Team:Tec-Monterrey/Description

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• Overview
• Module 1
• Module 2
• Module 3

Overview

Monterrey is one of the leading industrialized cities in Mexico, but this growth has led to many environmental and health issues. The metropolitan area is considered as the most polluted in Latin America in terms of air quality (Clean Air Institute). One of the main reasons is the high concentration of PM10 in the atmosphere, particles so small that they are able to penetrate deep into the lungs and cause a wide variety of pulmonary and heart diseases, leading to a premature death. It is estimated that industrial activities and “primitive” e-waste recycling techniques are among the main factors for the release of these type of particles into the atmosphere [3]. Due to these alarming problematics, our main motivation for the project is to create a novel synbio system to improve the e-waste recycling technology and make it a feasible approach to reduce the pollutants, and generating a business model with the recycling of these valuable metals.

E-waste contains valuable metals as well as potential environmental contaminants. Effective recycling technology, that recovers the valuable materials with minimal environmental impact is quite expensive. As a result, most of the electronic waste is dumped in landfills where it is burned with other wastes generating dioxins, hydrogen chloride and other compounds that directly affect the environment. [4]

The main benefit of using recycled metals over their virgin materials is the significant energy saved, almost 95%![2] Additionally, e-waste represents a rich source of precious metals compared to their primary ores. For example, one ton of e-waste from PCs contains more gold than the one recovered from 17 tons of gold ore [1].

For these reasons we decided to make a project based on a eco-friendly bioleaching technology using two modified bacteria, Acidithiobacillus ferrooxidans and Chromobacterium violaceum, for recovering metals from the e-waste.

References

[1] Khaliq, A., Rhamdhani, M., Brooks, G., & Masood, S. (2014). Metal Extraction Processes for Electronic Waste and Existing Industrial Routes: A Review and Australian Perspective. Resources, 3(1), 152–179. MDPI AG. Retrieved from http://dx.doi.org/10.3390/resources3010152

[2] Clean Air Institute, (2012). Air Quality in Latin America: An Overview. http://www.cleanairinstitute.org/calidaddelaireamericalatina/cai-report-english.pdf

[3]Gangwar, C., Singh, A., Kumar, A., Chaudhry, A. K., & Tripathi, A. (2016). Appraisement of Heavy Metals in Respirable Dust (PM 10) Around E-Waste Burning and Industrial Sites of Moradabad : Accentuation on Spatial Distribution , Seasonal Variation and Potential Sources of Heavy Metal, 10(6), 14–21. http://doi.org/10.9790/2402-1006021421

[4]Robinson, B. H. (2009). E-waste: An assessment of global production and environmental impacts. Science of The Total Environment, 408(2), 183-191. doi:10.1016/j.scitotenv.2009.09.044

Bioleaching Experiments

Our first microorganism, A. ferrooxidans is currently used in a few mining companies for the recovery of Zn, Ni and Cu from the residual dirt and rocks of their processes. A. ferrooxidans can recover these metals thanks to its extracellular matrix, which has an environment that facilitates electron interchange. The enzyme tetrathionate hydrolase (TetH), which is produced inside the bacteria and then taken out to the external matrix, constantly converts tetrathionate to two molecules: thiosulfate and elemental sulfur.
The reaction that takes place is illustrated in the following figure:

Figure 1. TetH is involved in the electron transfer for A. ferrooxidans. The reaction increases the acidity of the media and releases sulfates, which chelate metals.

For the bioleaching process, e-waste was washed three times with distilled water to diminish the natural basic pH of the metals. The protocol for the bioleaching experiments consisted in adding 1 g of slag per liter of medium in a flask containing A. ferrooxidans in a set exponential phase. The flasks were incubated for 15 days, taking aliquotes the days 3, 6, 9, 12 and 15 to analyze the metal content with ICP-OES, then the data would be compared to the original ewaste and the metals solubilized were calculated.

After being washed three times with distilled water, the solid residues of the bioleach treatment or the acid medium treatment were transferred to the Chromobacterium violaceum alkaline bioleaching phase of our MetalEca process.

A. Ferrooxidans is an acidophilic bacteria that turns its own culture media into a more acidic environment to satisfy its growing conditions. We added bromocresol green (0.5% V/V) on the petri dishes in order to identify the colonies and calculate the colony-forming unit of each experimental group.

Tetrathionate is a key intermediate during RISC oxidation, hydrolyzed by tetrathionate hydrolase (TetH), and used as sole energy source. The overexpression of TetH in A. ferrooxidans leads to have a high expression of sulfates in the culture media, with more activity, more protons are released to the media and leads to a greater solubilization of metal ions.

