effect of heavy metals and bfrs concentration on the

EFFECT OF HEAVY METALS AND BFRS CONCENTRATION ON THE ADAPTATION OF ACIDITHIOBACILLUS FERROOXIDANS FOR THE LED WASTE BIOLEACHING F. POURHOSSEIN , S.M. MOUSAVI* * Biotechnology Group, Chemical Engineering Department, Tarbiat Modares University, Tehran, Iran * Corresponding author. Tel: +98-21-82884917, Fax: +98-21-82884931, Email: [email protected] (S.M. Mousavi)

This study is the first research aimed to observe the combined effects of heavy metals and brominated flame retardants (BFR) in light-emitting diode (LED) waste on the bioleaching bacteria community. Microbiology management is one of the most important part to search in bioleaching process. In this study Acidithiobacillus ferrooxidans adapted to LED powder for increasing its tolerence to high concentration of heavy metals and BFRs. Adaptation of the bacteria strain was performed through a serial sub-culturing procedures. Adaptation experiments with LED powder in the presence of a mixture toxic substance showed that A.ferroooxidanse could tolerate up to 20 g/l LED powder. The growth characteristics reflected by pH, Eh, Fe3+ concentration, specific growth rate and tolerance index. The results indicated that the both BFRs and heavy metals could cause toxicity response. Generally, the high concentration of LED powder (≥20 g/l ) resulted in obvious inhibitory effects. In order to analyze the influence of the adaptation in metals recovery efficiency in the bioleaching process, the adapted and non-adapted A. ferrooxidans inocluated to 20 g/l LED powder. By comparing the results obtained from bioleaching with adapted and non-adapted bacteria, a reduction 30% and 15% in copper and nickel extraction was observed, respectively. It was suggested that positive effect of pre-adaptation on metals recovery from LED waste.The FT-IR spectra of residue shows effects of H2SO4 on the fate of BFRs in bioleaching processes. Keywords: Light emitting diode, heavy metals, brominated flame retardant, adaptation, bioleaching

1. INTRODUCTION 1.1 LED waste generation and problem Nowdays,The electronics industry has changed the world and without these products, the modern life is not possible(Sarath et al., 2015).The total global amount of electronic waste (E.waste) in 2014 was estimated to be around 40 million tons(Baldé, 2015). The behavior with electrical equipment waste has created many concerns in both developed and developing countries(Veit and Bernardes, 2015). Proceedings Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium/ 2 - 6 October 2017 S. Margherita di Pula, Cagliari, Italy / © 2017 by CISA Publisher, Italy

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

The generation rate of lamp waste is growing at an alarming rate. In 2014, the total amount of lamp waste was 1 milion tones in the world, most of these waste was produced in Asia. In generally, it can be resulted that the bulb manufacturing industry was the major consumers of many rare and precious metals among other(Baldé, 2015). A light-emitting diode (LED) is one of the most important electronic waste in the world that has grown rapidly in recent decades(Lim et al., 2010).LED is widely used in automotive, electronics and lighting display applications. LEDs have been replacing with other lighting source becouse of it has distinctive advantages such as environmental friendly, better energy efficiency and cost-effective(Wilburn, 2012).The market share for LED represents under 12 percent of the global lighting In 2012 and is expected to hit 80 percent by 2020 which leads to generation of a large volume of LED waste (Stephen Lacey, 2013). LED contains both valuable and hazardous metals and polymers result in affectthe environment in two approaches: first through reducing natural raw material source (precious and rare metals) and second they have considerable toxic chemical composition and heavy metals that are harmful for human and ecosystem(Veit and Bernardes, 2015). There is no exact number on the amounts of discarded LEDs annually. In general, LED is considered toxic because of a mixture of inorganic compounds such as copper, iron, aluminum, croumium, nickel, zinc, arsenic and various types of BFRs compound(Lim et al., 2010).Tetrabromobisphenol-A (TBBPA) is widely applied as BFRs in LED that often as addetives added to plastic polymer (Chang et al., 2012).TBBPA has been shown in vitro to bind to estrogen hormone receptors at high concentrations and cause other effects on hormone sensitive parameters(Gosavi et al., 2013). In recent decade, the population of the enviromental with toxic and dangerous heavy metals is one of the most important issue. In biological systems, metal ions reaction to cell componenet such as DNA and causes structural changes(Tchounwou et al., 2012). Accordinog to the above commens, In the several environmental protection agencies LED defined as hazardous waste(Lim et al., 2010).Therefore, the improperly disposal of LED waste resulting in adverse impact on human health and the enviroment. 1.2 LEDs waste management Several management tools have been applied for E-waste management which can be improve waste disposal. Directive 2002/95/EC known as RoHS(Restriction of Certain Hazardous Substances) is one of the main law that have been performed to prevent or reduce the use of toxic substances in electronics equipment. Factually,This resolution banned manufacture for use of hazardose substance to make product and the manufacture being responsible for E-waste recycling(Hester and Harrison, 2009; Veit and Bernardes, 2015).The Basel ban convention are established for transboundary movements of hazardous waste and its deposits. According to this Convention electric and electronic equipment waste (WEEE) must be appropriate landfill and recovery. Life cycle analysis has shown that priority of the WEEE waste management methods is through reuse and recycle(Jadhao et al., 2016; Li et al., 2013; Ongondo et al., 2011). WEEE disposal is most important concern in worldwide. Most of WEEE disposal in landfill or incineration which can be made serious environmental contamination. The flame-retardants containing bromine component presense in most of WEEE composition, when burning can generated harmfull emissions such as dioxins and furans. Nowadays, research and industrial processes are usually planned to recover the metal fractionin from E-waste due to the economical benefits(Kaya, 2016; Veit and Bernardes, 2015). The management of LED waste is important due to decreasing environmental effects and

