start-up, adjustment and long-term performance of a two-stage bioremediation process, treating real...

Minerals Engineering xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Minerals Engineering

journal homepage: www.elsevier .com/locate /mineng

Start-up, adjustment and long-term performance of a two-stagebioremediation process, treating real acid mine drainage, coupled withbiosynthesis of ZnS nanoparticles and ZnS/TiO2 nanocomposites

http://dx.doi.org/10.1016/j.mineng.2014.12.0030892-6875/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Universidade do Algarve, Campus Gambelas, FCT –Edifício 8, Lab. 2.35, 8005-139 Faro, Portugal. Tel.: +351 289 800 900x7634.

E-mail address: [email protected] (M.C. Costa).

Please cite this article in press as: Vitor, G., et al. Start-up, adjustment and long-term performance of a two-stage bioremediation process, treating rmine drainage, coupled with biosynthesis of ZnS nanoparticles and ZnS/TiO2 nanocomposites. Miner. Eng. (2015), http://dx.doi.org/1j.mineng.2014.12.003

G. Vitor d, T.C. Palma a,d, B. Vieira d, J.P. Lourenço c,d, R.J. Barros b,d, M.C. Costa a,d,⇑a Centro de Ciências do Mar, CCMAR, Portugalb Centro de Investigação Marinha e Ambiental, CIMA, Portugalc Centro de Investigação em Química do Algarve, CIQA, Portugald Faculdade de Ciências e Tecnologia, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal

a r t i c l e i n f o a b s t r a c t

Article history:Received 31 July 2014Revised 19 November 2014Accepted 5 December 2014Available online xxxx

Keywords:BioremediationAcid mine drainageSulphate-reducing bacteriaZnS nanoparticlesZnS/TiO2 nanocomposites

Acid mine drainage (AMD) generation is a widespread environmental problem in Europe, includingPortugal. Previous experience has shown that a combined process consisting of an anaerobic sulphate-reducing bioreactor, following neutralization with calcite tailing, produces water complying with legalirrigation requirements from synthetic AMD. Aiming the treatment of real AMD a new bioreactor wasinoculated with a SRB enrichment obtained from sludge from a local WWTP anaerobic lagoon. In the ini-tial batch phase, sulphate supplementation was needed to achieve high sulphate-reducing bacteriacounts before continuous feeding of AMD was started. The system quickly achieved good performance,proving it is easy to start-up. However, this time the neutralization step failed to keep bioreactor affluentpH higher than 5 for longer than three weeks. This was due to armouring of calcite by precipitates of var-ious metals present in AMD. A new configuration, replacing a packed-bed column by a shallow contactbasin, proved to be more robust, avoiding clogging, short-circuiting and providing long-term neutraliza-tion. The treated effluent, with excess of biologically generated sulphide, was successfully used to synthe-size zinc sulphide nanoparticles, both in pure form and as a ZnS/TiO2 nanocomposite, thus proving thefeasibility of coupling an AMD bioremediation system with the synthesis of metal sulphide nanoparticlesand nanocomposites.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Formation of Acid Mine Drainage (AMD) has become a majorenvironmental problem throughout Europe, and in particular inSpain and Portugal, where numerous abandoned mines exist inthe Iberian Pyrite Belt (Martins et al., 2010).

AMD results from exposure of sulphide minerals to air, wind andrain, generating accumulation of waters with high sulphate andmetals concentrations and with very low pH. One example of sucha source of soil, surface and waters pollution is the abandoned cop-per mine at S. Domingos, in Southeast Portugal, which AMD showspH values ranging between 2.0 and 2.5, and high concentrations ofsulphate ion and of metals, such as iron, copper and zinc.

Traditional remediation processes for AMD, based on chemicalneutralization and hydroxide precipitation, present several

disadvantages, namely high cost, lack of effectiveness in the reme-diation of sulphate and the formation of large volumes of sludge,which require disposal (Chatterjee and Dasgupta, 2005).

Hence, alternative bioremediation processes have been devel-oped, namely processes based on Sulphate-Reducing Bacteria(SRB).

SRB use sulphate as the terminal electron acceptor during themetabolism of organic matter, resulting in the production of H2S(1):

SO2�4 þ 2CH2O! H2SðgÞ þ 2HCO�3 ð1Þ

The generated sulphide ion, then causes the precipitation of themetal ions as sulphide (2):

M2þ þH2S!MSð#Þ þ 2Hþ ð2Þ

where M2+ represents the dissolved metal.The processes based on SRB have proven to be a viable alterna-

tive to traditional methods (Veeken and Rulkens, 2003; Kaksonen

eal acid0.1016/

http://dx.doi.org/10.1016/j.mineng.2014.12.003

mailto:[email protected]


http://www.sciencedirect.com/science/journal/08926875

http://www.elsevier.com/locate/mineng



Fig. 1. Schematic representation of the bioremediation system used: 1. Metal-contaminated effluent inlet; 2. Pumping stage (peristaltic pump); 3. Neutralizationstage; 4. Upflow Anaerobic Packed-Bed Reactor (UAPB); 5. Treated effluent outlet(still with excess dissolved sulphide); 6. Reaction vessel for nanomaterials synthesis(optional).

2 G. Vitor et al. / Minerals Engineering xxx (2015) xxx–xxx

and Puhakka, 2007; Huisman et al., 2006; Tabak et al., 2003), sincethey are more cost-effective, produce lower quantities of sludgeand are very effective in sulphate ion remediation.

A combined process previously developed by members of thisresearch team, consisting of an anaerobic sulphate reducing biore-actor, following pH neutralization in a calcite tailing column, pro-duces water complying with legal irrigation requirements whentreating synthetic AMD, simulating that from S. Domingos mine(Martins et al., 2010). That process was designed to use locallyavailable wastes and/or natural cheap materials, in order to obtainboth an economically viable and environmentally sustainabletreatment process. Also, it was designed to be as simple as possible,working at ambient temperature and pressure.