FUR/FURBOX

Although iron is the most abundant transition metal on Earth, its solubility is very low at neutral pH in aerobic environments. In such environments, Fe(II) is generally not available because it rapidly oxidizes to Fe(III), which precipitates as insoluble ferric ion complexes. (Quatrini, 2005). Thanks to the acidic conditions of A. ferrooxidans Fe(II) is stable and Fe(III) is more soluble than at standard pH (7.0)

Notes
Phase I: Acidithiobacillus ferrooxidans was obtained from the Geomicrobiology Laboratory of the Autonomous University of San Luis Potosí, which was isolated from a mine in Durango, Mexico. The medium contained the following ingredients (g/L): 0.2 - ammonium sulfate, 0.5 - magnesium sulfate, 0.25 - calcium chloride, 3 - dipotassium hydrogen phosphate and 0.005 - iron sulfate. The agar medium was made with the same concentrations, only 12.5 g/L was added. Both mediums were adjusted to a pH of 4.0 and autoclaved at 121 Celsius for 15 min.

A first experiment was carried to evaluate the growth of the bacteria in liquid medium with elemental sulfur and sodium thiosulfate. Group 1 had 10 g/L of elemental sulfur, group 2 had 10 g/L of sodium thiosulfate and group 3 had 5 g/L of elemental sulfur + 5 g/L of sodium thiosulfate.

The groups of the experiment consisted in: (1) Control medium without bacteria, (2) Control medium with bacteria without genetic modifications, (3) Bacteria with tetH overexpression. It was expected that the curve of the group 3 had the biggest slope value.

Unfortunately three weeks after our first inoculum we lost our strain due to a failure in the pH measurements, when we tried to inoculate them again to fresh media cells would not grow. It was found that the pH of the original strain had come down to a pH of 0.63, thus all organic material was dissolved despite A. ferrooxidans being an acidophile.

Aim of the module: gold & silver solubilization

With the acid oxidation process used in Module I, metals such as copper, zinc and nickel are solubilized by A. ferrooxidans, but valuable metals like gold and silver are not. However, the recovery of these last two metals is vital in making the process economically viable, due to their high market value and relative scarcity. An effective way to solubilize them is by bioleaching with a cyanide-producing organism such as C. violaceum.

Process description

Why Chromobacterium violaceum

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why we chose C. violaceum

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Why we chose to use C. violaceum for this process.

C. violaceum has several characteristics that make it ideal for our project:

• It is more suited for the project compared to other bacteria that could also be used for cyanide bioleaching. (e.g. Pseudomonas fluorescens)
  • It was shown by Pham & Ting (2009) that gold recovery with C. violaceum is more effective than with P. fluorescens as long as the e-waste has been pre-oxidized, as we have designed in Module I.
• It has already been studied as a bacteria of biotechnological interest because of its adaptability to different stresses. Chief among these characteristics, its resistance to heavy metals and ability to grow using many different carbon sources stand out, as was described by the Brazilian National Genome Project Consortium (2003), the team that first reported sequencing its genome.
• As it is a native cyanide producer, it is also highly resistant to it (Niven, Collins & Knowles, 1975).
• It is relatively easy to manage in the lab (similar enough to E. coli as to allow the use of very similar lab protocols)
  • It grows in LB media, with observed population kinetics similar to E. coli.
  • It was transformed successfully with a pretty standard electroporation protocol (Broetto et al, 2005).
  • We were able to transform it using iGEM’s standard PSB1C3 plasmid with a calcium competency protocol for E. coli

Close

How will we improve the process?

In order to improve the efficiency of the process, we will modify C. violaceum using two basic constructs:

1. A gold/copper-sensitive cyanide producing system
   • Though C. violaceum is a native cyanide producer, this basal production is relatively low and regulated by quorum sensing.
     • In a previous study, cyanide production was successfully uncoupled from quorum sensing regulation by transformation with hydrogen cyanide synthase (hcnABC) under pARA regulation (Tay et al, 2013).
   • pGolS is a promoter that has been used previously by other iGEM teams. Specifically, Team York 2013. This promoter is induced by gold, through the binding of transcriptional activator GolS with gold, which later binds to the recognition site of the promoter allowing for higher expression of genes in the presence of gold.
   
