Hexavalent chromium [Cr(VI)] contamination in soil primarily stems from industrial activities within key chromium-using sectors such as tanning, electroplating, and chromium salt production, as well as subsequent inputs from human-induced pollution. Chromium-containing compounds play an important role in industries such as electroplating, chemical manufacturing, printing, and leather processing (Enterline 1974). The hexavalent chromium, Cr(VI), is the most toxic and carcinogenic amongst all the different forms of chromium, due to its high solubility in water, rapid permeability through biological membranes, and subsequent interactions with intracellular proteins and nucleic acids (Das 2015). The 50% lethal dose of Cr(VI) in mammalian cells is as low as 0.15 μg/mL, much lower than the hygienic standard for drinking water of China, which was 0.05 mg/L (2006). Being a major pollutant in China’s industrial wastewater and a first-class carcinogen, Cr(VI) can lead to serious impacts on the environment, economy, and human health if not properly purified. However, the current physical methods in China to purify and detect Cr(VI) are often at a high cost and with relatively low efficiency. Specifically, detecting Cr(VI) demands expensive equipment and requires professionals to analyze the data. Furthermore, it was found that the market price of a common chemical compound, calcium polysulfide, costs 15 RMB per 500g. For a factory that produces 70 tons of 50mg/L Cr(VI) wastewater daily, approximately 11-15kg calcium polysulfide is needed for purification, costing at least 330 RMB a day. Furthermore, concentrations of chromium in wastewater often exceed China’s regulation for Cr(VI) concentrations in industrial wastewater, <1.5 mg/L, thereby making companies unable to send their wastewater directly to a filtration plant to filter for them.
Figure 1. Map of Hexavalent Chromium Pollution Status
Cr(VI) is also listed by the U.S. Environmental Protection Agency (EPA) as one of 129 priority pollutants and ranks among the 14 most toxic heavy metals. It is internationally recognized as a heavy metal carcinogen. Hexavalent chromium ions pose significant health hazards to humans. Workers directly exposed to chromium, residents living near factories, particularly those near smelters and chromate production plant landfills, may be exposed to elevated chromium levels through inhalation, skin contact, and consumption of groundwater (Zhang et al. 2011; Hayes 1988). Hexavalent chromium compounds possess strong oxidizing properties and can enter the human body through skin contact, inhalation, and oral ingestion. They pose hazards to the skin, mucus membranes, respiratory tract, and digestive system, with potential carcinogenic effects (Langard 1990). Due to its high bioavailability, hexavalent chromium is classified as an ingestible toxin/inhalation poison and presents persistent environmental hazards (Sharma et al. 2012). Inhalation primarily causes lung carcinogenesis (Hayes 1988); skin contact induces ulcers or allergic reactions, leading to dermatitis and eczema; ingestion may cause vomiting, abdominal pain, and potentially genetic defects (Zhang et al. 2011). Hexavalent chromium compounds exhibit carcinogenic effects in the body and cause numerous other health issues. Inhaling certain higher concentrations of hexavalent chromium compounds can induce a runny nose, sneezing, itching, nosebleeds, ulcers, and nasal septum perforation (Pesch et al. 2013). Ingestion of excessive chromium doses can cause kidney and liver damage, nausea, gastrointestinal irritation, gastric ulcers, convulsions, and even death. Residual hexavalent chromium in leather can be absorbed through the skin and respiratory tract, leading to gastrointestinal, hepatic, and renal impairment. It may also damage the eyes, causing retinal hemorrhage, optic nerve atrophy, and other conditions (Gherardi et al. 2007; Hayes 1988). Consequently, it has been classified by the International Agency for Research on Cancer as a human occupational carcinogen. For non-occupational populations, the primary route of exposure to hexavalent chromium (Cr(VI)) is oral ingestion. Consuming water contaminated with hexavalent chromium ions or ingesting foods enriched with these ions through bioaccumulation in the food chain poses significant health risks (Patton et al. 2007). For instance, in the late 1990s, China witnessed a village where 37 individuals developed poisoning symptoms from drinking water contaminated with hexavalent chromium ions. Workers at a Jinjiang leather factory also suffered food poisoning after consuming brown sugar contaminated with chromium. The impact of Cr(VI) on human health has garnered extensive global attention.
Going back to the topic, we were inspired by the Chromium slag contamination that occurred in Yunnan, China, in 2011. A total of 140,000 tons of chromium slag were illegally dumped by two drivers from a company called Luliang Peace Chemical into nearby villages. The slag was stacked along the Nanpanjiang River, a major source of drinking water to Guangdong Province. In recent decades, dozens of heavy metal contamination incidents have occurred in China, with over 10 cases of Cr(VI) contamination. Therefore, we realized the urge to develop more accessible and less expensive methods to help foster Cr(VI) purification and detection, preventing further Cr(VI) contamination in China. In addition, our leader is from Yunnan, so he felt the immense responsibility to use the power of synthetic biology to support his own hometown in managing Cr(VI) more efficiently.
