IMPLEMENTATION
Interfering Compounds and Removal Strategies
Impurities present in metal-finishing effluent have the potential to create an environment that is hazardous and degrading to proteins, thereby impeding the function of our product. Outlined below are common contaminants and known mechanisms which threaten the integrity of the system, as well as potential removal methods that may be implemented in the process before our system is employed.
Oil and grease may be found in electroplating effluent at a concentration of up to 10 mg/L (World Bank Group, 1998). The interface between the water and oil in the effluent may cause mechanical strain on the proteins, resulting in denaturing of the secondary or tertiary structures (Thomas & Geer, 2010). Additionally, from our discussion with stakeholders from Aevitas Hazardous Management, oil would need to be removed from the effluent before passing it through our packed column. Oil and grease can be removed by separating the oil phase by a pH and/or temperature change (Condorchem Envitech, 2019) or precipitated out to avoid interference (J.A. Livingston, personal communication, October 16, 2020).
Cyanide may be found in electroplating wastewater at a concentration of up to 0.2 mg/L (World Bank Group, 1998). Although our method of protein delivery does not rely on living organisms, cyanide has a high affinity for transition metals and sulfane compounds, which may interfere with the efficiency of our metal binding proteins (Botz, 2001). Cyanide can be removed from effluent through methods such as chlorination or ozonation (Chiu et al., 1987).
Fluoride is a potential contaminant that may be found at a concentration of up to 20 mg/L in effluent (World Bank Group, 1998). Fluoride is not known to cause significant damage to most organic molecules (Salwiczek et al., 2012). If further research reveals significant risk of damage to the column or proteins, fluoride may be removed through precipitation using calcium in the presence of aluminum and iron cations (Grzmil & Wronkowski, 2006).
Trichloroethane and trichloroethylene may be found at concentrations of up to 0.05 mg/L (World Bank Group, 1998). These compounds, along with other volatile organic contaminants, may be removed through methods such as regenerative thermal oxidation, recuperative thermal oxidation, regenerative catalytic oxidation, gas phase advanced oxidation, and biofiltration (Condorchem Envitech, 2016).
Phosphorus may be found at a concentration of up to 5 mg/L (World Bank Group, 1998). There are a wide range of technologies available to remove phosphorus from wastewater, such as chemical precipitation, biological treatment, adsorption processes, constructed wetlands, and a number of wastewater-and-sludge-based methods (Wei et al., 2008).
With these removal methods already widely implemented in the metal-finishing industry, a response to any of these commonly found contaminants is highly feasible before the effluent is passed through our packed column for metal ion recovery.
Alternative Purification Strategies
Since the intended column reactor design will require large amounts of protein, a plan for large-scale purification of the fusion proteins is required for implementation. Depending on the required amount of protein determined from our process design model, nickel affinity chromatography may prove to be too expensive. Therefore, we propose two alternate protein purification strategies that could be used separately or in tandem if switching is required further on.
Cellulose Catch Purification
Since the fusion proteins already require immobilization to the cellulose matrix, it would be advantageous to perform the purification and immobilization in one step. Fortunately, purification using a cellulose binding module (CBM) has already been described in literature (Shoseyov et al., 2006; Guerreiro et al., 2008; Rodriguez et al., 2004). More specifically, the same CBM2a (Xyn 10a) has been previously used as a cost-effective and high-capacity affinity chromatography system for the purification of recombinant protein (Rodriguez et al., 2004). It resulted in a product purity of greater than 95% and over 34 ± 3 times the concentration of the recombinant protein from the original culture (Rodriguez et al., 2004).
Cellulose is attractive as an affinity purification matrix since it is inexpensive, chemically and mechanically stable, and highly specific (Rodriguez et al., 2004). The process would involve application of clarified cell lysate to an equal volume of cellulose followed by equilibration at 4 °C for 4 hours (Rodriguez et al., 2004). The cellulose and attached fusion proteins are then recovered by centrifugation and washed four times before being packed into a column for use in capturing metal ions (Rodriguez et al., 2004). Since the fusion proteins are to be used in an immobilized state in this case, they do not need to be eluted from the column as described in the literature and the system can be used as is.
