BACKGROUND
What are nanoparticles?
According to the National Nanotechnology Initiative (NNI), “[n]anotechnology is the understanding and control of matter at dimensions of roughly 1 to 100 nanometers (nm).” Thus, by extension, nanoparticles (NPs) are defined as matter of roughly 1 to 100 nm in at least one dimension (Vert et al., 2012).
Why use nanoparticles?
The small size and high surface-area-to-volume ratio of NPs make them ideal for novel applications in many fields such as medical imaging, drug delivery, cosmetics, clothing, personal care and filtration. Currently, the Woodrow Wilson International Center of Scholars and the Project on Emerging Nanotechnologies (PEN) lists around 2000 consumer products containing nanomaterials in over 20 countries, with silver, carbon, titanium, silicon, zinc and gold being the most common materials used in products (Vance et al. 2015). For example, silver NPs (AgNPs) are commonly incorporated into sportswear, taking advantage of its antimicrobial properties to suppress odors (Ahamed et al. 2010). TiO2 and ZnO NPs are often used as the primary UV blocking agent in sunscreens because of their transparent appearance, smooth application and broad spectrum UV protection (Lewicka et al. 2013).
Figure 1-1 TiO2 NPs present in foundation (bright white dots). Sample: Emily C. SEM Imaging: Laurent H., Jesse K.
Figure 1-2 ZnO NPs present in sunscreen (bright white dots). Sample: Emily C. SEM Imaging: Laurent H., Jesse K.
What are some problems with nanoparticle usage?
When evaluating the potential risks of NPs to the environment or to human health, two main factors should be considered: likelihood of exposure and toxicity. While long term effects of NPs are largely unknown due to the relative novelty of nanotechnology, numerous in vitro and in vivo studies point to potentially negative health and environmental effects.
The first part of “risk” concerns the probability of exposure to NPs. According to the PEN, NPs are most commonly used in “Health and Fitness” products-- comprising 42% of the recorded products (Vance et al. 2015). Some examples of this category include sportswear, sunscreens and cosmetics. For instance, AgNP is a common coating on antimicrobial sports fabrics (Ahamed et al. 2010); however, it has been shown to be released from fabrics when incubated in artificial sweat (Kulthong et al. 2010). This suggests that AgNPs can fall out during exercise, making dermal exposure to AgNPs very likely. TiO2 and ZnO NPs are commonly found in makeup and sunscreens (figures 1-1 and 1-2), which are applied topically and worn for long periods of time. The recent increase of nanomaterial usage in consumer products has led to higher exposure rates.
The small size of NPs make them useful in consumer products, but also more reactive and often more toxic than larger, bulk-sized chemicals. A number of factors affect toxicity, such as composition, surface characteristics and shape, but size seems to have the most direct impact on toxicity. When tested in E. coli, toxicity dramatically increased when exposed to small silver particles under 10 nm (Ivask et al. 2014). In another study with crustaceans, algae and protozoa, CuO NPs were shown to be up to 5000 times more toxic than microparticles (i.e. particles between 0.1 um and 100 um) (Bondarenko et al. 2013; Vert et al., 2012).
Knowing that size is directly related to toxicity, several studies have examined both the environmental and health effects of commonly used small (< 35 nm) NPs. ZnO NPs can inhibit root growth in common wetland plant species (Yin et al. 2012). Fathead minnow (Pimephales promelas) embryos experience death or growth abnormalities after exposure to various concentrations of AgNPs (Laban et al. 2010). Furthermore, in vitro studies have reported negative effects on human cells. Paddle-Ledinek et al. found that antimicrobial wound dressings containing AgNPs are cytotoxic to skin cells (keratinocytes); the authors noted “disordered” morphology and decreased cell proliferation, viability and metabolism just 3 hours after exposure (2006). Xu et al. have also reported that 20 nm AgNPs can potentially cause detrimental effects to neuronal development; exposure reduced cell viability of premature rat neurons and triggered degeneration of mature rat neurons (2013).
As nanotechnology becomes an integral part of our daily lives, nanomaterials and their wastes are expected to enter—and likely are already polluting—our natural environment due to inadequate disposal methods. It is estimated that about 95% of AgNPs and TiO2 NPs used in consumer products end up in wastewater (Mueller & Nowack 2008). Considering that most households in the US are connected to the public sewers, Holder et al. estimates that up to 2.7 tons of AgNPs and 229.3 tons of TiO2 enter municipal wastewater treatment plants (WWTPs) annually in the US (2013).