Figure 2. Image of Yunnan Chromium Sludge Pollution Incident(GREENPEACE, 2011)
Therefore, it is necessary to develop accurate, cost-efficient, and user-friendly methods for detecting and degrading Cr(VI). To achieve this aspiration, our team aims to utilize synthetic biology to establish an alternative method of detecting Cr(VI) and converting it into the non-toxic Cr(III) through genetic engineering in E. coli. Our final bacterial strains (which produce proteins that degrade Cr(VI) will be integrated with our hardware machine. This constructs an automatic system of Cr(VI) detection and degradation, increasing both the efficiency and convenience of operation.
- We employed synthetic biology techniques to construct a biosensor capable of detecting Cr(VI), while simultaneously developing an engineered bacterial strain capable of degrading Cr(VI). We then validated whether this integrated detection-degradation engineered microorganism could resist, detect, and degrade Cr(VI).
- Design a hardware device based on microbial degradation technology to achieve rapid detection and efficient degradation of Cr(VI). Featuring a modular design that integrates detection, degradation, control, and filtration systems, it completes the entire process from detection to degradation within a short timeframe. Suitable for both laboratory and on-site emergency treatment.
Figure 3. Experimental Technology Roadmap
Figure 4. Hardware Device 3D Modeling Diagram
- Cr(VI) detection system
- Cr(VI) reduction system
We constructed several recombinant plasmids for chromium detection systems and chromium reduction systems. The details in each design characteristic of our recombinant plasmids create the foundation for our future experiments.
For the Cr(VI) detection system, we constructed pET28a-chr-T7-amilCP. This plasmid series contains the amilCP gene, which codes for the blue pigment protein amilCP that is derived from the coral Acropora millepora, serving as a foundation for developing biosensors.
When Cr(VI) from the environment enters the engineered bacterial cell, it is sensed by the ChrB sensor protein. If Cr(VI) is present, ChrB undergoes a conformational change and dissociates from the p_chr promoter, thereby activating transcription. This initiates the first level of transcription, where the T7 RNA polymerase gene is transcribed and translated, producing large amounts of T7 RNA polymerase protein. The T7 RNA polymerase then specifically recognizes and binds to the T7 promoter, triggering the second and stronger transcription stage. At this stage, the amilCP reporter gene is transcribed, leading to the production of a large amount of the blue chromoprotein amilCP. As a result, the bacterial cells turn blue, and the intensity of the color correlates with the concentration of Cr(VI). In the absence of Cr(VI), the p_chr promoter remains repressed, no transcription occurs, and the cells remain colorless.
Figure 5. Diagram of Hexavalent Chromium Detection System
For the Cr(VI) reduction system, we constructed the pSC101-ChrR, pSC101-YieF, and pSC101-ChrR-YieF, three target plasmid recombinants. ChrR, YieF, and ChrR-YieF (a combination of both) are genes coding for chromium reductase proteins. They catalyze the reduction of Cr(VI) to Cr(III) by mediating the transfer of electrons from electron donors (NADH or NADPH) via FMN/FAD cofactors to Cr(VI), thereby reducing Cr(VI) to Cr(V).
ChrR: ChrR is a dimeric flavoprotein that catalyzes both one- and two-electron transfer to Cr(VI) with the momentary formation of Cr(V), and lastly reduces it to Cr(III). Starting from Cr(VI), Chromate reductase can catalyze the reaction of transferring two electrons: one electron to Cr(VI) to form Cr(V), while the other electron generates ROS. The second electron transfer fully reduces Cr(V) to Cr(III). This process can generate reactive oxygen species, or ROS, unstable molecules that are oxidative.
Figure 6. Diagram Display of ChrR Degrading Cr(VI) to Cr(III)
YieF: YieF catalyses direct Cr(VI) reduction to Cr(III) via four electron transfer, in which three electrons are utilised in the reduction of Cr(VI) and the rest are transferred to oxygen. This also generates ROSs, but comparatively less than those generated by ChrR.
Figure 7. Diagram Display of YieF Degrading Cr(VI) to Cr(III)
E. coli BL21(DE3): E. coli BL21(DE3) is a type of E. coli that specializes in expressing large amounts of protein and preventing their automatic degradation. For our Cr(VI) Detection groups, BL21(DE3) has a mutation in the lon and ompT genes. This results in a lack of the lon and ompT proteases within the E. coli, which allows for increased protein expression. BL21(DE3) hence provides moderate expression of genes with strong E. coli promoters, such as the rhaB promoter - the one on our recombinant plasmids.
Restrictive Promoter Rhamnose: α-and β-L-rhamnopyranose is a reducing aldehyde sugar that is also the inducer sugar for the rhaBAD promoter. Our 3 target genes coding our degradation proteins (ChrR, YieF, and ChrR+YieF proteins) contain the rhaBAD promoter. We induced the expression of our target degradation proteins.
Note: Though rhamnose is a reducing sugar that can be a reducing agent for metal ions, at biological standard conditions, this chemical reduction is negligible. According to a recent paper, at pH 3-7, only one Cr(V) (oxidized chromium) signal was detected, indicating a negligible reduction rate at such conditions.