A similar bioremediation system was started-up aiming nowthe treatment of real AMD. The problems faced when real andmore contaminated AMD is used and the adaptations required tosolve them are relevant issues reported in this study.

Batch studies (Costa et al., 2012, 2013) have demonstrated thatSRB generated sulphide is capable of precipitating metals in aque-ous solution and that careful control of the reaction conditions willyield nanosized precipitates. As the effluent produced in the biore-mediation system described still presents an excess of sulphideion, which in turn presents its own environmental hazards(Mandal et al., 2006; Labrenz and Banfield, 2004), the systemdescribed in this paper, was designed to use this excess sulphidefor synthesizing specific metal sulphides, under controlled condi-tions, in an attempt to produce functional nanoparticles and nano-composites. These, in turn, would have further applications, assemiconductors or as photocatalysts, among others (Fang et al.,2011; Stengl et al., 2008), thus increasing the economic and envi-ronmental advantages of the bioremediation process described.Thus, also reported in this paper are the difficulties presented bythe addition of the biosynthesis stage, the solutions implementedto overcome those difficulties and the characterization of the zincsulphide nanoparticles and respective the nanocompositessynthesized.

2. Materials and methods

2.1. AMD composition

The AMD used to feed the bioremediation system was collectedin July 2013 in the stream of S. Domingos (N: 37�3903800 W:7�3001800), located in the village of S. Domingos, Mértola, SoutheastPortugal, where the abandoned copper mine of S. Domingos islocated. Its composition is indicated in Table 1.

2.2. Bioremediation system set-up

The lab scale bioremediation system initially set in operationwas composed of two glass column reactors: a calcite tailing col-umn, and an Up-flow Anaerobic Packed Bed reactor (UAPB).Fig. 1 shows a diagram of the bioremediation system set-up. Thecalcite tailing was used as a pH neutralizing and buffer material.The calcite column was packed with a mixture of coarse sandand small pieces of calcite tailing, in a 2:1 (w/w) ratio, obtainedfrom a marble stone cutting and polishing industry, having itscharacterization been previously reported (Barros et al., 2009).The UAPB was packed with 800 g of coarse sand (inside a plastic

Table 1Metal composition, sulphate concentration and chemical parameters of the AMD collected

Eh (mV) pH [SO2�4 ] (mg/L) [Fe] (mg/L) [Cu] (mg/L) [Zn

+505 2.22 4148 413 44 133

Please cite this article in press as: Vitor, G., et al. Start-up, adjustment and long-mine drainage, coupled with biosynthesis of ZnS nanoparticles and Znj.mineng.2014.12.003

mesh sleeve) and 30 mL of inoculum (corresponding to 10% v/v),by adding alternating layers of inoculum and coarse sand(�10 mL inoculum for each addition), until all the inoculum wasadded, after which the remaining coarse sand and Postgate B med-ium (Postgate, 1984) was added, taking care that no air remainedin the reactor. The SRB inoculum used was obtained from sludgecollected in the wastewater treatment lagoon of the wastewatertreatment plant of Faro Nascente, located near the city of Faro,Algarve, South Portugal.

The dimensions and operating conditions of the system aredescribed in Table 2.

During the first 21 days (period of acclimatization) the UAPBoperated in batch conditions to promote bacterial growth. Thetreatment of real AMD was only initiated when the Colony-Form-ing Units (CFU) reached the value of about 5.5 � 105 CFU/mL ofSRB. Then, AMD was added in a continuous regime, at a flow rateof 43 mL/day (yielding a hydraulic residence time of about 7 daysin the UAPB). 6 g/L of ethanol was supplemented to the AMD fedto the system, as carbon source for the SRB.

The hydraulic residence time of the UAPB was selected in accor-dance to previous studies (Martins et al., 2010) to assure that theconcentrations of sulphate and dissolved metals in the treatedAMD were lower than the Maximum Recommended Values (MRV)for irrigation waters according to Portuguese legislation(Decreto-Lei n� 236/98, 1998).

2.3. Coupled biosynthesis system

The biosynthesis system consisted of a reaction vessel (Schottflask, 1L), for collecting the bioremediation effluent, containing aZn(II) solution under agitation (Velp Scientifica ARE magnetic stir-rer). In order to prevent possible contamination of the synthesizedparticles (either with bacteria or other metal sulphides from thebioreactor dragged by the effluent), a two stage filtering systemwas implemented. It consists of a coarse glass wool pre-filter in aglass column (14 cm long, 2 cm diameter), followed by a 0.2 lmpore size syringe filter (Whatman Puradisc 25 AS), placed immedi-ately before the reaction vessel.

in S. Domingos stream and used in the present work.

] (mg/L) [Al] (mg/L) [Cd] (mg/L) [Ca] (mg/L) [Mg] (mg/L)

332 0.9 42 113

term performance of a two-stage bioremediation process, treating real acidS/TiO2 nanocomposites. Miner. Eng. (2015), http://dx.doi.org/10.1016/



Table 2Dimensions and operating conditions of the bioremediation system.

Parameters Calcite tailing column UAPB

Diameter (cm) 5.5 5.5Height (cm) 15 35Working volume (mL) 88 300AMD flow rate (Q) (mL/d) 43 43Hydraulic residence time (HRT) (d) �2 �7Ethanol added (g/L) 6 6

G. Vitor et al. / Minerals Engineering xxx (2015) xxx–xxx 3

Care was taken to minimize sulphide losses along the effluentline. Having deemed the use of glass tubing too prone to otherproblems (i.e., breaking during interventions to the system), theeffluent line was adjusted to have as few junctions as possible,while keeping the line (PTFE tubing, 3 mm inner diameter) as shortas possible (�50 cm).

Zinc solutions for the synthesis were made dissolving hepta-hydrate zinc sulphate (ZnSO4, 7H2O, >99.5%, Panreac) in ultra-purewater, yielding a concentration of �100 mg/L Zn2+ (�1.53 mM),which were deaerated in an ultrasound bath (J.P. Selecta Ultra-sons-H) for 15 min, immediately prior to synthesis start.

