Hexavalent chromium (Cr(VI)) is a ubiquitous heavy-metal contaminant that threatens soil and aquatic environments and has been shown to be carcinogenic, cytotoxic, and mutagenic even in low concentrations [1,2]. The reduced form of trivalent chromium (Cr(III)), however, is 10 to 100 times less toxic than Cr(VI) and is classified by the EPA as non-carcinogenic [3]. For this reason, reduction of Cr(VI) to Cr(III) is a common treatment method for Cr(VI)-contaminated water. Other methods consist of removal treatments and/or containment technologies [3].

Cr(VI) may occur naturally in the environment (see Figure 1), but larger concentrations usually result from human-caused contamination. The most common anthropogenic chromium sources include leather tanneries, textile industry, cooling tower blow-down and electroplating factories [4]. Environmental conditions that favor Cr(III) oxidation (such as high level of manganese oxides in the soil or high pH) may also contribute to increased Cr(VI) concentration [5].

Figure 1: Chromium cycling in the environment. Chromium is most abundant in the environment in its trivalent and hexavalent forms. Image modified from [5]

From 2013-2015, the Environmental Working Group (EWG) analyzed 60,000 nationwide water samples from local water utilities and found that more than 75% of them contained the carcinogenic Cr(VI) [6]. The only federally enforceable drinking water standard for chromium is mandated by the EPA as a maximum contaminant level (MCL) of 100 ppb total chromium in drinking water (see Table 1). This MCL was established in 1991, is currently under review, and includes chromium in both its hexavalent (highly toxic) and trivalent (much less toxic) forms [7]. Some states, like California, have set MCLs lower than the EPA for both Cr(VI) and total chromium. California in particular has also established a public health goal for Cr(VI) that is a remarkable 5,000 times lower than the EPA guideline to promote a de minimis lifetime cancer risk from exposure [8,9]. The nationwide EWG study estimated that water servicing more than 218 million Americans exceeded this public health goal for Cr(VI), which means that more than two thirds of the American population could be at cancer risk from a lifetime consumption of contaminated drinking water.

As well-hydrated students in Houston, Texas, we were surprised to find that Cr(VI) levels in Houston are also elevated: the average level is 0.75 ppb with a range of 0-6.7 ppb [10].While still below the EPA MCL of 100 ppb, this level is 37.5 times higher than the recommended 0.02 ppb benchmark. This startling statistic has motivated the Rice iGEM team to pursue a project to synthetically engineer bacteria to reduce Cr(VI) to Cr(III) in Cr(VI)-contaminated sources of drinking water.

The majority of drinking water in Houston and in the US – about 87% and 80%, respectively – is sourced from surface waters such as rivers, lakes, reservoirs, and oceans [11, 12]. Every day in Houston, 239 million gallons of wastewater are collected, treated, and subsequently returned to these surface waters, making wastewater an excellent target for pre-emptive remediation of drinking water. Wastewater is also a good target for Cr(VI) remediation because it shows higher levels of Cr(VI) contamination than drinking water [13]. In one study, the influent and effluent Cr(VI) levels for a wastewater treatment facility in Nacogdoches, Texas, were measured to be 1.9 ppb and 1.0 ppb, respectively, whereas the average drinking water level for the City of Nacogdoches is only 0.065 ppb [14,6].

In order to remediate Cr(VI)-contaminated wastewater, our team seeks to build a Cr(VI)-reducing Shewanella oneidensis with enhanced chromium permeability and a built in “kill switch” such that the bacterium will perish after it has degraded the environmental hazard to an acceptable threshold level. The full genetic circuit that we will transform into our bacteria is detailed in Figure 2 below. To address the issues of membrane permeability to chromium, we will increase expression of a membrane bound sulfate transporter system (cysPUWA), the natural entry point for chromate uptake in poisoned cells. We will introduce chromate-reducing and kill switch functionality by building a genetic circuit containing a chromate-sensing promoter (Pchr) which is repressed by a chromate-binding repressor (chrB), a chromate reductase (chrR6), and a toxin (BamHI) (see Figure 3 for the expected response of our circuit in both low and high levels of chromate).

Figure 2: Full genetic circuit diagram. The top-most portion represents the chromate-sensing, chromate-reducing, and “kill switch” components. The bottom-most portion represents the sulfate/chromate transport system components which will increase chromium permeability in the cell.

Figure 3: Chromate-sensing circuit expresses chromate reductase in the presence of chromate and a suicidal toxin in the absence of chromate (*For initial testing we will use the fluorescent protein mcherry as a stand-in for the BamHI toxin in the kill switch.)

Essentially, this circuit creates an artificial “addiction” by imposing a chromium requirement for survival and triggering death in low levels of chromium. When chromium is present, the circuit is in the “survival state” – chromium binds to the chrB repressor and derepresses expression of chromate reductase and the lacI inhibitor, the latter of which represses toxin expression. When chromium is not present, the circuit enters the “death state” – the chrB repressor binds the chrP promoter, preventing expression of lacI and thus derepressing expression of the toxin. Additionally, by placing the toxin under transcriptional control of a lac regulated promoter, we allow for the failsafe option of killing the cell upon introduction of IPTG.

After Cr(VI) is reduced to Cr(III) by bacteria, it can either bind to the functional groups on the cell surface or form nucleation sites for further precipitation. For Shewanella oneidensis, reduced Cr(III) precipitates on the outer surface of the cell. In case of intracellular reduction by other types of bacteria, Cr(III) either binds to the carboxyl, hydroxyl or amide groups on the inner cell surface or precipitates inside the cell in a form of Cr(OH)3. Figure 4 shows an overview of the chromate processes in a natural bacterial cellular environment.

Figure 4: Intracellular Chromate Processes. Image modified from [16]

The most common methods for Cr(VI) removal from water are reduction-precipitation, ion exchange filters, and reverse osmosis filters [16]. Although capable of decreasing Cr(VI) concentration, all methods have significant disadvantages. Reduction-precipitation can function only in a narrow range of temperatures and pH not always compatible with a given water source [4]. Ion exchange filters have high maintenance cost and must be replaced regularly. Reverse osmosis filters, while being an affordable option for separate households, are prohibitively expensive for large-scale operation [17]. Other techniques either have low efficiency of Cr(VI) elimination from water or are not sufficiently cost-effective [4].

The benefit of our solution is that low production cost of synthetic bacteria allows for a large-scale implementation of Cr(VI) bioremediation system. Introducing modified bacteria directly to the wastewater will allow for the removal of Cr(VI) from large volumes of water before it ever accumulates in surface waters and is subsequently supplied to a consumer.