PEG Precipitation
PEG precipitation is a protein mass-purification method that would reduce costs and increase the efficiency of our purification process when scaling up production of our packed column reactor. This method could also be used to recover proteins from the column once it is saturated by precipitating them out without denaturation.
Generally, PEG precipitation involves adding PEG into a buffer solution such that the concentration (% PEG) is just less than enough to precipitate protein, thereby precipitating out impurities (Ingham, 2004). The solution is incubated to promote precipitation, then centrifuged so the supernatant (with protein) can be recovered (Ingham, 2004). This process is repeated, but with % PEG being just enough to precipitate the protein. Centrifugation will yield protein in the pellet, along with other impurities that precipitate with the protein (Ingham, 2004). To separate the protein from these impurities, the process is repeated with an additional ligand. When the protein binds to the ligand, the protein-ligand complex precipitates at a lower % PEG than the unbound protein (Ingham, 2004). Following the same principle, the process is repeated at a % PEG that is just enough to precipitate the protein-ligand complex, which should result in a pure protein pellet after centrifugation (Ingham, 2004).
PEG precipitation is advantageous in that it does not require stringent temperature control; as long as the solution is appropriately buffered, the proteins will precipitate nicely (Ingham, 2004). As a result, PEG precipitation is unlikely to subject proteins to harsh conditions that may denature them (Ingham, 2004). PEG precipitation is also faster than other precipitation methods, such as ethanol precipitation or ammonium sulfate precipitation, which makes it appealing to use in industry to scale up the protein purification process (Ingham, 2004). However, the appropriate parameters of the PEG precipitation protocol (ex. % PEG, molecular weight of PEG, ligand) must be experimentally determined and optimized for each protein, therefore increasing time, effort, and cost.
Hazard Considerations
Since our final packed column does not contain any living organisms, biocontainment strategies do not need to be implemented. The other materials in our column are also not a biological or environmental hazard because the metal-binding proteins would be considered non-hazardous and safe to dispose of normally by typical laboratory standards and cellulose, being a natural derivative of wood, decomposes easily in the environment (Sample Preparation Laboratories, n.d.). Most types of similar chemical-biological waste are not classified as hazardous by regulatory bodies such as the EPA (National Research Council, 1995).Therefore, no specialised containment/disposal protocols need to be implemented and the use of our system should not pose any significant biological chemical hazards.
We designed an example industrial process suitable for treating copper-containing wastewater from a typical semiconductor manufacturing facility. Taking the results of our modelling and engineering work into consideration, we designed and scaled a packed column reactor that would be suitable for treating this waste stream. We could use a similar approach to design a packed column reactor for any type of wastewater and treatment rate.
Although further experiments and analysis are required, we can use our current knowledge to suggest how we would integrate our waste treatment technology into current industrial processes.
Interfering Compounds and Removal Strategies
Metal finishing waste streams contain a wide variety of components. Many of these components could chemically or physically damage the proteins found in our packed column reactor, or cause the fine pores of the reactor to become clogged. Many established treatment methods exist for removing these compounds. For these reasons, our packed column should be one of the last steps in the waste treatment process, after other contaminants have already been removed.
Outlined below are common contaminants and known mechanisms which threaten the integrity of the system, as well as potential removal methods that could be used to pre-treat the waste stream before our packed column.
Oil and grease may be found in electroplating effluent at a concentration of up to 10 mg/L (World Bank Group, 1998). The interface between the water and oil in the effluent may cause mechanical strain on the proteins, resulting in denaturing of the secondary or tertiary structures (Thomas & Geer, 2010). Additionally, from our discussion with stakeholders from Aevitas Hazardous Management, oil would need to be removed from the effluent before passing it through our packed column. Oil and grease can be removed by separating the oil phase by a pH and/or temperature change (Condorchem Envitech, 2019) or precipitated out to avoid interference (J. Livingston, personal communication, October 16, 2020).