Synthesis of composites was performed by adding 0.06 mg ofcommercial titanium (II) oxide (TiO2) powder as substrate to themetal solution, (Evonik Industries Aeroxide P25) per 50 mL ofmetal solution (Costa et al., 2013), prior to deaeration. Accordingto the manufacturer information the particle size of the TiO2 usedwas 21 nm (Evonik Industries, 2007).

The synthesized particles were separated by centrifugation at4000 RPM (�2469 RCF) for 15 min (Hettich Zentrifugen Rotofix32A), washed with 70% ethanol and dried under vacuum (desicca-tor connected to a vacuum pump).

At this moment, five ZnS synthesis were completed, as well asfive more for the ZnS/TiO2 composite.

2.4. Analytical methods

Periodically, samples were collected upstream and downstreamof the UAPB (i.e., between the neutralization stage and the UAPB,and after the UAPB, respectively), being this last one the treatedeffluent.

Oxidation reduction potential (ORP) and pH were measuredimmediately after sample collection, using a pH/Eh Meter (GLP21, Crison).

Sulphide concentration was also measured immediately aftersampling using a UV–Visible spectrophotometer (DR 2800, Hach-Lange) by the Methylene Blue Method (665 nm, Hach-Lange).

Samples were then centrifuged (4000 RPM, 5 min), decanted toclean containers and acidified (HNO3 6 M), after which sulphateconcentration was measured by UV/Visible spectroscopy at450 nm (Hach-Lange) using the sulfaVer4 method (Hach-Lange).

Metals concentration (Fe, Cu and Zn) was determined by flameatomic absorption spectroscopy (Flame-AAS) using a ShimadzuAA-680 model spectrometer. For each sample, five readings weretaken, and the results were critically treated and only accepted ifa reasonable standard deviation (<5%) was achieved.

Aluminium concentration of selected samples was also mea-sured by UV/Visible spectroscopy at 522 nm (Hach-Lange) usingthe AluVer3 method (Hach-Lange).

SRB in the UAPB column samples were enumerated by thethree-tube Most Probable Number (MPN) assay, with serial dilu-tions in Postgate E medium (Postgate, 1984). The MPN tubes wereincubated at room temperature (±21 �C), for 7 days.

The synthesized particles were analysed by X-ray Diffraction(XRD), using a PANalytical X’Pert Pro powder diffractometer, oper-ating at 45 kV and 40 mA, with Cu Ka radiation filtered by Ni. XRD


patterns were recorded using an X’Celerator detector, with a stepsize (2h) of 0.016�, and a time per step of 50 s. Peak analysis andcrystalline phase identification were conducted using the High-Score Plus software, with the ICDD PDF-2 database.

Scanning Electron Microscopy (SEM), coupled with an Energy-Dispersive X-ray Spectroscopy (EDX) analyser was used for themorphological characterization of the synthesized particles. AFEG-SEM Hitachi S4100 microscope, operating at 25 kV, and a Bru-ker EDX detector were used for this purpose. The samples wereprepared by deposition of the precipitate directly onto the carbontape, which was then coated by carbon evaporation (EmitechK950X).

Zeta potential measurements were made for the synthesizedZnS particles and ZnS/TiO2 composite, as well as for the usedTiO2 alone, using a Malvern NanoZS Zetasizer working at 148 V.Each sample was suspended in aqueous solution, with pH valuesranging between about 2 and 8, adjusted using NaOH and HCl solu-tions, and subject to ultrasounds for 1 min. These suspensionswere then used for the zeta potential measurements in a foldedcapillary cell.

3. Results and discussion

3.1. Acclimatization period

During acclimatization, SRB populations were enumeratedalong time. In the initial batch phase, sulphate supplementationwas required to achieve SRB counts of 105–106 CFU/mL before con-tinuous feeding of AMD was started. In fact, according to our pre-vious experience (Martins et al., 2011), the number of SRB reachedin the bioreactor after 21 days of acclimatization (approximately5.5 � 105 CFU/mL), is enough to assure the effectiveness of the sub-sequent treatment. Hence, the MPN of SRB was the main criterionused to determine the best moment to initiate AMD treatment.Another important parameter considered during the acclimatiza-tion period was sulphate concentration (as it is consumed by theSRB over time). Care must be taken not to allow the sulphatesource to be exhausted, as it would limit bacterial growth of theconsortium. After 21 days, the sulphate initially present in the bio-reactor was almost entirely consumed, with the production ofabout 160 mg/L sulphide. Redox potential values were decreasingalong time, also indicating bacterial reduction of sulphate and pHvalues remained constant, near 7.

3.2. Performance of the bioremediation system

The system has been in continuous operation for 339 days, withvery good results in terms of AMD treatment. However, althoughthe system has proven to be effective, it has become evident thatthe AMD neutralization step does not perform adequately for longperiods. In fact, pH values for the AMD leaving the neutralizationcolumn were not up to the UAPB’s needs for longer than about3 weeks, after which pH values dropped significantly, from about6.5 to values lower than 5, which are not suitable for SRB (Bartonand Hamilton, 2007; Jong and Parry, 2006). This event is probablycaused by two factors: one is the fact that with time the calcite tail-ing grains erode, releasing small particles which eventually clogthe pores in the neutralization column, the other and probablymore important, is the fact that during the neutralization step,there is some metal precipitation, possibly of metal oxides and/orhydroxides, which also tend to clog the pores in the column.

These two facts eventually lead to a ‘‘short circuit’’ in the neu-tralization column: not being able to flow through the calcite tail-ing grains, the AMD tends to flow around these, i.e., not allowingeffective contact between the AMD and the calcite, resulting in a




Fig. 2. ORP (A) and pH (B) values over time.

Fig. 3. Sulphate and sulphide concentrations over time.


detrimental effect on pH neutralization effectiveness. This problemdid not arise in previous studies (Martins et al., 2011), most prob-ably because the AMD used was a synthetic one, which had onlyiron, copper and zinc in similar concentrations and about 2.0 g/Lsulphate. Thus, it did not contain some of the elements presentin the real AMD from S. Domingos mine, such as aluminium (referto Table 1, Section 2.1), which, together with iron, easily precipi-tates as hydroxides (Xinchao et al., 2005).