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

increasing source of raw material. Currently, due to lighting plays important role to society, extensive research was done about this waste management in order to remove toxic impact on human health and enviromental in both nationl and international levels, in order to abtain this aim it is nesseray employed certain sterategy. The LED waste has heterogeneous nature and it has major effect on processing, recycling and other parts of waste management process. But, the management of this waste is still deficient in the majority of countries around the world. 1.2.1 LEDs bioleaching There are three main technologies: pyrometallurgy, hydrometallurgy and biohydrometallurgy for metal recycling from electronic waste. Bioleaching is one of the specific green techniques which successfully applied for metal recovery from electronic waste(Xu and Liu, 2015). Bioleaching is the dissolution of solid metallic compounds from their mineral sources through the natural ability of microbes to generate oxidizing agents such as iron (III) ions and/or protons in the system(Erüst et al., 2013).Compared to common extraction methods, bioleaching is more environmentally-friendly, economical, energy efficient and simple to implement; thus it is the most promising technology for recovery of valuable and heavy metals from solid waste(Asghari et al., 2013).The iron (II) oxidizing bacteria (Acidithiobacillus ferrooxidans), is one of the important microorganism which used for metals recovery from electronic waste(Erüst et al., 2013). A. Ferrooxidans reoxidation ferrous iron to ferric iron by chemical action under acidic conditions and low oxygen. Then, insoluble metals are oxidaized to soluble metal ions by biologically generated ferric iron[mishra2005]. Microbilogy management is one of the most important part to search in bioleaching process(Jang and Valix, 2017). One weak point in bioleaching process is bactrial sensivity to metallic ions concentrations(Astudillo and Acevedo, 2008).The microorganism have a biophasic response to against heavy metals. Several heavy metals such as Ni,Cu,Zn,… are essential for bactrial growth but beyond certain concentration become toxic and have negative affect on bacterial activity and metals leaching efficiency[valix2003]. When metals concentration increasing over acceptable levels the bactrial uses resitant mechansim for survive because of high metal concentration interrupt cell function throughout damage to bactrial cell membrane and change nucleic acids structure and enzymes(Orell et al., 2010). Exposure of A. ferrooxidans to LED waste which containing elevated mixed heavy metals concentration result in inhibition of bactrial growth,especially at high pulp density, but A. ferrooxidans can be adapted to tolerate several heavy metals. thus, adapted A. ferrooxidans can grow at significantly higher pulp density due to active biochemical pathway which allow to bacterial cell continue to growth, compared with the same non-adapted bacteria(Molaei et al., 2011). zhung et al. (2014) investigated the TBBPA had an adverse impact on the structure and function of microbial community.The results suggested that the soil microorganism population decreased with increasing in TBPPA concentration and high concentration of these substance can make inhibitory effect (Zhong et al., 2014).The TBBPA exit at many electronic waste (at special LED), up to now there is no study of their impact on bioleaching micobes. According to the pervious investigiation, there is several methods for acidiphilis bactrial adaptation to sulfide minerals and concentrates containing high concentration of heavy metals. one of the most commen methods for A. ferroxidanse adaptaion to heavy metals is serial subculturing. Haghshenas et al. (2008) reported succesfully adaptation of A. ferooxidanse to high grade sphalerite concentrate by using serial culture methode in two diffrent modes. First they use diffrent concentration of Zn2+ ions for bacrerial adaptation and then the adaptated bactrial to Zn2+ ions transfer to media containg high concentration of sphalerite concentrate(4.5 wt/vol.% pulp density ). In the secod procedure the bactria cell adapted to different pulp density

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

of sphalerite concentrate. In finally the obtained result compared to each other and shows the first mode is more successfully than for Zn2+ recovery(Haghshenas et al., 2009). The adaptation criteria is important point in the adaptation process and based on the bacteria cells can be concerned as adapted. For example in the study by wang et al. (2013), the adaptation criteria was the bacterial cell number, when the cell density of a culture at the end of the culturing at a given pulp density reached 2×108 cells/mL (Wang et al., 2014).Hong et al. (2015) reported, the adaptation of mixed culture of acidophiles bacteria from refectory gold concentrate containing high concentration of Arsenic. The bacteria adaptation carried out according to the wang et al. (2013) procedure. The obtained results shows, adapted bacteria showed a peak maximum percentage of biooxidation and it can overcome negative impact of increasing in pulp density(Hong et al., 2016). This study is the first report that investigated A. ferrooxidans adaptation behaviour in simultaneous present of two different toxic component: heavy metals and BFRs which existence in all electronic waste( at specially LED). The large portion (>70% ) of LED waste included of BFR materials. The unpleasant aspect of BFRs existed in LED will be the major impediment in during bioleaching process and can reduced metals recycling efficiency. Thus, more systemic studies on the adaptation of A. ferrooxidans to electronic waste required. Considering the above comments, the main aim present study was to focuses on adaptation capacity of A. ferrooxidans in present high concentration of toxic substances for metals recovery from LED waste. 2. MATERIALS AND METHODS 2.1 LED waste preparation The several discarded pin-type LED, were collected from various repairer electronic shops in Tehran, Iran (Fig.1). The samples of LED were in different colours and luminous intensities which used as different application. The sample of LEDs were shredded with industrial crusher (IKA-Werke, Germany) and then samples were milled using a ball mill (Fristch; Germany) to achieve a fine powder and finally, screened through standard 200 mesh by electric vibrating.The sieved particle size was smaller than 70 µm(Fig. 1).