   • Our construct is constituted by the pgolS promoter and the coding sequence of CueR. As the pgolS promoter has a basal transcription level, some CueR would be constantly expressed. CueR is a transcription factor that stimulates the transcription at the pgolS promoter when bound to copper or gold, as was described by the iGEM York 2013. Thus, this construct increases its own expression through positive feedback in the presence of gold or copper through the production of a copper/gold sensitive protein. Its organism of origin is Salmonella typhimurium. We optimized the original sequence for Chromobacterium violaceum and used this system along with Hydrogen Cyanide Synthase operon hcnABC in order to create a self-regulatory system that activates in the presence of gold, increasing cyanide production and enabling the solubilization of this precious metal as [Au(CN)2]–.
2. Proton Pump
   • Hydrogen cyanide synthase generates HCN, not free CN- ions. Since free CN- ions are responsible for the reaction that solubilizes gold, we must take into account the dissociation of HCN. As HCN is a weak acid (pKa = 9.22[source?]), it requires a basic pH in order to dissociate. In the mining industry, it has been established that a pH > 9.4 is ideal (Marsden & House, 2006), as at lower pH values not enough HCN is dissociated; and at higher pH values other factors (such as salinity) become more important. [source?]
   • As shown in the modelling section, the overall gold solubilizing reaction generates H+. Thus, in order to mantain a pH = 9.5, a base has to be continously added to the reactor.
   • In order to improve the efficiency of C. violaceum’s activity under these alkaline conditions, we will introduce a proton pump. [explicación de por qué creemos que esto funcionaría, referenciada al paper de Suria]. Specifically, we will use the intermembrane protein Na(+)/H(+) antiporter NhaA, which is a proton pump from E. coli. It excretes one Na(+) ion in exchange for two H(+) external protons. This protein is active at alkaline pH.
   
   • Within our construct, the proton pump is regulated by a weak constitutive promoter from the Anderson collection, and the coding sequence is optimized for its expression in C. violaceum. We will also test it as a potential selectivity marker for C. violaceum under these alkaline conditions (pH=9.5).

Safety Considerations

Post-processing:cyanide degradation module

After gold is recovered in the third module, we will degrade the remaining cyanide, as it is too toxic to be released to the environment without previous treatment.

We will use a different transformant of C. violaceum that will overexpress cyanide hydratase, a cyanide degrading enzyme. This enzyme has been found in different cyanide-degrading fungi such as Aspergillus niger and Fusarium lateritium.

Cyanide hydratase has been successfully expressed in E. coli, where it retains its original activity. In fact, Brown, Turner, and O'reilly (1995) reported that a modified E. coli reached 5.5 times the cyanide degrading activity compared to that observed in the native F. lateritium.

References

1. Broetto, L., Cecagno, R., Sant'anna, F. H., Weber, S., & Schrank, I. S. (2005). Stable transformation of Chromobacterium violaceum with a broad-host-range plasmid. Appl Microbiol Biotechnol Applied Microbiology and Biotechnology, 71(4), 450-454. doi:10.1007/s00253-005-0140-5
2. Pham, V., & Ting, Y. P. (2009). Gold Bioleaching of Electronic Waste by Cyanogenic Bacteria and its Enhancement with Bio-Oxidation. AMR Advanced Materials Research, 71-73, 661-664. doi:10.4028/www.scientific.net/amr.71-73.661
3. Niven, D. F., Collins, P. A., & Knowles, C. J. (1975). The Respiratory System of Chromobacterium violaceum Grown under Conditions of High and Low Cyanide Evolution. Journal of General Microbiology,90(2), 271-285. doi:10.1099/00221287-90-2-271
4. Tay, S. B., Natarajan, G., Rahim, M. N., Tan, H. T., Chung, M. C., Ting, Y. P., & Yew, W. S. (2013). Enhancing gold recovery from electronic waste via lixiviant metabolic engineering in Chromobacterium violaceum. Scientific Reports, 3. doi:10.1038/srep02236
5. Marsden, J. O., & House, C. I. (2006). The chemistry of gold extraction. Littleton: Society of Mining Metallurgy and Exploration.
6. Perky, R., Browner, R., Dunnei, R., & Stoitis, N. (1999). Low pH cyanidation of gold. Minerals Engineering, 12(12), 1431-1440. doi:10.1016/s0892-6875(99)00132-6
7. Brown, D. T., Turner, P. D., & O'reilly, C. (1995). Expression of the cyanide hydratase enzyme from Fusarium lateritium in Escherichia coli and identification of an essential cysteine residue. FEMS Microbiology Letters, 134(2-3), 143-146. doi:10.1111/j.1574-6968.1995.tb07928
8. Brazilian National Genome Project Consortium (2003). The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proceedings of the National Academy of Sciences, 100(20), 11660-11665. doi:10.1073/pnas.1832124100
9. Rinágelová, A., Kaplan, O., Veselá, A. B., Chmátal, M., Křenková, A., Plíhal, O., … Martínková, L. (2014). Cyanide hydratase from Aspergillus niger K10: Overproduction in Escherichia coli, purification, characterization and use in continuous cyanide degradation. Process Biochemistry, 49(3), 445–450. http://doi.org/10.1016/j.procbio.2013.12.008

References

Centers for disease control and prevention. (2009). Biosafety in Microbiological and Biomedical Laboratories. (5th Edition ed.). United States: HHS Publication.

Norma Oficial Mexicana NOM-157-SEMARNAT-2009. Diario Oficial de la Federación, 30 de agosto de 2011.

Norma Mexicana NMX-AA-058-SCFI-2001. Diario Oficial de la Federación, 09 de julio de 2009.

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