# Team:Tec-Monterrey/Description

iGEM 2016 - Tec de Monterrey

#### 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 [2]. 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]

Monterrey is the leading city with a high concentration of PM10 particles among Latin American countries. [2]

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.

Bioleaching is the process of metal extraction undertaken by microorganisms. Its mechanism underlies on the transformation of solid metal compounds into soluble products that can be easily extracted and recovered, due to the catabolism of microorganisms.

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

The first microorganism implemented onto our three step synbio system, Acidithiobacillus ferrooxidans, is a chemoautotrophic bacteria that thrives in very acidic pH (1.5 to 3.0). It is commonly used by mining companies for the industrial recovery of metals such as Zn, Ni and Cu from residual dirt and waste materials from 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.

Overexpressing a specific gene in an organism, may affect cell’s metabolism and growth rate. Also the expression of the desired gene will tend to decrease over time. To solve this problem, we will use two control mechanism to inhibit gene expression when not needed: a temperature induced promoter (HSP) and an iron induced inverted operon (FUR/FURBOX).

#### HSP

Heat Shock Promoter is a σ32-dependent promoter for for the Afe_1437 gene in Acidithiobacillus ferrooxidans (Ribeiro, D. A.,2011). It is a putative promoter in Acidithiobacillus ferrooxidans. We retrieved this sequence from ferrooxidans’ genome and ran a nucleotide blast to find similitudes with other promoters just as done by the Ribeiro’s article.

#### FUR/FURBOX

Inverted operon with a Ferric Uptake Regulator & a LacI coding sequences. This inverted operon is expected to activate gene expression in presence of iron. We found this composite regulator on IGEM parts registry but then we made some changes, FUR and FURBOX nucleotide sequences were retrieved from ferrooxidans’ genome, these parts are smaller and proved better efficiency in A. ferrooxidans (Quantrini, R. 2005).

Figure 2. Functionality of FUR/FURBOX cassette. First, without the presence of iron, the lactose inhibits the pLac promoter. When iron is in a sufficient concentration, it makes a complex with the FUR protein which then binds to the FURBOX inhibiting LacI transcription, activating the pLac promoter and the transcription TeTH sequence for protein synthesis.

The characterization of the part would be done using an RFP coding sequence, instead of the production of TetH. For further information on these control mechanisms and the rest of our parts, please go to the Parts section of our wiki.

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)

#### CELL CULTURE

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.

#### OUR EXPERIENCE

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.

#### Overview

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.

#### 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.
• BLAST analysis revealed that the translated golS protein included in the part submitted by York 2013 has a 100% homology with CueR from Salmonella typhimurium. Thus, 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 iGEM York 2013. It follows that 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. We optimized the original sequence for C. 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), 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.
• 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. With the work of E. Padan et al. (2005), we found that monovalent antiporters play an important role in alkaline pH homeostasis, in addition to roles in Na+ and volume homeostasis. Similarly, Na+/H+ antiporters play essential roles in homeostasis of pH, Na+ and volume in bacteria. Thus, we reasoned that addition of a proton pump could improve survival under basic conditions by increasing proton transport across the membrane. 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).
3. #### 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. BPadan, E., Bibi, E., Ito, M., & Krulwich, T. A. (2005). Alkaline pH homeostasis in bacteria: New insights. Biochimica et Biophysica Acta - Biomembranes, 1717(2), 67–88. http://doi.org/10.1016/j.bbamem.2005.09.010
10. 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

#### Overview

After dissolving the metals in in modules I and II, it is necessary to precipitate them and to do this we require to reduce them to a neutral charge. Using the reduction potentials of the metallic ions in the solutions, we established a spontaneous reaction by coupling them with the oxidation of S2- from H2S into S0. To obtain this, we expressed the enzymes sulfur reductase and iron oxidase in E. coli. Sulfur reductase is used to catalyze the reduction of elemental sulfur in the presence of oxygen, with sulfite and hydrogen sulfide as products. Iron oxidase catalyzes the oxidation of Fe2+ to Fe3+, giving it a big enough reduction potential for its reduction to happen spontaneously. The electrical potential difference between the two electrodes can be used as power source to feed a small device or be stored in a battery for its later use.