In order to overcome the above mentioned problems, thepacked bed calcite tailing column was replaced by a new configu-ration neutralization stage. Thus, a shallow contact basin has beentested from day 180 after start-up of the system. It consists of acovered glass tank, 21 cm � 21 cm, filled with calcite tailing grains(in this case, it was deemed unnecessary to mix any coarse sand),into which the AMD (already with the addition of the used carbon-source, ethanol) is pumped. The same mass of calcite tailing pieceswas used as in the previous configuration, which yielded about2 cm height in the tank, with the level of AMD about 3–4 mmabove this level, resulting in a working volume of about 250 mL.Care is taken so that the fresh AMD is pumped into one end ofthe basin, entering at the bottom, and exits at the opposite end,in a point above the level of the calcite tailings. Flow rate was keptat the same value as previously. Since the tanks used are not air-tight, it has become necessary to add a second pumping stage, tocarry the neutralized AMD to the UAPB, an obvious disadvantagecompared to the previous configuration.

Visual inspection of the collected samples indicated that thisnew configuration also presents the advantage of dragging less cal-cite particles into the UAPB, thus allowing a more clarified (i.e., lessturbid) effluent.

3.2.1. ORP and pHFig. 2 shows the measured values of oxidation reduction poten-

tial (Fig. 2A) and pH (Fig 2B), in samples collected upstream anddownstream of the UAPB (i.e., between the neutralization stageand the UAPB, and after the UAPB, respectively). The zero valuein the x axis, corresponds to the day that the acclimatization periodwas considered complete and AMD supplying to the UAPB wasinitiated.

Since the initial purpose of the study was to evaluate the perfor-mance of the system in terms of AMD remediation, there are nodata for the neutralization stage before day 74 since system start-up. Those data only started to be collected after the first time thatclogging forced to replace the calcite tailing in the neutralizationcolumn. From that date, the calcite tailing material was replacedwhenever clogging was detected, which generally happened aboutevery three weeks. Due to rapid intervention (replacement of thespent calcite tailing for a new batch), those events did not consider-ably affect the pH of the neutralized AMD entering the bioreactor,since the pH values, although not ideal, never reached critical levelsfor SRB activity. The exception to this behaviour happened at day178, when a value below 5, and thus critical for SRB activity, wasobtained. This fact led to the implementation of the new neutraliz-ing configuration described in Section 3.2.

It becomes clearly evident that the new configuration for theneutralization stage has an obvious effect on the pH values of theneutralized AMD entering the UAPB. Since the alteration, pH valueshave increased to almost 8, and since then kept steadily above 6.5,which is optimum for SRB activity (Barton and Hamilton, 2007;Jong and Parry, 2006). The effect on ORP values is not so evident,although more stable results have been obtained since the alter-ation. It is also noticeable that this alteration does not seem to havea pronounced effect on the pH and ORP values of the effluent.

It should be emphasized that the pH of the treated effluentcomplies with the Portuguese legislation for irrigation waters(Decreto-Lei n� 236/98, 1998) during all the treatment process,


as it can be observed in Fig. 2B, taking into account the rangebetween the Maximum Admitted Values (MAV), i.e., the rangebetween the higher and lower values admitted) for this parameter.

3.2.2. Biological sulphate reductionAs already mentioned, SRB consume sulphate and produce sul-

phide. Fig. 3 shows a plot of the values measured for sulphate con-centration, in samples collected upstream and downstream of theUAPB, and values measured for sulphide concentration, in samplescollected downstream of the UAPB, only.

It becomes evident that there is a relation between sulphate andsulphide content in the effluent produced by the system, asexpected (since one is produced at the expense of the other). Also,it is noticeable that variations in the sulphate content of the trea-ted effluent can be, at least in part, attributed to similar fluctua-tions in the sulphate content of the neutralized AMD (notice that,




Fig. 5. Zinc concentration upstream and downstream of the UAPB, over time.


due to retention time, samples taken upstream in a given day areonly reflected in the treated effluent about seven days later).According to the results, half of sulphate removal was attainedimmediately in the neutralization stage.

The alteration of the neutralization step also contributed to amore stable sulphate reduction, with the values of this ion in thebioreactor effluent usually below 575 mg/L, the Maximum Recom-mended Value (MRV) of the Portuguese legislation for irrigationwaters (Decreto-Lei n� 236/98, 1998).

One further aspect to retain is the sharp decrease of sulphateconcentration of the effluent (and the corresponding increase insulphide) from day 241 on. This is the effect of the addition ofphosphorous and nitrogen (as a solution of KH2PO4 and NH4Clsalts) to the bioreactor, as supplements for bacterial growth. Ini-tially, this addition was done directly to the AMD. This processwas somewhat ‘‘trial and error’’; it was decided to add a quantityof these substances to the AMD, corresponding to 1/20th of theconcentration of equal volume of Postgate B medium (i.e.,0.025 g/L KH2PO4 and 0.05 g/L NH4Cl).

Although the results were promising, it quickly became evidentthat it also caused a huge biological growth (probably fungal, onvisual inspection) in the neutralization stage, with a detrimentaleffect on the neutralization performance. Hence, it was decidedto add these supplements directly to the UAPB (via the sample col-lecting tap), in the form of 2 mL doses of a solution with thedescribed concentrations of each salt. Studies are still underwayto determine the optimum schedule for the additions, in order to(eventually) implement a continuous line for feeding thesemicronutrients to the system.

3.2.3. Metals removalSince start-up, the system has proven to be very effective in

metals removal, even when treating such a contaminated AMDas that from S. Domingos mine. Iron, zinc and copper, which arethree of the main metals present in this AMD, decreased to concen-trations complying with Portuguese legislation for irrigationwaters (Decreto-Lei n� 236/98, 1998). This can be seen in Figs. 4and 5, which show the initial metal concentration of the AMDand the concentration of both the neutralized AMD and the efflu-ent, in iron and zinc, respectively, as determined by Flame-AAS.Also displayed are lines indicating Maximum Recommended Val-ues (MRV) as set by Portuguese legislation for irrigation waters(Decreto-Lei n� 236/98, 1998).

