Role of Bacterial Chromate Reductase in Bioremediation of Chromium-Containing Wastes

Chromium toxicity is a major environmental concern as it is the chief environmental pollutant released by paint, stainless steel, and mining industries. In nature, chromium exists in two stable valance states: Cr(VI) and Cr(III). Cr(VI) is highly toxic and soluble at neutral pH, whereas Cr(III) is insoluble at normal pH and is less toxic. Thus, it is essential to draw strategies for mitigation of Cr(VI), and microbial reduction of toxic Cr(VI) has been identified as a bioremediation technique not only to detoxify chromium but also to recover the non-toxic Cr(III) by physical means. Chromate reductase, the central enzyme involved in bioreduction of Cr(VI) to Cr(III) may be both intracellular as well as extracellular in nature. Most of the chromate reductase enzyme belongs to the oxidoreductase group such as nitroreductase, iron reductase, quinone reductase, hydrogenase, flavin reductase, as well as NAD(P)H-dependent reductase. Detailed analysis of the structure of the enzymes will help us in the suitable application of these enzymes in bioremediation of metal-contaminated wastes.

Chromium (VI) tolerance and bioaccumulation by Candida tropicalis isolated from textile wastewater

In the present study a yeast strain isolated from industrial wastewater, identified as Candida tropicalis, showed chromium (Cr) tolerance level up to 5 mM. Yeast grown in minimal salt medium containing Cr (VI) ions for 48 h and crude enzyme extracts were tested for chromate reductase activity. Optimum temperature and pH of chromate reductase were 30 °C and pH of 7. The enzyme activity was greatly enhanced in the presence of divalent metal cations. Total protein profile revealed some protein bands were present in hexavalent chromium [Cr (VI)] treated samples but were absent in non-treated samples, especially low molecular-weight protein bands in the mass range of < 25 kDa with greater intensity in Cr (VI) treated samples. Yeast cells were able to uptake Cr (VI) between 21 and 80 mg g− 1 within 2–12-d of time, indicating yeast strain promising potential for Cr (VI) removal from the wastewater. The present study results suggest that C. tropicalis is a suitable candidate for bioremediating chromium ions from the contaminated-environment.

NfoR: Chromate Reductase or Flavin Mononucleotide Reductase?

ABSTRACT Soil bacteria can detoxify Cr(VI) ions by reduction. Within the last 2 decades, numerous reports of chromate reductase enzymes have been published. These reports describe catalytic reduction of chromate ions by specific enzymes. These enzymes each have sequence similarity to known redox-active flavoproteins. We investigated the enzyme NfoR from Staphylococcus aureus, which was reported to be upregulated in chromate-rich soils and to have chromate reductase activity (H. Han, Z. Ling, T. Zhou, R. Xu, et al., Sci Rep 7:15481, 2017, https://doi.org/10.1038/s41598-017-15588-y). We show that NfoR has structural similarity to known flavin mononucleotide (FMN) reductases and reduces FMN as a substrate. NfoR binds FMN with a dissociation constant of 0.4 μM. The enzyme then binds NADPH with a dissociation constant of 140 μM and reduces the flavin at a rate of 1,350 s−1. Turnover of the enzyme is apparently limited by the rate of product release that occurs, with a net rate constant of 0.45 s−1. The rate of product release limits the rate of observed chromate reduction, so the net rate of chromate reduction by NfoR is orders of magnitude lower than when this process occurs in solution. We propose that NfoR is an FMN reductase and that the criterion required to define chromate reduction as enzymatic has not been met. That NfoR expression is increased in the presence of chromate suggests that the survival adaption was to increase the net rate of chromate reduction by facile, adventitious redox processes. IMPORTANCE Chromate is a toxic by-product of multiple industrial processes. Chromate reduction is an important biological activity that ameliorates Cr(VI) toxicity. Numerous researchers have identified chromate reductase activity by observing chromate reduction. However, all identified chromate reductase enzymes have flavin as a cofactor or use a flavin as a substrate. We show here that NfoR, an enzyme claimed to be a chromate reductase, is in fact an FMN reductase. In addition, we show that reduction of a flavin is a viable way to transfer electrons to chromate but that it is unlikely to be the native function of enzymes. We propose that upregulation of a redox-active flavoprotein is a viable means to detoxify chromate that relies on adventitious reduction that is not catalyzed.

Operational Characteristics of Immobilized Ochrobactrum sp. CUST210-1 Biosystem and Immobilized Chromate Reductase Biosystem in Continuously Treating Actual Chromium-Containing Wastewater

Cr(VI) detoxification by biotreatment is considered one of the most practical detoxification methods, especially at low-to-medium concentrations. Although the capabilities of chromium-reducing bacteria and related enzymes in removing Cr(VI) have been explored, little is known about their differences in engineering applications. In this study, Ochrobactrum sp. CUST210-1 was isolated and its chromate reductase identified and separated as biological elements in biosystems developed for Cr(VI) removal. Results indicate that intracellular Cr(OH)3(s) accounted for 88.01% of Cr(VI) reduction product, and a possible reduction mechanism was exposed. The chromate reductase in Ochrobactrum sp. CUST210-1 was ChrR protein, and its crystal structure was revealed. The toxicity of Cr(VI)-containing wastewater was decreased by 57.8% and 67.0% (at minimum) by the CUST210-1 strain and ChrR, respectively. The Ochrobactrum sp. CUST210-1 biosystem demonstrated good adaptability to pH (7–9), and the ChrR biosystem exhibited high removal efficiency (>98.2%) at a wide range of temperatures (25 °C–40 °C). The outlet Cr(VI) concentration of the CUST210-1 biosystem met the industrial discharge limit of 0.5 mg L−1 when the inlet Cr(VI) concentration in the actual Cr(VI)-containing wastewater was <430 mg L−1. The stricter water quality standard of 0.05 mg L−1 could be complied with by the immobilized ChrR biosystem when <150 mg L−1 Cr(VI) concentration was introduced. These developed biosystems can be used in the bioremediation of various Cr(VI)-contaminated wastewaters. Regarding capital costs, those of the CUST210-1 biosystem were higher. To our knowledge, this is the first report comparing differences in the economic and operational characteristics of bacteria and enzyme biosystems for Cr(VI) removal.