Fig. 1 LED and its powder used in bioleaching experiment 2.2 Microorganism and culture condition In the present study, A. ferrooxidans as ironoxidizing bacteria was obtained from the Iranian Research Organization for Science and Technology (IROST) in Tehran, Iran. A. ferrooxidans was cultured in 9K medium contains basal salts solution 3 g (NH4)2SO4, 0.5 g

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

K2HPO4, 0.5 g MgSO4. 7H2O, 0.1 g KCl, 0.01 g Ca(NO3)2 and 44.22 g FeSO4 .7H2O as an energy source. The medium pH was adjusted at 2 by H2SO4. A. ferrooxidans 2% (v/v) was incubated in 250 mL Erlenmeyer flask containing 100 mL of the medium and shaken at 140rpm in an orbital shaker at 29 oC. 2.3 Bacterial adaptation protocol and bioleaching experiment The A.ferrooxidanse needed to be adapted for increasing its tolerence to high concentration of heavy metals(Al, Cr, Cu, Ni and Zn) and BFRs in LED powder. The culture described in pervious section was adapted to 2% (w/v) pulp density of LEDs waste. Adaptation of the bacteria strain was performed through a serial sub-culturing procedures. In this study, the adaptation criterion was bactrial cell concentration. First A. ferrooxidans was adapted to LED waste at low pulp density 1 g/l , when bactria cell concentration in culture reached 5×107 gradually exposing them to growth medium containing higher LED waste concentrations 5,10,15,20,25 g/l for 30 days. At the end of expriments the final culture has containing highest LED waste concentration which can be tolerate by A.ferrooxidans . One-step bioleaching of 2% (w/v) LED waste was carried out by inoculating 5×107 cell/ml adapted and non-adapted bactria to LED waste. The pH of culture was fixed to 2.0 with H2SO4. All Bioleaching expriment was performed at 30 °C, 140 rpm. After the desired bioleaching time, metal concentration monitored by taking sample from each flask and filtered through filter 0.45µm and analyzed by Inductively Coupled Plasma Optical Emission Spectrometers (ICP-OES) using standard procedures. 2.4 Analytical method The indection of bacterium response to heavy metals was mapped by calculating growth tolerance index (TI) with time. According to the follwing equation the tolerance index was measured from growth rate of the bacteria in the presence of the various heavy metals divided to a control, which contained no heavy metals(Valix and Loon, 2003). cellcount(cell / ml)withHM TI = cellcount(cell / ml)withoutHM Maximum specific growth rate (µmax) was calculated from the exprimental data of logarithmic phase in A. ferrooxidans growth curve using the following equation:

⎛X⎞ ⎟/t ⎝ X0 ⎠

µ max = ln ⎜

Oxidation–reduction potential(ORP) was measured with Eh meter(Metrohm, Swiss). The pH of the reaction solution was determined with a (Metrohm,Swiss).The bacteria free cell number in the liquid solution was calculated through direct counting using Neuber counting chamber with phase contrast microscope (Carl Zeiss, Germany). The analysis of Ferric iron concentrations in bioleaching solution were performed by spectrophotometeric method using 5-sulfosalicylic acid at a wavelength of 500 nm (Karamanev et al., 2002).

Metals concentration in bioleaching solution and digests of LED powder were determined by by inductively coupled plasma optical emission spectrometer (ICP-OES; Vista-pro, Australia). The X-ray diffraction (XRD) (X’Pert MPD; Philips; the Netherlands) was used to analyses chemical and mineralogical of LED powder with Co Karadiation, a tube voltage of 40 kV and 30 mA of current. The first identification of BFR and several impurity in LED powder sample was conducted by acombination of techniques: (1) Fourier transformed infrared spectroscopy FT-IR (400–4000 cm-1)( Frontier,Perkin-Elmer, USA) were identified polymer (blend) types by spectra library. (2)

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

The Raman spectrum of the sample was measured with an excitation wavelength of 532 nm between (100-4200 cm-1)and a spectral resolution of 4 cm-1with a power of 30 mW on Almega Thermo Nicolet Dispersive Raman Spectrometer. We compared results for raw LED sample before and after bioleaching process. The morphological study of the virgin and residual LED samples was performed by scanning electron microscopy (SEM) and Energy Dispersion Spectroscopy (EDS) under high vacuum conditions. The both samples were coated with thin layer of gold for observation. The SEM observations show morphological and chemical surface changes occurring in raw sample during bioleaching process. 3. RESULT AND DISCUSSION 3.1 LEDs waste component The LED consist of various recyclable fractions such as metals, plastics and ceramics. According to the wet chemical method, plastics are the frist largest component by amount up to 70% of total LED weight and the remaining mass is derived from the metals and ceramics(see Fig. 2a). The concentration of plastics in LED waste is not negligible. The LEDs can contain various polymers such as Epoxy resin, polypropylene (PP), high impact polystyrene (HIPS), acrylonitrile-butadiene-styrene (ABS) and polycarbonate (PC) blends on their composition. In generally, BFRs are the key additives in polymers that extensively used to increase their fire resistance(Han et al., 2007). There is brominated polystyrene, tetrabromobisphenol-A(TBBPA), decabromodiphenyl ether(DecaBDE) and brominated polystyrene as brominated flame retadents in polycarbonate, epoxy resin and high impact polystyrene repectively(Morf et al., 2005). The BFR use as polymer cover in LED. In generally, any inorganic and non-metalic material defined as ceramic material such as silica and other oxides(Ba, Ti, Mg, Nb, Zn, Ta oxides). The ceramics use in electronic equipment, beacuse of their special peroperties such as semi-conducting, superconducting, ferroelectricity and superesets properties. The silicon microchips is using in LED result in the ceramic materials such as sillica or silicon can be found in LED (Veit and Bernardes, 2015).