No plot is presented for copper, as this metal precipitates veryeasily, and most of the determinations conducted, even for theneutralized AMD, actually fell below the concentration of the low-est standard used for calibration, and thus are well below the Max-imum Admitted Value (MAV) of 5.0 mg/L set in the Portugueselegislation for irrigation waters (Decreto-Lei n� 236/98, 1998).

Fig. 4. Iron concentration upstream and downstream of the UAPB, over time.


Also monitored were the concentration values for aluminium,since this metal is also present in the AMD from S. Domingos minein high concentration (see Table 1, Section 2.1). The valuesmeasured are indicated in Fig. 6.

It becomes evident that the new configuration of the neutraliza-tion stage has also increased the extent of metals precipitationduring neutralization.

Iron content, in particular, appears to have been most affected.Before the change of the neutralization system, the concentrationof this metal after the neutralization step was well above 5 mg/L,the Maximum Recommended Value (MRV) for irrigation waters,according to the Portuguese legislation (Decreto-Lei n� 236/98,1998). After the alteration, the concentration of this metal in theneutralized AMD was consistently lower than this limit, oftenreaching values below the concentration of the lowest standardused for AAS calibration (0.5 mg/L).

Zinc precipitation was also improved with the new neutraliza-tion system. Although much lower, zinc concentration in the neu-tralized AMD never quite reached below the MaximumRecommended Value (MRV) of the Portuguese legislation for irri-gation waters (2 mg/L), although values below the MaximumAdmitted Value (10 mg/L) have been reached (Decreto-Lei n�236/98, 1998). In any event, this is not problematic, since the finaleffluent, downstream of the UAPB, always had zinc concentrationsbelow this limit, even with the initial neutralization configuration.

Taking into account the concentration values of aluminium inthe effluents collected upstream and downstream of the UAPB itis obvious that the neutralization step is sufficient to remove mostof the aluminium present in the AMD. In fact, although the initialconcentration of aluminium in the AMD is as high as 332 mg/L,in most of the samples analysed the aluminium concentration

Fig. 6. Aluminium concentration upstream and downstream of the UAPB, overtime.





has already dropped below the MRV set for aluminium in the Por-tuguese legislation for irrigation waters of 5 mg/L (Decreto-Lei n�236/98, 1998), thus proving the efficiency of the system. In anyevent, with either of the two neutralization configurations tested,aluminium content in the final effluent was consistently keptbelow the MRV for irrigation waters.

3.3. Coupled biosynthesis of zinc sulphide nanoparticles andnanocomposites

As described in Section 2.3, the biosynthesis of zinc sulphidewas accomplished by simply allowing the effluent resulting fromthe bioremediation process to flow into a reaction vessel, contain-ing the metal ion to precipitate. As the effluent slowly flows to thereaction vessel, and as the sulphide present in the effluent contactswith the metal ions in the solution, there is a very large (local)excess of metal ions in relation to the sulphide ions. This favoursthe formation of nanosized particles, as opposed to larger crystal-lites (Costa et al., 2012, 2013). The reaction was allowed to con-tinue, until enough volume of effluent had flown to the reactionvessel to yield a 2/1 ratio between the sulphide concentrationadded and the zinc concentration initially present, in order to guar-antee that all the metal is precipitated (Costa et al., 2013).

As mentioned in Section 2.3, care was taken in the setup of thesynthesis system, to minimize sulphide losses along the effluentline. However, these attempts were not entirely successful. In fact,samples collected right at the end of the UAPB and at the end of theeffluent line, indicate that over one third of the sulphide present atthe end of the UAPB is lost, probably in the junctions used in theeffluent line. In any event, the sulphide that does reach the reactionvessel has proven to be sufficient for the intended purposes, andnear total precipitation of the metal in the solution was achieved.

It has become apparent that one critical aspect to the system’sperformance is the filtration step used to avoid the contaminationof the precipitate. Even with the use of the coarse glass wool pre-filter, the 0.2 lm filter very rapidly becomes clogged, which leadsto slower flow of the effluent, with consequential greater loss ofsulphide content, and in extreme cases leading to internal pressurebuild-up and rupture of the UAPB (the top lid lifts, causing spillsand exposing the SRB to open air). To minimize this, a schedulewas set for timely filter replacement; every 3–4 days, a new0.2 lm syringe filter was installed (with glass wool replacementwhenever found necessary, by visual inspection).

Of the several ZnS synthesis made, the average mass obtained(after centrifugation washing and vacuum drying) was 0.068 g.The visual aspect of the synthesized ZnS particles matches theexpected: Pure ZnS is a white-to-yellow solid (Lohninger, http://www.almacgroup.com, 2011), in accordance to the light beige col-our of the synthesized ZnS. The mass of ZnS/TiO2 compositeobtained in each synthesis was much larger, as expected, averaging0.718 g. The ZnS/TiO2 composite presents a white colour, which ischaracteristic of the used TiO2.

Over 90% of the initial zinc present in the solution was removedas ZnS or ZnS/TiO2, which is in accordance with results reported byother authors for zinc sulphide precipitation using biogenic sul-phide (Esposito et al., 2006; Bijmans et al., 2009; Mokone et al.,2010; Costa et al., 2012).

Table 3 shows the typical parameters of the zinc solution mea-sured before and after the synthesis (i.e., in the initial Zn2+ solutionand in the supernatant liquid, prior to precipitate separation). The

Table 3Parameters measured in the zinc solution before and after precipitation.

Eh metal solution (mV) Eh supernatant (mV) pH metal

+305 �198 5.47


sulphide concentration indicated refers to the supernatant aftersynthesis, as there is no sulphide present in the initial zincsolution.

