Biomining

System Design

A Separation Problem

Our access to easily extractable copper is gradually diminishing as demands for copper continues to grow worldwide. To meet these demands, non-traditional, metallurgically challenging deposits are expected to become more prevalent, thereby forcing us to deal with more complex, and lower-grade ores containing higher levels of impurities. Arsenic-challenged deposits is a concern for the copper mining industry as arsenic produces hazardous fumes and oxide dusts during the smelting process. Smelting with arsenic therefore poses a significant risk to the health of the workers and to the environment. Furthermore, safe removal and disposal of stabilized arsenic is often difficult and costly. With stricter legislation in place, the mining industry is facing increasing pressure to progressively reduce the amount of allowable arsenic concentrations for smelters. Consequently, fines and penalties are set for arsenic concentrations exceeding 0.2%, while ores past 0.5% arsenic concentrations are rejected by smelters. Therefore, there is increasing precedence for removing arsenic-containing minerals such as enargite (Cu3AsS4) from relevant sulfide minerals, such as chalcopyrite (CuFeS2), during the flotation process. However, separation is often conflicted with shared flotation properties between enargite and the associated valuable minerals. The mining industry has developed several methods for dealing with arsenic impurities, which includes precipitation with scorodite and pressure hydrometallurgical procedures (150 C and 1380 kPa) for processing high concentration of arsenic while extracting copper in parallel. However, many procedures requires an enormous amount of energy or additional acidic chemicals to help selectively separate arensic-containing minerals in the ore mixtures.

Our solution: Mineral Binding

We, the UBC iGEM team, feel that separation and enrichment can be done in other ways that does not rely on energy or chemically-intensive methods. Our synbio solution involves the use of small surface heptapeptides and octapeptides, which have previously been demonstrated in M13 bacteriophage by Curtis et al. to selectively bind to chalcopyrite(3). Three peptides, labelled WSD-1 (TPTTYKV), WSD-2 (DSQKTNPS), and WSD-3 (DPIKHTSG), have been identified for binding. However, operating with bacteriophage is not feasible for large scale operations in mining as it is difficult to scale up titers to compensate for the smaller surface area available for binding in bacteriophage. Our idea is to operate these peptides in bacteria which have a larger surface area and are much more responsive to stimuli. These peptides must be accessible on the surface. Therefore, we have chosen Caulobacter crescentus as our chassis given that it contains a S-protein layer in which we can express our peptides. A developed kit for protein secretion and display of peptides on the cell surface S-layer in Caulobacter crescentus (via cloning to the S-layer gene sequence) can be found within Caulobacter S-layer Kits.

Figure 1. Curtis et al. screened a random heptapeptide library through a series of ‘biopanning’ assays for peptides that could be expressed on M13 bacteriophage and selectively bind to chalcopyrite. These peptide-containing bacteriophage have the potential to be utilized to separate chalcopyrite from enargite in ore slurries.

Figure 2. Our project adapts these chalcopyrite-binding peptides (WSD-1 (TPTTYKV), WSD-2 (DSQKTNPS), and WSD-3 (DPIKHTSG)) by cloning them into the S-protein layer of ''Caulobacter crescentus''.

Our solution: Mineral Separation

As a proof of concept, we have chosen to express these peptides to selectively bind to chalcopyrite, which will then fall out of solution by aggregation using two interacting coiled-coil peptides expressed on the S-layer of Caulobacter crescentus. These coiled-coil peptides (K-coil: BBa_K1189012 , E-coil: BBa_K1189013, K-coil-His: BBa_K1189010, and E-coil-His: BBa_K1189011 )were constructed by iGEM Calgary 2013. In this model, Caulobacter A will contain chalcopyrite-binding peptides bound to chalcopyrite elusively while holding a K-coil interaction peptide. By addition of Caulobacter B which holds the E-coil interaction peptide, Caulobacter A will coalesce with Caulobacter B by E-coil and K-coil interactions, forming a greater mass that will selectively sink in a froth floation solution. Alternatively, we have considered floating bound chalcopyrite using gas vesicle proteins (BBa_I750016) developed by iGEM Groningen 2009.

Figure 3. K-coil and E-coil interaction peptides are expressed on the S-protein layer in two strains of ''Caulobacter crescentus''.

Figure 4. ''Caulobacter'' A will contain chalcopyrite-binding peptides bound to chalcopyrite elusively, while holding a K-coil interaction peptide. Addition of Caulobacter B which holds the E-coil interaction peptide will create an interacting link with ''Caulobacte''r A, forming a mass that will fall out of solution

Binding Assay

To observe whether our peptides are capable of binding to chalcopyrite, we incubated 40um and 100um size chalcopyrite with our peptide expressing ''Caulobacter crescentus'' which were then passed through a 40um size cell strainer and subsequently washed with buffer and eluted with elution solutions. The elutions were then plated on YPD plate. Colonies grown on YPD plate suggest that the Caulobacter crescentus were bound to the chalcopyrite as they were present after the wash and elution steps.

Figure 5. Figure 5 Binding assay by incubating peptide-containing ''Caulobacter crescentus''with chalcopyrite and subsequent wash and elution steps to remove unbound bacteria.

Results

Biomining precipitation assays

Before attempting to demonstrate binding with peptide inserted into the S-Layer, we tested binding with WT cells. After having observed binding, we then hypothesized that the fucose layer in Caulobacter was potentially involved in binding. Since there was a fucos knockout was available, we ere able to confirm fucos binding. Moreover, this observation demonstrated that a precipitation assay was feasible when mixing cells with Chalcopyrite.

As a preliminary test, we observed increased Chalcopyrite precipitation with Caulobacter cells expressing a Chalcopyrite binding peptide in the S-layer. While non-quantitative, the data showed noticeable differences in precipitate settle when the peptide expressed in various locations in the S-layer. See the video below.

Figure 7.Various peptides from Curtis et al. are displayed in different positions in the S-layer of Caulobacter cells. The cells were mixed with calcopyrite and precipitate settling was observed.

Biomining protein assays

In an attempt to support the above observation, we incubated ''Caulobacter'' cells with Chalcopyrite and separated the Chalcopyrite by centrifugation. The supernatant and cells adhered to Chalcopyrite (eluted after several buffer washes) were assayed for protein concentration as a read out for biomass. For fucose containing cells and cells expressing Chalcopyrite binding peptides in the S-layer, we expected protein concentration to decrease in the supernatant and increase on Chalcopyrite as compared to controls.

Figure 8. Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite. Peptides are expressed in wild type Caulobacter cells.

Figure 9. Bradford assay for protein from cells in supernatant and adhered to Chalcopyrite. Peptides are expressed in fucose knockout cells.