30% metals

70% plastic

Fig. 2 Materials fraction in LED The threeteen metals were identified in LED powder using inductively coupled plasma (ICP) after chemical digestion. The metal concenteration shows in Table1. The result show iron is the most widely used metal in LED with more than 90% of total metalic weight. Thus, the LED may be considreded as the most important source of base (Al,Cu,Fe,Pb,Sn, Zn) ,percious(Ag, Au) and rare metals(Ga, As, In).This waste represents a considerable amount of heavy metals such as cooper, nickel, coroum, arsenic, lead that this metals can have hazardous nature and

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

hampered the bacteria growth and metabolism during bioleaching process. The CHNS elemental Analyzer(ECS 4010, Costech, Italy) was used to analyze the amount of carbon, hydrogen, nitrogen and sulfur (CHNS) in LED powder .The average elemental carbon, hydrogen nitrogen and sulfure content of the LED powder were 46.5,0.05,0.22,7.79% (w/w),respectively. Table1. Metal concentration in LED powder Metals ppm Silver 17.818 Aluminum 3.5484 Barium 0.23976 Chromium 2.1246 Copper 47.734 Iron 3108.6 Gallium 4.0095 Indium 1.2364 Nickel 18.174 Lead 2.9088 Zinc 0.61993 Arsenic 1.2703 Gold 0.60924

mg/kg LED 1781.8 354.484 23.976 212/46 4773.4 3108600 400.95 123.64 1817.4 290.88 61.993 127.03 60.924

3.2 Effect of adaptation on bacterial growth A. ferrooxianse kinetic parameter ( µ max ) obtained from corresponding growth curve shows in Table. 2 The result shows increasing in pulp density concentration led to increasing lag time in bacterial growth curve. The high pulp density concentration has negative effect on A.ferrooxidans cell growth, and with increasing pulp density in range of 0-20 g/l led to an increase in the lag time from 2 day (for pure culture) to 9 days for 20 g/l pulp density and as well as decreasing in specific growth rate from 0/676 to 0/122 (1/day) for 0 and 20 g/l pulp density, respectively. It is most likely due to the inhibitory effect and toxicity of various hazardous compounds such as heavy metals, phenols, and BFRs in LED powder. So that by increasing pulp density, A.ferooxidanse need more time to grow. In pulp density≥ 25 g/l LED, the concentration of both chemical and metals component for the bacteria was completely toxic and the growth of the bacteria stopped. In the bioleaching of high grade sphalerite concentrate by A.ferooxidanse the specific growth rate decreased as pulp density increased, the specific growth rate decreased from 0.047 1/h for the control to 0.018 1/h for 60 g/l pulp density (Haghshenas et al., 2009). An increase in the lag time by increasing in electronic waste concentration has also been reported in the ten et al. (2003) study in bioleaching of electronic scrap material by Aspergillusniger and Penicillium simplicissimum(TEN WEI, 2003). Table2. A. ferrooxianse kinetic parameter Pulp density ( g/l ) 0 2.5 5 10 15 20

Lag time ( day ) 2 4 6 6 8 9

µmax ( 1/h ) 0.0282 0.0216 0.0135 0.011 0.0096 0.005

µmax ( 1/day ) 0.678 0.519 0.324 0.268 0.232 0.122

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

3.3 Acid production during adaptation The largest part of LED powder is plastic. It has different organic fraction including aromatics and BFRs which led to alkaline nature. When LED powder stirred in distilled water its pH was neutral and the alkalinity nature was not exhibited. This event was only shows when the LED stirred in acidic medium that some alkaline materials transfer into the medium through the action of acid(Ilyas et al., 2007; Ilyas et al., 2010). In this study the pH profile studied under two different conditions. The effect of pulp density on acid production during adaptation shows in Fig. 3a. Two reasons can be considered for the sharp increase in pH at the beginning of the reaction: 1- The alkaline compounds in LED use of release protons in medium result in increasing pH value. For higher pulp density, this increase in pH value is higher because of by increasing in pulp density the concentration of alkaline substances increased. In maximum pulp density (25 g/l ) after one day the pH value increases to 4(Işıldar et al., 2016). 2- Ferrous metal is largest metalic component by amount up to 90% of total LED metals weight. A. ferooxidanse oxidation ferrous ion (Fe2+) to ferric ion (Fe3+) by using protons and increasing pH value according to the following equation (Bajestani et al., 2014). After 48 hour of growth period the pH value for all pulp density induced because of two reason first the ferric ion hydrolysis in aqueous solution (Eq. (2) and (3)) and generated H+ and next reason is the extracellular polymeric substances (EPS) present on the surface of A. Ferrooxidans were mainly contained OH and –COOH functional group. As can be seen in Equation (4) EPS make complex with ferric ion and released H+ into solution(Nie et al., 2015). After 12 days the pH value was stabilizes in range of 2 to 2/5 until end of bioleaching process. It's contributed to formation jarosite from competing reaction for the hydrolysis. The influence of adaptation on pH of system is shown in Fig. 3b. The initial pH of leaching system for adapted and non-adapted culture was adjusted to 2. The results obtained for pH showed a similar trend for both the adapted and non-adapted conditions at the beginning of the process and, the pH increased from 2 to 4. When bioleaching is carried out after 30 days of reaction time pH value for adapted culture was ≤2 but for non- adapted culture higher pH values were obtained. This increase of pH confirms that adaptation does effect on the pH of the system. Adapted A. ferrooxidans has higher acid production rate because of adaptation increased bacterial tolerance against alkaline components in LED powder. In other hand the adapted cells have more positive group on their surface which tend to decreased metal citations and removed negative bacteriostatic effects of heavy metals. Actually adaptation allowed bacterial cell to growth in medium rich of metals and other toxic components (Jang and Valix, 2017).