As expected, the ORP of the initial zinc solution is positive, dueto the high metal content, and is markedly negative in the end ofthe synthesis, due to the removal of the dissolved metal. pH isalmost invariant, and the sulphide ion concentration in the finalsupernatant, given the low solubility of ZnS, indicates that thereaction is complete.

3.3.1. Characterization of the obtained precipitatesSeveral techniques were used to characterize the chemical nat-

ure, morphology and size of the precipitates obtained, consideringthat those properties are important for eventual subsequentapplications.

3.3.1.1. X-ray Diffraction (XRD). Fig. 7 shows the XRD patterns of theobtained precipitates and composites. Fig. 7A corresponds to theTiO2 used as support for the composites, Fig. 7B corresponds tothe synthesized ZnS particles and Fig. 7C corresponds to thesynthesized ZnS/TiO2 composites.

The diameter of the particles (Dp) was calculated using theScherrer equation (Scherrer, 1922), based on the form factor (K,0.94) and considering the most intense peak observed for eachcrystalline structure present. However, the definite size of thedifferent materials prepared was established by electronicmicroscopy.

As expected (both from data supplied by the manufacturer andfrom previous studies (Costa et al., 2012), the used TiO2 presentedpeaks characteristic of both anatase and rutile structures, whichindicates that a mixture of both morphologies is present, and theaverage TiO2 crystallite size was estimated at about 20 nm, usingthe Scherrer equation (Scherrer, 1922).

According to the results presented in Fig. 7B, hexagonal ZnS isthe predominant crystalline phase present in the analysed precip-itate, unlike what was found in previous batch tests by Costa et al.(2012). These authors reported the synthesis of cubic-structuredZnS nanoparticles, also from biologically generated sulphide. Thisis most likely due to the different experimental conditions duringthe synthesis, which indicates that manipulating these parameterswill probably allow some control over the nature of the synthe-sized particles, not only in terms of particle size, but also of theircrystalline structure.

Using the Scherrer equation (Scherrer, 1922), the average ZnScrystallite size was estimated as ranging between about 29 and39 nm.

In the XRD diffractogram obtained for the compositessynthesized (Fig. 7C), characteristic peaks for anatase- and rutile-structured TiO2 (both tetragonal) are clearly identifiable, as wellas peaks characteristic of hexagonal zinc sulphide. However, themost intense peak for ZnS partially overlaps the one for rutile. Assuch, it becomes impossible to use the Scherrer equation to esti-mate the particle size of the ZnS/TiO2 composite. In the compositediffractogram (Fig. 7C), the peak at the angle of about 47�, althoughattributed to anatase, is in fact probably masking the lower inten-sity hexagonal ZnS peak, which was expected from the ZnS diffrac-togram displayed in Fig. 7B. Closer inspection of this peak shows adeformation, not present in the corresponding peak of the TiO2

diffractogram (Fig. 7A).

solution pH supernatant [S2�] supernatant (mg/L)

5.44 104.2




Fig. 7. X-ray powder diffraction patterns for the commercial TiO2 used as supportmaterial (anatase, ‘+’, JCPDS# 01-071-6411 and rutile, ‘#’, JCPDS# 01-084-1285) (A),for the precipitate (hexagonal ZnS, ‘⁄’, JCPDS# 01-089-2144) (B) and for thecomposite (C) synthesized.


3.3.1.2. SEM–EDX. Fig. 8 shows a SEM image and the correspondingEDX analysis for the zinc sulphide particles synthesized. Predomi-nantly spheroidal morphology is observed for these particles and,as expected, the presence of zinc and sulphur was confirmed bythe EDX analysis, approximately in a 1:1 ratio, indicating the syn-thesized particles are indeed ZnS. There appears to be some clus-tering of the particles, which range between about 30 and 50 nm,in reasonable agreement with values estimated by the Scherrerequation.

Fig. 9 shows SEM and EDX results for the samples synthesizedin the presence of TiO2. The SEM images reveal that the compositenanocrystallites present average diameter ranging between 30 and50 nm. Again, a spheroidal morphology was observed for the syn-thesized particles. EDX analysis confirms the presence of titaniumand also of zinc and sulphur, with these, again, in a 1:1 ratio,approximately.

Taking into account that no elements other than Zn and S or Zn,S and Ti were detected in the ZnS nanoparticles and in the respec-tive TiO2 nanocomposites, the results suggest that they should bepure enough, for potential future applications.

These results are very similar to ones reported previously forthe synthesis of zinc nanoparticles and nanocomposites in batch,using bio-generated sulphide (Costa et al., 2012).

A large variety of methods have been used for the production ofmetal sulphide nanoparticles, including high gravity (Chen et al.,2004), hydrothermal processes (Liu et al., 2009), solid–liquid

Fig. 8. SEM image of the ZnS particles synthesiz


chemical reactions (She et al., 2010), single-molecular precursordecomposition (Monteiro et al., 2004), to name only a few. Someof these research teams have reported the production of ZnS parti-cles ranging between 5 and 10 nm in diameter (Liu et al., 2009).ZnS nanoparticles with average diameter close to those reportedin our research have been obtained by other research teams ofabout 27 nm (Chen et al., 2004). However, both of these examplesuse either a hydrothermal process or high pressure methods. Onthe contrary, the method here described uses biologically gener-ated sulphide, at room temperature and atmospheric pressure,avoiding the use of toxic and expensive chemicals and/or othersophisticated apparatus, which, upon reaching the final goalintended in the development of this process (installation of fullscale remediation plants on situ), would represent a clear economicadvantage over other processes, as it would require almost nomonitoring (i.e., manpower) after initial setup, ensuring the pro-cess’ lower cost in the long term.

On the other hand, this synthesis step also helps to minimizethe release of excess sulphide to the environment, which is themajor hazardous by-product of the bioremediation system understudy.

