Genetically Engineering Bacillus subtilis with a Heat-Resistant Arsenite Methyltransferase for Bioremediation of Arsenic-Contaminated Organic Waste

Abstract

cOrganic manures may contain high levels of arsenic (As) due to the use of As-containing growth-promoting substances in animal feed. To develop a bioremediation strategy to remove As from organic waste, Bacillus subtilis 168, a bacterial strain which can grow at high temperature but is unable to methylate and volatilize As, was genetically engineered to express the arsenite Sadenosylmethionine methyltransferase gene (CmarsM) from the thermophilic alga Cyanidioschyzon merolae. The genetically engineered B. subtilis 168 converted most of the inorganic As in the medium into dimethylarsenate and trimethylarsine oxide within 48 h and volatized substantial amounts of dimethylarsine and trimethylarsine. The rate of As methylation and volatilization increased with temperature from 37 to 50°C. When inoculated into an As-contaminated organic manure composted at 50°C, the modified strain significantly enhanced As volatilization. This study provides a proof of concept of using genetically engineered microorganisms for bioremediation of As-contaminated organic waste during composting.A ppropriate recycling of organic wastes to agricultural land can increase soil organic matter, improve soil microbial communities, and provide valuable nutrients such as nitrogen and phosphorus. In China, for example, annual production of organic waste produces more than 60 million tons of nutrients (expressed as the sum of N, P 2 O 5 , and K 2 O), but only about half is recycled to agricultural land (1). Inappropriate disposal of organic waste causes eutrophication and water pollution in China (2). One reason for the lack of recycling of organic waste is concern about elevated levels of pathogens and organic and inorganic contaminants. Heavy metals and metalloids, such as zinc (Zn), copper (Cu), and arsenic (As), are often overused in animal feed in China, resulting in high concentrations of these inorganic contaminants in some animal manures (3, 4). For example, a recent national survey of the organic manure composts conducted by our laboratory (unpublished data) showed that 13% of the samples exceeded the As limit for organic manure (15 mg kg Ϫ1 ) (5), with the highest concentration reaching 72 mg kg Ϫ1 . Aromatic organoarsenicals such as roxarsone (4-hydroxy-3-nitrophenylarsonic acid) have been added to animal feed for disease control and growth promotion since the middle of the 1940s (6, 7). Although organoarsenicals have low toxicity, their main degradation product (inorganic As) is highly toxic to animals and humans (8). The use of organoarsenicals (mainly roxarsone) has been halted in the United States and the European Union, but farmers continue to use these substances in China. Recently, Yao et al. (9) reported the presence of organoarsenicals in a quarter of 146 animal feed samples collected in Guangdong Province, China, with average concentrations of roxarsone and arsanilic acid of 7.0 and 21.2 mg As kg Ϫ1 , respectively. In our national survey of organic manure composts, only inorganic As and small amounts of dimethylarsenate [DMA...

Efficient Arsenic Methylation and Volatilization Mediated by a Novel Bacterium from an Arsenic-Contaminated Paddy Soil

“…29 As speciation analysis was performed using high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC–ICP-MS) as described previously. 31 Samples were analyzed directly for the quantification of As(V), MAs(V), and DMAs(V) and after reaction with 20% (v/v) H 2 O 2 to oxidize As(III) to As(V) to separate and quantify the coeluted TMAs(V)O and As(III).…”

Arsenic methylation by a novel ArsM As(III) S‐adenosylmethionine methyltransferase that requires only two conserved cysteine residues

“…The resulting supernatants were filtered through 0.22 lm MCE syringe filters (ASD, China). The filtered solutions were analyzed for As species using HPLC-ICP-MS as described previously (Huang et al, 2015). The cell morphology was observed by SEM (S-3000N, Hitachi).…”

Bioelectrochemical and Conventional Bioremediation of Environmental Pollutants

“…In a recent study, petroleum hydrocarbons were most rapidly biodegraded when the C:N:P ratio of was 100:15:1 [10]. Bioremediation using genetically modified organisms (GMOs) has shown promise but the ecological effects and broader implications of releasing genetically modified microbes are the subject of considerable debate [88,89].…”