Fe 2+ + O 2 + H + → Fe3+ + H 2 O

(1)

Fe3+ + 2H 2 O → Fe(OH) 2 + 2H +

(2)

Fe3+ + 3H 2 O → Fe(OH)3 + 3H+

(3)

Fe3+ + 3 EPS − H → Fe(EPS)3 + 3 H +

(4)

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

(a) 3.8

5g/l 10g/l

pH

3.3

15g/l 20g/l

2.8

25g/l

2.3

1.8

0

5

10

15

20

25

30

35

Time (day)

Fig. 3 the change of pH over time (a) for various pulp density (5,10,15,20 g/l )(b) for adapted and non-adapted A.ferrooxidans at 20 g/l LED powder 3.4 Influence of adaptation on the Fe Oxidation Activity of A. ferrooxidans Oxidation-reduction potential The ORP is most important parameter that indicated bacterial activity in bioleaching process. The effect of different pulp density concentration on value of ORP during adaptation process shows in Fig. 4a the LED powder has immediate effect on ORP value at the beginning of the process when LED powder addition to bacterial culture the value of ORP dropped because of there is large amount of acid consuming material (Fe2+ ≥90%metalic weight and alkaline material≥70% of total LED weight) in LED powder. The value of ORP impressively descended with addition of the pulp density from -37 for 5 g/l pulp density to -470 for 20 g/l pulp density because of in higher pulp density there is more acid consuming material (TBBPA and Fe)(Bryan et al., 2015). After 48 h the ORP value rapidly increased with the oxidation of Fe2+ to Fe3+ by A.ferrooxidans activity. The need time for reach to maximum ORP was increased by increasing in pulp density from 6 to 12 days for 5 and 20 g/l, respectively. With over time the value of ORP for all pulp density was stabilizes in range of 500-560 mv until end of bioleaching process (30 days). At pulp density 25 g/l the heavy metal and other toxic component was beyond the microorganism inhibitory concentrations so disrupt the integrity of

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

cellular membrane and affect its population in leaching environment. A. ferrooxidanse hasn’t any activity in 25 g/l LED powder and the maximum ORP value was 360 mv. Fig. 4b shows, the ORP trend for Adapted and non-adapted bacteria with time. The ORP trend for adapted bacteria tends to increase with time and after 9 days reach to maximum value (546 mv) but the maximum ORP value for non- adapted bacteria after 30 days was 372 mv. Higher values of ORP represent a higher oxidation rate from Fe 2+to Fe3+, which implies a higher adapted bacterial activity in comparison to nan- adapted bacteria in the system. Ferric ion concentrations data presented in Fig. 4c shows the effect of various pulp density on Fe2+ oxidation with time during adaptation process. The Fe3+ concentration regeneration was higher rate at lowest pulp density (5 g/l) compared to higher pulp densities (10-15-20-25 g/l ).the time needed to reach maximum Fe3+ concentration were 6 and 9 days for 5 and 20 g/l LED powder ,respectively. The Fe3+ concentration observably increased with increasing in pulp density from 5 to 10 g/l, due to its heterogeneous nature and it contained significant quantities of iron. The results show that at pulp density≥25 g/l has inhibitory effect on Fe2+ oxidation rate by A.ferroxidanse and the maximum value of Fe2+ concentration was 1.547 g/l. a decrease in the Fe3+ concentration in the solution observed after 10 days can be attributed to the formation of a jarosite [Eq. (5)]. The Fe3+ concentration for adapted and non-adapted A.ferroxidans with time shows in Fig. 4d. The ferric ion concentration and ORP (Fig. 2b) has similar trend. The results shows that the Fe3+ concentration for adapted in comparison to non-adapted culture increases with time. When using adapted culture higher Fe3+ concentration (6.436 g/l) obtained after 6 days of process. The Fe3+ concentration difference between adapted and non-adapted A.ferroxidans proved that adapted A.ferroxidans have higher Fe2+ oxidation rate which implies a higher adapted bacterial activity in the system. The Fe3+ concentration was stable for non-adapted culture at 0/5 g/l in the first 12 days of process, continued by brief increase and reaching the highest concentration (1.5 g/l) at 20 days of process. After this point the ferric concentration was stabilizes until end of bioleaching process it's suggested that non-adapted A.ferrooxidans activity completely stopped.