3.3.1.3. Zeta potential. Zeta potential analysis is a technique fordetermining the surface charge of nanoparticles suspended in solu-tions (colloids). Nanoparticles have a surface charge that attracts athin layer of ions of opposite charge to its surface, which travelswith the nanoparticle as it diffuses throughout the bulk solution.The electric potential at the boundary of this double layer is knownas the f-potential of the particles, and has values typically rangingfrom +100 mV to �100 mV, also depending on the characteristicsof the solution (namely, pH).

The magnitude of a particle’s zeta potential is an indicator of thecolloidal stability (or lack thereof). Nanoparticles with zeta poten-tial value between �30 mV and +30 mV in a suspension will tendto aggregate, due to Van Der Waals inter-particle attractions, whilenanoparticles with f-potential values greater than +30 mV or lowerthan �30 mV will usually display a relatively high stability(Mandzy et al., 2005).

Fig. 10 shows a plot of the measured f-potential vs. pH, for theZnS and ZnS/TiO2 nanocomposite synthesized, and for the TiO2

used in the composite synthesis alone. Results obtained for TiO2

are different to ones found in literature (Suttiponparnit et al.,2011), yet very close, and the difference can probably be attributedto the fact that samples were prepared in NaCl solution, whereassamples in the present study were prepared in ultrapure water(in both cases, pH was corrected with HCl or NaOH solutions).

The results presented in Fig. 10 indicate that the ZnS nanopar-ticles synthesized are susceptible to forming clusters, in the whole

ed (A) and corresponding EDX analysis (B).




Fig. 9. SEM image of the ZnS/TiO2 synthesized (A) and corresponding EDX analysis (B).

Fig. 10. Plots of zeta potential vs. pH, for TiO2, ZnS and ZnS/TiO2 nanocomposite.


range of pH values tested, which may cause some difficulties intheir use as catalysts in photodegradation (Lin et al., 2013).

One of the reasons TiO2 was tested as coadjuvant in these stud-ies, was that, not only is it a good photocatalyst on its own, it alsodoes not tend to form particle aggregates at pH values lower thanabout 5.5. Results obtained for the ZnS/TiO2 nanocomposite showthat indeed the nanocomposite has a lower stability than pureTiO2, but far from the values obtained for the ZnS nanoparticles.For pH values below about 3.5, zeta potential values are aroundthe threshold of ‘‘stability’’. Also of note for use as photocatalyst isthe isoelectric point, the pH value at which f-potential reaches zero.The value measured for pure ZnS is about 3.7, with positive poten-tial values below this pH. On the other hand, the isoelectric point forthe ZnS/TiO2 nanocomposite (6.7) is very close to the one of TiO2

(6.6), again with positive potential values below this pH, allowingits application at higher (less acidic) pH values than ZnS.

The results presented in this paper confirm the possibility ofcoupling the bioremediation process to the synthesis of ZnS nano-particles and/or ZnS/TiO2 nanocomposites with potential interestas catalysts, by using the surplus bio-generated sulphide. Furtherstudies are underway to evaluate the possibility of synthesizingother metal sulphide nanoparticles and nanocomposites, and tocontrol the dimension of the particles produced, by manipulatingthe system’s operating conditions and/or reaction parameters.

4. Conclusions

Since start-up, the UAPB system described has been workingcontinuously for 339 days, fed with AMD from S. Domingos mine,supplemented with ethanol as carbon source.

During all this time, the performance of the system has beenvery good, yielding metals contents below (in some cases, well


below) Portuguese legal limits for irrigation waters. The samehas in general been observed for sulphate.

The new configuration tested for the neutralization stage hasproven to be more effective than the previous configuration,despite the obvious disadvantage of requiring an additional pump-ing stage. Not only is it easy to set up, it is simpler and faster toaccess if needed, reducing the time for any interventions requiringsystem interruption, and it is more stable in time, as pH valueshave remained steady for over two months. Finally, this configura-tion also has the added benefit of precipitating to a greater extentsome of the metals present in the AMD, thus contributing to theeffectiveness of the whole process.

It has also been shown that it is possible to implement a simplesystem (an ‘‘add-on’’ to the regular bioremediation system) to usethe excess sulphide produced in the bioremediation process forprecipitating metal sulphides, obtaining functional nanoparticlesand nanocomposites, under normal temperature and pressure con-ditions. With this, the environmental issue of the most problematicresidue produced in the bioremediation process, is not only mini-mized, it is also turned into an economic advantage, with theadded benefit of dismissing the use of expensive and toxic chemi-cals and/or any sophisticated apparatus.

Acknowledgments

Funding by Fundação para a Ciência e a Tecnologia (FCT)through Project PTDC/AAG-TEC/2721/2012 is acknowledged.

References

Barros, R.J., Jesus, C., Martins, M., Costa, M.C., 2009. Marble stone processing powderresidue as chemical adjuvant for the biologic treatment of acid mine drainage.Process Biochem. 44, 477–480.

Barton, Hamilton, E., 2007. Sulphate-reducing Bacteria – Environmental andEngineering Systems. Cambridge University Press.

Bijmans, M.F., van Helvoort, P.J., Buisman, C.J., Lens, P.N., 2009. Effect of the sulphideconcentration on zinc bio-precipitation in a single stage sulphidogenicbioreactor at pH 5.5. Sep. Purif. Technol. 69 (3), 243–248.

Chatterjee, J., Dasgupta, S., 2005. Visible light induced photocatalytic degradation oforganic pollutants. J. Photochem. Photobiol. C: Photochem. Rev. 6, 186–205.

Chen, J.F., Li, Y.L., Wang, Y.H., Yun, J., Cao, D.P., 2004. Preparation andcharacterization of zinc sulfide nanoparticles under high-gravity environment.Mater. Res. Bull. 39, 185–194.

Costa, J.P., Girão, A.V., Lourenço, J.P., Monteiro, O.C., Trindade, T., Costa, M.C., 2012.Synthesis of nanocrystalline ZnS using biologically generated sulfide.Hydrometallurgy 117–118, 57–63.

Costa, J.P., Girão, A.V., Lourenço, J.P., Monteiro, O.C., Trindade, T., Costa, M.C., 2013.Green synthesis of covellite nanocrystals using biologically generated sulfide:Potential for bioremediation systems. J. Environ. Manage. 128, 226–232.

