BIOALGA reactor to immobilise algae on a rotary drum. The reactor was operated with synthesised wastewater containing 3,000 μg/L ion cobalt and pH ranged between 8.6 and 8.9 in 20-day batch mode. The authors reported 94.5 % cobalt removal after 10 days. Al and Co do not have a significant role in metabolic function. The toxic effect of these elements can adversely affect cellular function. Consequently cells may exhibit resistance mechanisms to enable them to withstand high concentrations of such metals and hence exhibit low sorption capacities. The resistance mechanism employed may either prevent initial uptake of an ion or alternatively may provide a means of expelling the ion from the cell (Costley and Wallis 2001b). As explained previously, low pH results in the low removal efficiency of metals by biofilms (Ferris et al. 1989). The lower pH of 3 attributed significantly to the lower removal of some trace elements such as Al, Pb, Cr and Mo and higher removal of Co and Se in the reported research which demonstrated the pH-dependent binding of metal ions to the algal–microbial biofilm. The heavy metal ion loading capacity increased along with increasing pH as a result of heavy metal competition with hydrogen ions for the same binding sites (Say et al. 2003). The removal efficiency of the algal–microbial biofilm for removing the trace metals at both pH conditions was not comparable with the trend of major elements removal. A comparison between the removal percentages of major and trace elements demonstrates the lower removal for trace elements, except for Ag. As it was mentioned previously, the initial concentration of elements in the solution is an important factor in biosorption and the lower concentrations of the trace elements in Syn-AMD attributed in the lower removal of trace metals. In the reported study, the acidity and EC of the Syn-AMD decreased gradually, due to the biosorption or bioprecipitation of the positively charged ions in acidic solution. Additionally, micro-algal photosynthesis contributes to the gradual increase of pH, which is an advantage of using micro-algal treatment with live cells (Malik 2004). The correction of pH and EC in AMD treatment is one of the most important issues at mine sites before discharge to the environment (Lottermoser 2010).

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Temperature affects the biosorption rate metal ions, but to a limited extent under a particular range of temperature which varies for different biosorbents (Mack et al. 2007; Wang and Chen 2006). Munoz and Guieysse (2006) stated that the efficiency of microalgal-based treatments normally decreases at low temperatures. They observed that the removal efficiency doubled when the temperature increased from 25 to 30 °C using a symbiotic microcosm formed by the Chlorella sorokiniana and Ralstonia basilensis strains. However, the biosorption process is usually not operated at high temperature because it will increase the operational cost (Wang and Chen 2006). The results of the reported study substantiate the considerable potential of indigenous AMD biofilms for removing a variety of major and trace elements from AMD under acidic conditions, which is challenging and costly by using traditional chemical precipitation processes. The treatment results showed the resistive nature of these living microorganisms and their potential to be loaded with a variety of toxic heavy metals and elements from the acidic and multiion nature of AMD within 3–6 days. This environmentally friendly technology shows great promise for first or secondary treatment of AMD at mine sites. However, to develop a sustainable treatment system, sequential arrangements of PRBCs are required to maintain high removal, which is the topic of future research. Acknowledgements This work was financially supported by the R&D centre at the Sarcheshmeh copper mine in Iran and GHD Pty Ltd in Adelaide, South Australia. Special thanks go to Saeid Ghasemi and Afsar Eslami for their cooperation in undertaking work at the mine site and to Mohammad Reza Nikouei for his great assistance in the field, and to John Ewers and Joanne Princi for their cooperation at GHD. The authors would also like to thank Jason Peak, Jeffrey Hiorns and Michael Jung for constructing the PRBC in the School of Chemical Engineering workshop at the University of Adelaide. Additionally, the authors would like to thank Amir Ahmad Forghani from the School of Chemical Engineering, University of Adelaide for his analytical assistance.

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