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

Fig.4 ORP changes of bioleaching solution (a) in various pulp density(5-20 g/l),(d) for adapted and non-adapted A.ferrooxidans at 20 g/l LEDpowder, (c) the concentration of Fe3+ in bioleaching solution for various pulp density(5-20 g/l ), (d) the concentration of Fe3+ in bioleaching solution for adapted, non-adapted A.ferrooxidans and control at 20 g/l LEDpowder 3.5 Adaptive behavior of the bacteria The LED would be categorized as hazardous waste. The results of TCLP analysis suggested that the LED contains heavy metals such as lead, cooper, nickel and silver at exceeded regulatory limit (Lim et al., 2010). When the pulp densityof LED powder increases led to greater heavy metals toxicity in leaching solution (Santhiya and Ting, 2006). The accumulation of heavy metals beyond the tolerable concentration of microorganisms results in damage to the cell membrane and affects the cell population in the bioleaching environment. Considering all this, we need to new strain which can be tolerance high concentration of heavy metals, adaptation led to an increase in tolerance of the bacteria to high concentration of heavy metals. They are using different resistance mechanism against heavy metals. In generally, acidophilic bacteria uses genetic and biochemical resistance mechanisms to reduce negative heavy metals affect. They make active eflux of heavy metals from cytoplasm to periplasmic(Orell et al., 2010). Jong. et al (2017)studied resistance mechanism Acidithiobacillus thiooxidanse for arsenite toxic effect, the result shows A.thiooxidanse can efflux As3+ of its cell membrane by ArsB protein in order to decrease toxic effect(Jang and Valix, 2017). In pervious study reported that in environment with high concentration of copper, A. ferrooxisdanse expressed unknown proteins on its surface for survive in toxic condition as well as A. ferrooxidanse loses extrachromosomal structures in present high concentration of cooper (Orell et al., 2010). Adaptation is common strategy that provided tolerance profile for bacteria growth. In this method effect of 10 g/l pulp density on A. ferrooxidans growth over the time depicted in Fig. 5a A. ferrooxidans growth pattern was investigated by five stages as (a) lag phase that happen for 6 days beginning of the process (b) “continued with rapid growth up to day 11 (TI=0.55)” (c) “retarded growth: the growth was declined for 3 days” (d) “similar growth was observed between 14 to18 days (TI=0.5) and (e) “enhanced growth was show from 19 to 25 days (TI=0.8 -1). The TI of the similar growth phase ≤1. It is reasonable to conclude that present the heavy metal reduced adapted A.ferrooxidans growth and LED powder being most toxic. Fig. 5b presented the tolerance index of the A.ferrooxidans adapted to different concentration of LED powder. The

Sardinia 2017 / Sixteenth International Waste Management and Landfill Symposium / 2 - 6 October 2017

adaptation of A.ferrooxidans to LED powder resulted in an increase in the tolerance index of the bacteria with increasing concentration of pulp density. A.ferrooxidans cell concentration over time in various pulp density shows in Fig. 5c, For all pulp density a lag phase occurred at the beginning of inoculation, the highest lag time is related to the highest pulp density (20 g/l ).it can be seen from Fig. 4c the cell concentration increased very slowly during first 9 days then fixed from day 9 to day 24. The cell concentration started to increase that coinciding with increase Fe3+ concentration or ORP value (see Fig. 3). At 25 g/l LED powder no bacterial growth was observed because of high toxicity of LED for bacterial activity. Fig. 5d shows effect of adaptation on A.ferrooxidans growth behavior. The adapted bacteria cell concentration after along lag phase (9days) reach to 8×107 in present 20 g/l LED powder. The non-adapted culture grew slowly and they have no reproduced after 24 days of process and the highest cell concentration was 4×107. It is reasonable to conclude, when the bacteria were adapted changes many genetic systems and use new biochemical pathway to tolerate high concentration of heavy metals and other chemical toxic material to survive in adversely condition.

(b)

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Fig. 5 (a) growth phases of A.ferrooxidans in the present of 10 g/l LED powder, (b) tolerance index of A.ferrooxidans in different LED powder pulp density (5-20 g/l),(c) cell concenteration of A.ferrooxidans in bioleaching solution at different LED powder pulp density(5-20 g/l),(d) cell concentration of A.ferroxidanse in bioleaching solution for adapted, non-adapted A.ferrooxidans at 20 g/l LEDpowder

3.6 Bioleaching of the metals from LED waste Fig. 6a illustrated maximum extraction of the metals (cooper, nickel) in leaching with adapted and non-adapted culture. It is shown at the 20 g/l pulp density the adapted A.ferroxidanse leached approximately 0.96 g/l (100%) and 0.58 g/l (100%) cooper and Nickel, respectively. non-adapted culture represented a 30% and 15%decreasing in copper and Nickel extraction efficiency. It was suggested that positive effect of pre-adaptation on bioleaching metals from LED waste. The metals extraction occurs by adapted A.ferroxidanse according to following steps: At first step A.ferooxidanse produce Fe3+ as oxidizing agent as expressed byEq. (1). At

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second step Fe 3+ is responsible for oxidizing metals in the LED by Eq. (5). The ferric ion played an important role in the metals recovery by adapted A.ferooxidanse. In the bioleaching with adapted culture the system containing a high concentration of Fe3+ ions will furthered metals removal efficiency. In the leaching process with non-adapted A.ferroxidanse after for 12 days there is no significant increases in the Fe3+ concentration (see Fig. 4) .So that it is reasonable to conclude that metals was directly leached out chemically under Eq. (6). In fact, the hydroxide ions that formed are responsible for increase pH of value in non-adapted culture during bioleaching experiment (Fig. 2)(Chen et al., 2015). 2+

Fe 2 (SO4 )3 + M0 ⇔ M2 + + (SO4 ) + 2FeSO4 M [Cu, Ni ]

(5)

M 0 + H 2 O + O 2 ⇔ 2M 2+ + 4OH −

(6)