Cyanide may be found in electroplating wastewater at a concentration of up to 0.2 mg/L (World Bank Group, 1998). Although our method of protein delivery does not rely on living organisms, cyanide has a high affinity for transition metals and sulfane compounds, which may interfere with the efficiency of our metal binding proteins (Botz, 2001). Cyanide can be removed from effluent through methods such as chlorination or ozonation (Chiu et al., 1987).
Fluoride is a potential contaminant that may be found at a concentration of up to 20 mg/L in effluent (World Bank Group, 1998). Fluoride is not known to cause significant damage to most organic molecules (Salwiczek et al., 2012). If further research reveals significant risk of damage to the column or proteins, fluoride may be removed through precipitation using calcium in the presence of aluminum and iron cations (Grzmil & Wronkowski, 2006).
Trichloroethane and trichloroethylene may be found at concentrations of up to 0.05 mg/L (World Bank Group, 1998). These compounds, along with other volatile organic contaminants, may be removed through methods such as regenerative thermal oxidation, recuperative thermal oxidation, regenerative catalytic oxidation, gas phase advanced oxidation, and biofiltration (Condorchem Envitech, 2016).
Phosphorus may be found at a concentration of up to 5 mg/L (World Bank Group, 1998). There are a wide range of technologies available to remove phosphorus from wastewater, such as chemical precipitation, biological treatment, adsorption processes, constructed wetlands, and a number of wastewater-and-sludge-based methods (Wei et al., 2008).
With these removal methods already widely implemented in the metal-finishing industry, a response to any of these commonly found contaminants is highly feasible before the effluent is passed through our packed column for metal ion recovery.
pH Requirements For Our Reactor
As described on the modelling and engineering, our packed column requires that the incoming waste have a pH above 7.2. In order to achieve the correct pH, it might be necessary to adjust the pH of the incoming waste using a small volume of acid or base. Van Dijken et al. (1999) reports that metal finishing wastes are often near neutral pH, so minimal adjustment would be required. The pH adjusted wastewater could then be fed into the reactor.
Our model indicates that our reactor will remove nearly all of the copper from a given wastestream, leaving only about 1 µM dissolved copper. This is well below the USEPA regulatory limit of 1.3 mg/L copper (Al-Sayed et al., 2017). If no other harmful contaminants are present, the outflow of our treatment process could be released directly to the environment. Otherwise, additional treatment steps could be used before releasing the waste to the environment.
Once the reactor has been saturated with copper, we will elute the bound copper using a relatively small volume of a pH 4.8 solution. This will result in a concentrated and pure copper solution that can be re-used for other applications.
As determined by our model, a positive pressure of 0.126 bar is required at the reactor’s inlet so that fluid can flow through. This pressure would be applied using a pump just before the reactor. Since the pressure requirement depends on the nature of the reactor’s packing, the same pressure is required for both the binding and elution steps.
Proposed Daily Schedule of Operations
Based on our proposed reactor design and the rate at which it must treat wastewater, our process will be able to run continuously for about 5 hours before becoming saturated. Although further experiments would be required, we will generously allow 1 hour for the copper unbinding and elution step. This means that we could run four 5-hour cycles of our reactor each day, with one-hour gaps in between for metal unbinding and elution. These one-hour gaps could also allow time for other routine maintenance.
We assume that the waste stream we want to treat will be generated in a continuous manner over the entire day. However, since our process will have 1 hour gaps where it is not able to treat waste, it would be necessary to store any waste produced during this time in a holding tank. We have designed our reactor to function at such a rate that it can catch up on this lost time, emptying the holding tank and treating all new waste within the 5 hour cycle.
In the development of REMINE, it was crucial to analyze our final product in every step of the way, from a stakeholders’ perspective. Diverse discussions with industry representatives as well as our own critical thinking helped guide the thorough development of our product. Early discussions with Brian Montgomery in particular, a mining regulations lawyer, helped focus the scope of our project in a number of ways to optimize the feasibility of our project, specifically through elements that would increase its value in the eyes of stakeholders, when our project was specific to the field of mining.
Brian Montgomery aided us in recognizing the value of the metals left in our column; their disposal may come at a greater cost than their own value. He mentioned that there are no current effective methods of recovering the filtered metals, and if it could theoretically be recovered and sold for profit, REMINE would hold its place as a never-before seen innovation.
