Reducing cadmium in rice using metallothionein surface-engineered bacteria WH16-1-MT

Highlights

• The engineered WH16-1-MT strain increased Cd removal efficiency from medium and soil.

• WH16-1-MT improved plant height, panicle length and thousand-kernel weight.

• WH16-1-MT decreased the activities of antioxidant enzyme under Cd stress conditions.

Abstract

Cadmium (Cd) accumulation in rice grains poses a health risk for humans. In this study, a bacterium, Alishewanella sp. WH16-1-MT, was engineered to express metallothionein on the cell surface. Compared with the parental WH16-1 strain, Cd²⁺ adsorption efficiency of WH16-1-MT in medium was increased from 1.2 to 2.6 mg/kg dry weight. The WH16-1-MT strain was then incubated with rice in moderately Cd-contaminated paddy soil. Compared with WH16-1, inoculation with WH16-1-MT increased plant height, panicle length and thousand-kernel weight, and decreased the levels of ascorbic acid and glutathione and the activity of peroxidase. Compared with WH16-1, WH16-1-MT inoculation significantly reduced the concentrations of Cd in brown rice, husks, roots and shoots by 44.0 %, 45.5 %, 36.1 % and 47.2 %, respectively. Moreover, inoculation with WH16-1-MT reduced the bioavailability of Cd in soil, with the total Cd proportion in oxidizable and residual states increased from 29 % to 32 %. Microbiome analysis demonstrated that the addition of WH16-1-MT did not significantly alter the original bacterial abundance and community structure in soil. These results indicate that WH16-1-MT can be used as a novel microbial treatment approach to reduce Cd in rice grown in moderately Cd-contaminated paddy soil.

Introduction

Environmental pollution with heavy metals such as cadmium (Cd), as a result of human activity, is becoming a global issue (Hu et al., 2020). Unlike some organic substances, heavy metals cannot be chemically or biologically degraded (Vilas-Boas et al., 2020). Therefore, the level of heavy metals in the environment can remain unchanged for long periods of time and can contaminate commonly consumed foods, such as fruits and vegetables (Ali et al., 2020). Rice is one of the most important food crops worldwide. However, due to rapid industrialization in recent years, Cd contamination in arable soils has become a significant problem in China (Hu et al., 2020; Pan et al., 2020). In China, the concentration of Cd in rice was reported to range from 0.01 to 5.50 mg/kg, with the highest median value of 0.73 mg/kg found in Hunan Province (Hu et al., 2016; Liu et al., 2016). Cd accumulation in various organs may lead to breast, kidney, liver, lung, pancreatic, prostate and skin cancers (Lee et al., 2018; Genchi et al., 2020). Hence, it is crucial that Cd contamination of rice is controlled to reduce these potential health risks (Hu et al., 2016; Liu et al., 2016).

To date, a range of physical, chemical and biological methods have been used to remediate Cd-contaminated soils (Hu et al., 2016). In Japan, some highly Cd-contaminated soils have been rehabilitated by replacement and removal, but this method has many disadvantages, including environmental disruption and high cost (Uraguchi and Fujiwara, 2012). In Taiwan, soil dilution and turnover have been used for some Cd-contaminated soils, but this method requires large quantities of fertilizer to restore the treated soils (Hseu et al., 2010). Phytoextraction using hyperaccumulator plants is an eco-friendly remediation method, but it is rather slow and sacrifices land productivity (Abe et al., 2011). In addition to the removal of Cd from contaminated soils, reducing the accumulation of Cd in rice grains is another method employed in paddy fields. Lime and calcium carbonate are used to increase soil pH and immobilize Cd in soil to reduce Cd accumulation in rice grains (Bian et al., 2016). The application of iron oxides can form iron plaques on the roots of rice to limit the uptake of Cd²⁺ (Liu et al., 2010). The application of zinc salts, which are chemically similar to Cd²⁺, can reduce Cd uptake by competing for Cd²⁺ transport pathways (Hu et al., 2016). Additionally, the application of animal manures, sewage sludge or biochar can immobilize metal cations in soil, resulting in lower Cd accumulation in rice grains (Cheng et al., 2007; Han et al., 2012; Bian et al., 2013). However, the physical, chemical and fertility properties of soils may be altered by the application of chemical agents.

Microbial bioremediation is an eco-friendly technology for the remediation of heavy metals from contaminated soils (Gupta et al., 2016). Bacteria use a variety of means, including accumulation, sorption and transformation, to detoxify heavy metals in contaminated soil (Nwaehiri et al., 2020). The surface display of proteins on live bacterial cells is a novel, cost-effective technique that can immobilize a protein on the cell, eliminating mass transfer limitations and increasing reaction rates (Liu et al., 2016). Cells that express metal-binding proteins on their surface can be used as an efficient strategy to adsorb heavy metals (Liu et al., 2016). For example, tethering the molybdate-dependent transcriptional regulator, ModE, on the cell surface enabled cells to adsorb molybdenum (MoO₄²⁻) (Nishitani et al., 2010). Yeast engineered to express the metal fixation motifs of a P1 ATPase were able to adsorb lead (Pb²⁺) (Kotrba and Ruml, 2010). A Saccharomyces cerevisiae strain modified to express the transcription factor, CadR, showed higher affinity for Cd²⁺ than the wild type strain (Tao et al., 2015).

Metallothionein (MT) is a histidine-free low molecular weight protein, rich in cysteine and non-aromatic amino acids (Romero-Isart and Vasák, 2002), that is widely found in bacteria, fungi and various animals and plants (Sun et al., 2016). Cyanobacterial MTs have higher affinities to Cd, Zn and Cu ions than mammalian MTs (Shi et al., 1992). The abundance of cysteine residues in MT allows it to bind metals by mercaptide bonding (Cobbett and Goldsbrough, 2002). MT plays crucial roles in metal homeostasis and detoxification in animals, plants and microorgansims (Cobbett and Goldsbrough, 2002). In this study, two tandem repeats of bacterial MT were expressed on the surface of Alishewanella sp. WH16-1. The capacity of this engineered strain, WH16-1-MT, to reduce Cd concentrations in media and rice was investigated. Compared with the parental strain WH16-1, WH16-1-MT showed higher capacity for Cd²⁺ removal from media. Inoculation with WH16-1-MT significantly reduced Cd accumulation in rice. The residual amounts of Cd in brown rice met the national food safety standard in China (0.2 mg/kg) (GB 2762–2017). Our findings provide an environmentally-friendly approach for the bioremediation of environments contaminated with Cd.