Based on the result obtained, adapted A.ferrooxidans had higher metals bioleaching rates compared to the non-adapted strain. It is can be concluded that the adaptation may affect the efficiency of metals extraction from electronic waste. The LED powder contained mixed heavy metal and other toxic material in their component that led to grater bacteriostatic effect. It has been pointed out that in several study the A.ferrooxidans uses several resistance mechanism in order to survive in toxic condition. In the bioleaching process of LED waste Fe2+, Cu2+ and other metals as well as polymeric material such as BFRs are generated and released in to bioleaching solution. When the concentration of metals elevated acceptable levels for A.ferroxidanse can be deteriorated cell membrane or inactive enzyme, and virtually cellular function can be interrupted. Considering all this, the bacteria uses resistance mechanism in order to survive in extreme conditions and reduced metals negative effect. The adaptation to the LED powder enhancement in ability of the A.ferroxidanse cells to survive in this condition and metals recovery effiency increase by bacterial activity. Fig. 6b shows the metals removal efficiency as a function of time by adapted A.ferrooxidans into 9K medium containing 20 g/l LED powder. The lowest metal extraction is related to early 6 days process, after 9dayes, the Fe3+ concentration and value of ORP reached to 6.436 g/l and 546 mv which is corresponded to significant bacterial population in solution. After days 12, the metal extraction was rose from 30%, 40% to 66%, and 80% for cooper, nickel, respectively. After this point the Fe3+ concentration and pH began to decrease which led to an increase of metals recover. At the end of process, approximately 100%cu, 100%Ni leached from 20 g/l LED powder.

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Fig. 6(a) metals recovery from LED waste at 20 g/l pulp density by adapted and non-adapted A. ferrooxidans, (b) metals recovery efficiency over time by adapted A.ferrooxidans at 20 g/l LED powder 3.7 Characterization of LED powder before and after adaptation process XRD analysis the XRD pattern of the LED powder before and after bioleaching process are shown in Fig 7. The ICP data substantiated that there are high concentration of iron, silver and copper in LED powder. Thus, it is reasonable to conclude the XRD pattern confirmed the result that obtained from ICP analysis in Table 1. XRD patterns show crystalline phases for LED powder these marked peaks suggested the present of iron, silver, silica and tin (Fig. 7a). The iron has two major peaks at 52.752 and 77.442. The peak located at 44.546 and 37.358 is related to silver and tin. The existence of silica in LED powder monitoring by peaks at 35.810. After bioleaching process (30 days) the reddish brown participate was obtained in bioleaching with adapted A.ferrooxidans. The X-ray diffraction was developed to further characterization the composition of the precipitate at both condition (with adapted and non-adapted A.ferrooxidans). In accordance to the results obtained, Goethite and jarrosite with the formula (FeO(OH)),KFe3(SO4)2(OH)6 were the magnetically dominant phases after bioleaching. The formation of Iron oxide hydroxide and Iron hydroxysulfates can be expressed as Eq. (7) and (8)(Silva et al., 2015). Fe(OH)2+1/4 O2 FeOOH+1/2H2O 3+

Fe(OH) 3 + SO24− + Fe + H2O + K + → KFe2 (SO4 )(OH)6 + H +

(7) (8)

In the Precipitate with adapted bacteria the jarosite phases was more than non-adapted bacteria participate. It is believed that the adapted bacteria could be generated the higher Fe3+ concentration in bulk solution (Fig.3). The Geothite phases in participate for both adapted and non-adapted condition was almost the same.it can be concluded that the adaptation has no significant effect iron oxide hydroxide generation. It can be concluded that all of Fe2+ concentration oxidation to generated FeOOH was carried out by chemically reaction under Eq. (6)(Lane, 2007).

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Iron Silver Tin Silica

Jarosite Goethite

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Jarosite Goethite

Fig.7 XRD pattern of (a) virgin LED powder, (b) the reddish brown precipitate obtained after bioleaching with adapted A.ferrooxidans, (c) the reddish brown precipitate obtained after bioleaching with non-adapted A.ferrooxidans at 20 g/l pulp density FTIR analysis the analytical result from infrared spectra (100-4000) of LED powder and residual after bioleaching process with adapted and non-adapted A.ferrooxidans shows in Fig. 8a. FTIR analysis of LED powder before bioleaching process revealed present of PC and TBBPA as polymers in their structure. The Raman spectroscopy (Fig. 8b) has also been developed for postulates IR spectra pattern. Raman activity show excellent agreement with the IR intensities for LED powder before bioleaching process. The C-X[X=cl, Br, I] vibration of commonly shows multiple weak bands in the range of 480–1129 cm-1. The bands observed in 560.77 is unambiguously assigned to C-Br band in TBBPA Structure as well as Raman bands at 181 and 632 confirms the C-Br vibration. The. The C-H band in methyl groups of TBBPA was observed at 2955 cm-1. The C–H in plane and out plane vibrations related to benzene in TBBPA shows with frequency1011, 1043, 1183and 829.96 cm-1. The C-O stretching vibration of phenol are often in the region 1200-1300 cm-1 and the O-H bending and stretching vibration occur in the range 1260-1420 cm-1 and 3200-3650 cm-1 respectively. In this study, the frequency at 1236 cm-1 and 1362cm-1 and 3654cm-1 as assigned the C-O and the O-H, bonds in TBBPA. It is interesting to note, the two peaks at 3059 and 3584 in Raman spectra unambiguous assigned to O-H vibration. The C–C stretching vibration of benzene normally appeared in the region of 1400–1650 cm. in this study, hence four C–C bending vibrations clearly can be seen at 1458, 1510, 1583, 1608 cm-1. Nevertheless, the peaks at 1598cm-1 was observes in Raman spectra that related to C-C band in TBBPA. The deep band at 1720 cm-1is attributed to O=C (carbonyl group) in poly carbonate (Qiu et al., 2013; Taurino et al., 2010). The infrared spectral of LED powder after bioleaching with adapted and non-adapted A.ferrooxidans shows in Fig.8c. It can be found from obtained result that adaptation has no significant effect on organic component and non-metallic part of LED powder. In generally, the comparison between adapted and non-adapted IR spectra allows to result in that adapted and non-adapted IR spectra after 30 days bioleaching were almost overlapping but it is reasonable to conclude there was some changes in the absorption band peaks However, the characteristic peaks after bioleaching process indicate significant changes of C-Br, C-O and O-H stretch (in phenol group ) and C-C stretch (in benzene ring )at TBBPA structure and O=C stretch in carbonyl functional group in poly carbonate. This findings indicated that the oxidation of TBBPA and PC with H2SO4 occurred in during bioleaching process (Zhong et al., 2014).