Once the project direction shifted towards metal recovery, the electronics manufacturing industry seemed like a natural fit for REMINE. Due to the industry’s diversity, to simply say that REMINE was a ‘good fit’ for electronics manufacturing was not good enough. What started out as an answer to our implementation issue quickly gave way to more questions: what type of electronics would REMINE be best suited for, and where along the manufacturing process could REMINE best be implemented? For this decision to be finalized, a variety of different avenues were explored and assessed.
Initially, electronic waste recycling seemed like a promising area to focus on. Recycling copper is up to 85% more energy efficient than producing it from primary ores (Koch et al., 1997). This was an encouraging statistic, but upon further inspection, REMINE did not prove to be compatible with this type of process. Currently, most electronics recycling methods utilize hydrometallurgical processes which primarily aim to precipitate solid copper from wastewater to form a sludge, during the recovery process (Koch et al. 1997), (Işıldar et al., 2018). This posed a major challenge, as having copper ions in solution is critical to the proper functioning of the REMINE column. Furthermore, electronic waste recycling is predominantly composed of physical waste which is not suited for our system.
Electronic waste recycling was not the only idea discarded due to solubility issues, electroplating faced the same unfortunate outcome. Initially, this process seemed like a good fit for REMINE’s implementation, due to the fact that plating typically includes copper, among other metals (Rajendran et al., 2019). However, the process of recovering copper from electroplated objects involves precipitation, coagulation, flocculation and sedimentation (Rajendran et al., 2018), (Designs, n.d.). On the flip side, the recycling industry presented an interesting case of business ethics that our team further analyzed through independent research and stakeholder consultation. Furthermore, communicating with stakeholders from Aevitas Hazardous Waste Management confirmed that precipitation was an industry standard as a way of allowing the safe disposal of the metals in solution. Conversely, REMINE aims to replace precipitation steps altogether, by recovering the copper present in the solution. Electrowinning and electrorefining were also discarded due to the use of electrolysis as an industry standard (Fatimah, 2020), (Burshai, 2016). The electrolysis process is cheap, efficient and leads to solid deposits of pure copper, so REMINE was once again left without holes to fill.
While investigating electroplating, another issue arose: pH level. Many metal recovery processes use a specific pH as part of the precipitation procedure and often lower it to very acidic levels (Rajendran et al., 2018), (Designs, n.d.). For example, in the case of electroplating, prior to precipitating the desired metal(s), the pH is often around 3.5 (Rajendran et al., 2018). Even if REMINE had no involvement in the precipitation process, another challenge to overcome was going to be finding a process involving a pH at which the proteins could function.
Despite the setbacks endured, the beauty of the electronics manufacturing industry is that, above all else, it is diverse. After countless hours researching, a feasible option was finally found.
Metal finishing is a process similar to electroplating, where the surface of an object is altered to improve durability and appearance (Stubbings, 2019). The process of metal finishing generates wastewater, and the current procedures for managing this wastewater are not very efficient( United States Environmental Protection Agency, 2019). Part of the inefficiency of this process is the drag caused by the wastewater having to travel through different tanks before it has been fully processed (Calnan, 2009). Current metal finishing procedures require wastewater to travel through multiple pH altering tanks, in order to precipitate the metals out of solution (Calnan, 2009). However, having wastewater with dissolved copper ions in solution is exactly what REMINE needs. REMINE could easily fit into the metal finishing wastewater treatment process by being inserted after the first pH adjustment tank. This tank could be used to make the pH suitable for the proteins in the column and then the protein-friendly wastewater could be pumped through REMINE, allowing for copper to be recovered and reused, instead of undergoing further downstream wastewater treatment processes. After the copper is recovered, the wastewater could continue through the normal treatment process and be safely discarded.
While the path to metal finishing was rocky, gaining insight into different areas of the electronics manufacturing industry, even those that were not compatible with REMINE, allowed us to find an area to implement our project that has real benefits for the industry, financial feasibility and that would be compatible with a bioreactor such as REMINE.
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