Current Solutions
There are no existing government policies that specifically regulate the usage and disposal of NPs, as various institutions in the world still regulate them under existing bulk chemical guidelines (Breggin et al. 2011). Recently, however, it has been shown that NPs behave radically differently and have significantly elevated risks compared to bulk-sized chemicals. In the US, there is a 100 kg annual production threshold regulating bulk chemical production, over which manufacturers need to inform the EPA of their activities (American Bar Association 2006). Since nanomaterials possess different properties as bulk chemicals, these outdated laws allow many companies to evade regulations and sell NP-containing products to customers without oversight (Holder et al. 2013). In response to this, we have developed our own policy recommendations for lawmakers around the world. Check it out here.
Despite the need to prevent NP pollution, current municipal WWTPs do not have specialized procedures to remove NPs in wastewater. Instead, NPs are subject to the conventional procedures used to treat larger particulates: sedimentation with the use of flocculants. While this process removes some NPs, complete NP removal has not yet been achieved. A study monitoring an Arizona WWTP found that while sedimentation is effective at filtering large aggregates (72% removal rate), most small-sized TiO2 NPs (41% removal rate) can still pass through the WWTP and enter major water systems downstream (Kiser et al. 2009).
The same is true at our municipal WWTP in Dihua, Taipei—there is no process targeting small NPs. When we visited, they gave us a sample of treated effluent that is released into the Tamsui River. We imaged this sample using a scanning electron microscope and found NPs in the effluent (figure 1-3), indicating NPs are indeed getting released into the Tamsui River, a major recreational spot for Taipei city dwellers. As such, the development of an effective and energy-efficient method to remove NPs in WWTPs worldwide is critical to stop NP pollution of water bodies and reduce public health and environmental hazards.
Figure 1-3 Effluent water from Dihua WWTP observed under SEM. Significant amount of aggregated NPs (bright white dots) indicate that our local WWTP was unable to completely remove NPs. SEM Imaging: Laurent H., Jesse K.
OUR SOLUTION
Our goal is to efficiently remove NPs from wastewater systems to prevent NP pollution. What makes this simple goal difficult, however, is that NPs “come in all shapes and sizes” (literally). This means approaches that exploit highly specific properties of one type of NPs are inefficient, because these approaches will not work for other types of NPs. In response to this, we’ve devised a two-pronged approach to maximize the types of NPs that can be captured.
Proteorhodopsin (PR) Membrane Receptor
Most NPs found in consumer products or created for research have a “coating”—known as capping agents—on their surface to prevent aggregation. This makes capping agents a unifying property across many different types of NPs. We learned that citrate is the most common capping agent used by industry (Levard et al. 2012), and a membrane protein called Proteorhodopsin (PR), found in marine proteobacteria is capable of binding to citrate. Our goal is to express PR on the membrane of E. coli to bind and hold onto citrate capped NPs (CC-NPs). Ideally, E. coli expressing PR will be able to bind to CC-NPs, and like many other microbes used in the wastewater treatment process, will increase in size and weight, such that existing infrastructure in WWTPs can filter them out before NPs get released into natural water bodies.
Sticky Biofilm
What about NPs that are not capped by citrate? To trap an even wider variety of NPs, both capped and uncapped, we used a general approach that does not target the specific characteristics of any NP. We were originally inspired by a study using jellyfish mucus to trap and take out gold NPs from water after a short mixing period. However, since very few studies have examined this novel use, we ran into a wall trying to find the genes responsible for jellyfish mucus production.
We soon turned to another slimy material naturally produced by many species of bacteria: extracellular polymeric substances (EPS) in biofilms. Our preliminary tests and literature research show that biofilms can also trap NPs and pull them out of solution, similar to jellyfish mucus (Patwa et al. 2015).
We learned that E. coli produces biofilms through two curli operons, which can be regulated by two proteins, OmpR and CsgD. There are many other regulatory mechanisms regulating E. coli biofilm synthesis, but since biofilm formation is commonly associated with diseases such as Urinary Tract Infections (UTIs), we avoided genes associated with virulence (Fattahi et al. 2015). We also chose to use a safe and common laboratory strain—E. coli K-12—as our chassis (Environmental Protection Agency 1977). Our goal is to increase biofilm yield in a common laboratory strain E. coli K-12 by constitutively overexpressing OmpR and CsgD.
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