Decreto-Lei n� 236/98, A.X. (01 de 08 de 1998). Diário Da República — I Série-A N176. Lisbon, Portugal: Imprensa Nacional – Casa da Moeda, E. P.

Esposito, G., Veeken, A., Weijna, J., Lens, P.N., 2006. Use of biogenic sulphide for ZnSprecipitation. Sep. Purif. Technol. 51 (1), 31–39.


http://refhub.elsevier.com/S0892-6875(14)00399-9/h0005
























Evonik Industries, 2007, https://www.aerosil.com. Retrieved 07 29, 2014, from<http://www.novochem.ro/letoltes/aeroxide%20tio2%20p25%20en.pdf>.

Fang, X., Zhai, T., Gautam, U.K., Li, L., Wu, L., Bando, Y., et al., 2011. ZnSnanostructures: from synthesis to applications. Prog. Mater. Sci. 56, 175–287.

Huisman, J., Schouten, G., Schultz, C., 2006. Biologically produced sulphide forpurification of process streams, effluent treatment and recovery of the metals inthe metal and mining industry. Hydrometallurgy 83, 106–113.

Jong, T., Parry, D.L., 2006. Microbial sulfate reduction under sequentially acidicconditions in an upflow anaerobic packed bed bioreactor. Water Res. 40, 2561–2571.

Kaksonen, A., Puhakka, J., 2007. Sulfate reduction based bioprocesses for thetreatment of acid mine drainage and recovery of metals. Eng. Life Sci. 6, 541–564.

Labrenz, M., Banfield, J.F., 2004. Sulfate-reducing bacteria-dominated biofilms thatprecipitate ZnS in a subsurface circumneutral-pH mine drainage system.Microb. Ecol. 47, 205–217.

Lin, Y., Bai, H., Lin, C., Wu, J., 2013. Applying surface charge attraction tosynthesizing TiO2/Ag composition for VOCs photodegradation. Aerosol AirQual. Res. 13, 1512–1520.

Liu, J., Ma, J.F., Liu, Y., Song, Z.W., Sun, Y., Fang, J.R., et al., 2009. Synthesis of ZnSnanoparticles via hydrothermal process assisted by microemulsion technique. J.Alloys Comp. 486, L40–L43.

Lohninger, H., 2011, http://www.almacgroup.com. Retrieved 07 29, 2014, from<http://www.vias.org/genchem/inorgcomp_zincsulfide.html>.

Mandal, D., Bolander, M.E., Mukhopadhyay, D., Sarkar, G., Mukherjee, P., 2006. Theuse of microorganisms for the formation of metal nanoparticles and theirapplication. Appl. Microbiol. Biotechnol. 69, 485–492.

Mandzy, N., Grulke, E., Druffel, T., 2005. Breakage of TiO2 agglomerates inelectrostatically stabilized aqueous dispersions. Powder Technol. 160, 121–126.

Martins, M., Santos, E.S., Faleiro, M.L., Chaves, S., Tenreiro, R., Barros, R.J., et al., 2011.Performance and bacterial community shifts during bioremediation of acid


mine drainage from two Portuguese mines. Int. Biodeterior. Biodegr. 65 (7),972–981.

Martins, M., Santos, E.S., Pires, C., Barros, R.J., Costa, M.C., 2010. Production ofirrigation water from bioremediation of acid mine drainage: comparing theperformance of two representative systems. J. Cleaner Prod. 18, 248–253.

Mokone, T.P., van Hille, R.P., Lewis, A.E., 2010. Effect of solution chemistry onparticle characteristics during metal sulphide precipitation. J. Colloid InterfaceSci. 351 (1), 10–18.

Monteiro, O.C., Neves, M.C., Trindade, T., 2004. Zinc sulfide nanocoating of silicasubmicron spheres using a single-source method. J. Nanosci. Nanotechnol. 4 (1–2), 146–150.

Postgate, J.R., 1984. The Sulphate-Reducing Bacteria, second ed. CambridgeUniversity Press.

Scherrer, W., 1922. A theorem on lattice and volumes. Math. Ann. 86, 99–107.She, Y.Y., Yang, J., Qiu, K.Q., 2010. Synthesis of ZnS nanoparticles by solid–liquid

chemical reaction with ZnO and Na2S under ultrasonic. Trans. Nonferrous Met.Soc. China 20, S211–S215.

Stengl, V., Bakardjieva, S., Murafa, N., Housková, V., Lang, K., 2008. Visible-lightphotocatalytic activity of TiO2/ZnS nanocomposites prepared by homogeneoushydrolysis. Microporous Mesoporous Mater. 110, 370–378.

Suttiponparnit, K., Jiang, J., Sahu, M., Suvachittanont, S., Charinpanitkul, T., Biswas,P., 2011. Role of surface area, primary particle size, and crystal phase ontitanium dioxide nanoparticle dispersion properties. Nanoscale Res. Lett. 6, 27.

Tabak, H., Scharp, R., Burckle, J., Kawahara, F., Govind, R., 2003. Advances intreatment of acid mine drainage and biorecovery of metals: 1. Metalprecipitation for recovery and recycle. Biodegradation 14, 423–436.

Veeken, A., Rulkens, W., 2003. Innovative developments in the selective removaland reuse of heavy metals from wastewaters. Water Sci. Technol. 47, 9–16.

Xinchao, W., Roger, C., Viadero, J., Karen, M., 2005. Recovery of iron and aluminumfrom acid mine drainage by selective precipitation. Environ. Eng. Sci. 22 (6),745–755.


https://www.aerosil.com

http://www.novochem.ro






















http://www.almacgroup.com

http://www.vias.org/genchem/inorgcomp_zincsulfide.html












































start-up, adjustment and long-term performance of a two-stage bioremediation process, treating real...

Documents

anaerobic sulphate

synthetic amd

drainage amd generation

sulphate supplementation

use sulphate

continuous feeding of

treatment of real amd

amd showsph values