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Some of these bands at 560.77, 1236, 1362, cm-1 related to C-Br,C-O,O-H completely disappeared and the absorbance C-C and C-H stretch (the specified peak at1510 and 2955 cm1 ) significantly decreased after bioleaching with both adapted and non-adapted bacteria because of the isopropyl group were oxidized to carboxylic by H2SO4 during bioleaching reaction. It has been explained Geothite and jarosite formed from ferrous and ferric ion in during bioleaching process. In addition to, some of Ferric ion produce more-complex with sulphate for instance Carphosiderite and Parabutlerite.The 437.45 and 630.12 cm-1 peaks is related to the Fe-O vibration in the Goethite (Blanchard et al., 2014). The Jarosite exhibits three bonds at 1001cm-1, 1082cm-1 and 1194cm-1 (Lane, 2007). The deep vibration was observed at 1425cm1 in IR spectra after bioleaching process is related to other Iron hydroxysulfates ( Carphosiderite and Parabutlerite)(Lane, 2007). The long-wave, broad bond at 3408 cm-1 is attributed likely to O-H vibrations due to high concentration of jarrosite and Goethite and other Iron hydroxide complexes in LED residue after bioleaching (Prasad et al., 2006).

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Fig.8 (a) FTIR spectra of virgin LED powder, (b) FT-Raman spectra of virgin LED powder, (c) FTIR spectra of precipitate obtained after bioleaching with adapted A.ferrooxidans and nonadapted A.ferrooxidans at 20 g/l pulp density

SEM analysis Scanning electron microscopy was carried out to examine the surface morphology of the LED powder before and after bioleaching process. The color of LED powder with bioleaching changed from gray to reddish brown. Comparison of raw LED powder and residue resulting from bioleaching with adapted and non-adapted A.ferrooxidans when exposure to 20 g/l pulp density is shown in Fig. 9. It accordance to the SEM observation the LED powder having clean and smooth surface before bioleaching reaction, whereas after 30 days of bioleaching with adapted A.ferrooxidans the SEM images(Fig. 9b) indicated that the LED powder particle totally shrinking to smaller size by Fe3+ as oxidation agent that generated by adapted bacteria. The most interesting result was that formation of poly porous layer on the surface of LED particle it is due to the adapted A.ferrooxidans during bioleaching reaction attached in these small particle and increasing porosity and roughness of surface LED powder. The sample of LED powder bioleached with non-adapted bacteria is shown in Fig. 9C. Larger particle size were seen in bioleached LED powder with non- adapted bacteria due to the lower content of oxidation agent in non-adapted culture. According to the growth result (Fig4) the bacteria population in non-adapted culture is too low, thus the porous layer resulting from bacterial attachment on LED particle surface was not identified in residue after bioleaching with non-adapted bacteria.Thus it is reasonable to conclude that bacteria adaptation has significantly effect on LED particle for metals recovery. In this case the adaptation plays most important role for direct bioleaching from LED powder.

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(a)

(b)

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(c)

Fig. 9 SEM images of (a) LED powder, (b) the precipitate obtained after bioleaching with adapted A.ferrooxidans , (C) the precipitate obtained after bioleaching with non-adapted A.ferrooxidans in 20 g/l pulp density 4. CONCLUSION In this paper indicated adaptation enhanced tolerance of A.ferrooxidans to LED waste which containing high concentration of toxic material (heavy metals and BFRs) in the bioleaching process. Various analytical techniques were used to monitor the compositions of LEDs. Initially, the chemical structure of plastic material was identified by Fourier transform infrared spectroscopy and FT-Raman spectra. Polymeric contaminants of these plastics, in particular BFRs were detected in LED powder.The tolerance index was use to indicated effect of heavy metals in bacterial growth and tolerance behavior with increasing pulp density.The results indicated that the both BFRs and heavy metal could cause toxicity response. Generally highdose of LED powder resulted in obvious inhibitory effects. In the present of 20 g/l LED powder the growth characteristics (pH, cell concentration, Fe3+ concentration, Eh) of adapted and nonadapted A.ferrooxidans was analysed. The results show that adaptation can be used as an efficient tool for enhancement of A.ferrooxidans activity in bioleaching solution containing high concentration of toxic substance. By comparing the results obtained from metals bioleaching with adapted and non-adapted bacteria, a reduction 30% and 15% in copper and nickel extraction was observed, respectively.thus, adaptation have positive effect in bioleaching process REFERENCES Asghari I., Mousavi S.M., Amiri F., Tavassoli S. (2013). Bioleaching of spent refinery catalysts: A review. Journal of Industrial and Engineering Chemistry. vol. 19, 1069-1081. Astudillo C., Acevedo F. (2008). Adaptation of Sulfolobus metallicus to high pulp densities in the biooxidation of a flotation gold concentrate. Hydrometallurgy. vol. 92, 11-15. Bajestani M.I., Mousavi S., Shojaosadati S. (2014). Bioleaching of heavy metals from spent

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