Conclusion: Ultimately, our experiments are heavily interlinked with the scientific mechanisms behind the promoters and target genes in our recombinant plasmids. Some major mechanisms that marked transitional stages in our experiment are listed above. We have a clear logic behind our series of experiments, and they were used to either verify DNA/protein presence or test the specific resistance/detection/degradation of Cr(VI). Our results can be clearly listed out as (1) the highest concentration at which our proteins could still optimally perform; (2) the best time for degradation to happen; and (3) the specific protein with the best reduction (degradation) results.
It consists of three main chambers: Degradation, Detection, Precipitation and Filtration. Initially, the water samples go through a primary filtration zone, where sands and rocks are filtered out of the sample, and the remaining liquid is stored in the Degradation Chamber. Once the hardware is activated, a small portion of the sample flows down to the Detection Chamber. If not present, or if present at low concentrations, the remaining sample in the Degradation Chamber will flow to the Precipitation and Filtration Chamber. For a sample that has been confirmed to require degradation, E. coli containing Cr(VI) degradation genes will be released from the bacterial storage zone beside the Degradation Chamber, initiating a thermometer, along with the heating pads and fans, and mixed thoroughly in the water sample, which thus degrades the Cr(VI) in the water sample to Cr(III).
After the degradation process is complete, bacteria will be killed by UV lighting in the Degradation Chamber, and a resulting small portion of the post-treatment sample will flow back down to the Detection Chamber. Once again, the sample is mixed with bacteria released from the bacterial storage zones, and shortly disposed of after detection is complete. Similarly, if Cr(VI) is detected, the degradation process repeats. If not, the valve to the Precipitation and Filtration Chamber will open, allowing the water sample to flow to that area, whilst the previous valve closes.
The water sample reaching the Precipitation and Filtration Chamber should fulfill one of the two conditions of water flowing to this area. At the preliminary stage of detection, the water that initially was detected not to contain Cr(VI) will flow to this area and immediately pass through the final filter, where this water will be collected. For samples that passed through the secondary detection and were already degraded, a pH detector in the Precipitation and Filtration chamber inspects the sample’s pH. Acidic samples whose pH is below the usual 7~8 value will have NaOH added to them, neutralizing the sample as the mixer mixes the NaOH thoroughly. The remaining precipitate and the dead E. coli containing the degradation genes will be filtered by the final filter, and the resulting water will be free of both bacteria, precipitate, and excessive Chromium.
Figure 8. Integrated System Design Diagram for Detection and Degradation
- By using a biosensor based on pigment protein amilCP to detect the presence of Cr(VI), and using the spectrophotometric method of diphenylcarbazide to determine the concentration of reduced Cr(VI).
- By employing synthetic biology, utilize soluble chromium reductase flavoproteins ChrR and YieF to effectively reduce Cr(VI).
- Based on a safe microbial method, we will achieve more efficient, convenient, and environmentally friendly methods to remediate the toxicity of Cr(VI).
- Baldiris, Rosa, et al. “Reduction of Hexavalent Chromium and Detection of Chromate Reductase (ChrR) in Stenotrophomonas Maltophilia.” Molecules, vol. 23, no. 2, 13 Feb. 2018, p. 406, https://doi.org/10.3390/molecules23020406.
- Das, Manimita, et al. Microbial Chromium Degradation: Biological Evolution, Mitigation, and Mechanism. 2015.
- Gherardi, M., Gatto, M.P., Gordiani, A., Paci, E., and Proietto, A. (2007). [Occupational exposure to hexavalent chromium during aircraft painting]. G Ital Med Lav Ergon 29, 553-555.
- Hayes, R.B. (1988). Review of occupational epidemiology of chromium chemicals and respiratory cancer. Sci Total Environ 71, 331-339.
- Hegewald J et al. Systematic review and quantification of respiratory cancer risk for occupational exposure to hexavalent chromium. Int Arch Occup Environ Health 86, 957-960.
- https://www.greenpeace.org.cn
- Jia, Hepeng. “Chromium Slag Contamination in China.” Chemistry World, 19 Sept.2011,www.chemistryworld.com/news/chromium-slag-contamination-in-china/3001010.article.
- Langard, S. (1990). One hundred years of chromium and cancer: a review of epidemiological evidence and selected case reports. Am J Ind Med 17, 189-215.
- Patton, G., Dauble, D., and McKinstry, C. (2007). Evaluation of early life stage fall chinook salmon exposed to hexavalent chromium from a contaminated groundwater source. Environ Monit Assess 133, 285-294.
- Pesch, B., Weiss, T., Pallapies, D., Schluter, G., and Bruning, T. (2013). Re: Seidler A, Janichen S,
- Sharma, A., Singh, K., and Almasan, A. (2012). Histone H2AX phosphorylation: a marker for DNA damage. Methods Mol Biol 920, 613-626.
- Zhang, X.H., Zhang, X., Wang, X.C., Jin, L.F., Yang, Z.P., Jiang, C.X., Chen, Q., Ren, X.B., Cao, J.Z., and Wang, Q., et al. (2011). Chronic occupational exposure to hexavalent chromium causes DNA damage in electroplating workers. BMC Public Health 11, 224.