#### Recovery of metal through spontaneous reactions

Figure 1. Metals recovered with Acidithiobacillus ferrooxidans. The solubilised metals are going to be then used in the galvanic cell, but they will have to reduced or oxidised first.

Figure 2. Metals recovered with Chromobacterium violaceum.The solubilised metals are going to be then used in the reduction half-cell.

From module I, elemental sulfur is produced due to the metabolism of A. ferrooxidans. The composite part BBa_K2055025 uses the T7 system in order to express the enzyme sulfur reductase, which catalyzes the simultaneous oxidation and reduction of elemental sulfur in the presence of oxygen, with sulfite and hydrogen sulfide as products.

Figure 3. Sulfur reductase generator. The composite part is used to express the enzyme sulfur reductase.

From module I, ions of Fe+2 are produced due to the metabolism of A. ferrooxidans. The composite part BBa_K2055425 uses the T7 promoter in order to express the enzyme iron oxidase, which catalyzes the oxidation of Fe+2 to Fe+3.

Figure 4. Iron oxidase generator. The composite part is used to express the enzyme iron oxidase.

In a galvanic cell, the element with the higher (or more positive) reduction potential gets reduced and the one with the lower (or more negative) reduction potential gets oxidized. From Table 1, it can be observed that copper, silver and gold be reduced to a neutral charge spontaneously when coupled with the H2S half-cell. Iron can also be reduced this way but only from Fe3+ to Fe2+. In the case of nickel and zinc, they can’t be reduced by sulfur oxidation, they needs to be recovered by using an electric current or in another cell arrangement. The energy produced when reducing copper, silver, gold and iron (Fe3+ to Fe2+) can be used to reduce the rest of them or further reduce iron. (Figure 5)

Reaction Reduction Potential (V)
$$\text{S}_{(s)} + 2\:\text{H}^{+}_{(aq)} + 2\:\text{e}^{-}\;\rightarrow\;\text{H}_2\text{S}_{(aq)}$$ $$+\:0.14$$
$$\text{Fe}^{3+}_{(aq)} + \text{e}^{-}\;\rightarrow\;\text{Fe}^{2+}_{(aq)}$$ $$+\:0.771$$
$$\text{Fe}^{2+}_{(aq)} + 2\:\text{e}^{-}\;\rightarrow\;\text{Fe}_{(s)}$$ $$-\:0.44$$
$$\text{Cu}^{2+}_{(aq)} + 2\:\text{e}^{-}\;\rightarrow\;\text{Cu}_{(s)}$$ $$+\:0.34$$
$$\text{Ag}^{+}_{(aq)} + \text{e}^{-}\;\rightarrow\;\text{Ag}_{(s)}$$ $$+\:0.80$$
$$\text{Au}^{+}_{(aq)} + \text{e}^{-}\;\rightarrow\;\text{Au}_{(s)}$$ $$+\:1.68$$
$$\text{Zn}^{2+}_{(aq)} + 2\:\text{e}^{-}\;\rightarrow\;\text{Zn}_{(s)}$$ $$-\:0.763$$
$$\text{Ni}^{2+}_{(aq)} + 2\:\text{e}^{-}\;\rightarrow\;\text{Ni}_{(s)}$$ $$-\:0.25$$

Table 1. Table of standard reduction potentials.

Figure 5. Possible reactions occurring in the cell. In the left side, the oxidation of H2S to elemental sulfur occurs. In the right side, the reductions occur and the recovery of metals occur.

References
1. Çolak, D., et al. (2015). Biochemical characterization of wild-type and mutant (Q9F and S21Y/V22D) iron oxidases isolated from Acidithiobacillus ferrooxidans M1. Turkish Journal of Biology, 2016(40), 166-173.
2. He, Z., et al. (2000). Cloning and heterologous expression of a sulfur oxygenase/reductase gene from the thermoacidophilic archaeon Acidianus sp S5 in Escherichia coli. FEMS Microbiology Letters , 2000(93), 217-221.
3. Smccd. (n. a.). Expanded Reduction Potential Table. Retrieved October, 2016, from http://accounts.smccd.edu/batesa/chem220/reference/Exp-Reduction.pdf
4. The UniProt Consortium. UniProt: a hub for protein information. Nucleic Acids Res. 43: D204-D212 (2015)

Brought to you by iGEM Tec-Monterrey 2016