Arsenic transforming abilities of groundwater bacteria: combined use of strain 2WW and goethite in metalloid removal

The revised drinking water standard for arsenic imposes a 10 μg L-1 threshold limit (World Health Organisation), which has boosted research efforts to remove arsenic efficiently. Arsenic has a high affinity for adsorption to metal oxides, with arsenate [As(V)] being more effectively adsorbed than arsenite [As(III)]. Consequently the oxidation of As(III) to As(V) is a prerequisite for achieving arsenic concentrations below the threshold. Bacterial As(III)-oxidation is a promising step in the removal of arsenic from groundwater in combination with adsorbing materials such as goethite, as a sustainable alternative to the use of chemical oxidants. The aim of our work was to study the indigenous microbial communities of arsenic-rich groundwater from Lombardia (Italy) and to isolate As(III) oxidizing bacteria in order to test the feasibility of an arsenic removal process that comprises biological oxidation of As(III) to As(V) followed by adsorption to goethite. Water samples were collected from ten sites (six wells and four piezometers), chosen from the dataset of the Regional Agency for Health Prevention and Environmental Protection of Lombardia, and based on their different levels of arsenic content. Water samples were previously purged under controlled flow until stabilization of temperature, dissolved O2 and redox potential. pH and electrical conductivity (EC) were determined on refrigerated samples, within a few hours after collection. Iron, manganese and arsenic total contents were determined on acidified samples by ICP-MS (Agilent Technologies); for arsenic speciation, water samples were previously passed through a WATERS Sep-Pak_Plus Acell Plus QMA cartridge (Waters Corporation). Samples from piezometers showed lower pH values, significantly higher concentrations of total dissolved iron and manganese, and significantly higher values of electrical conductivity than samples from wells (p ≤ 0.05). The total arsenic concentration in groundwater samples ranged from 0.7 to 171 μg L-1. Samples from eight out of ten sites exceeded the 10 μg L-1 arsenic threshold (D.Lgs. 31/2001). In all the arsenic polluted samples, As(III) was dominant and the As(III)/As(V) ratio ranged from 4 to 7. Bacterial strains involved in the arsenic cycle, so far not been studied in groundwater in Italy, were isolated from successive enrichment transplants on As(III) or As(V) of groundwater samples. Twenty isolates able to reduce As(V) were retrieved from most of the sites. They were affiliated to different species of Pseudomonas, Achromobacter and Rhodococcus. They carried an ars detoxification system, as detected by PCR amplification of arsC and arsB genes for As(V) reductase and As(III) efflux pump, respectively. As(III) oxidising strains were more rare as only three isolates were able to produce As(V) from As(III). They belonged to Rhodococcus ruber, Achromobacter sp. and to Aliihoeflea aestuarii and aioA gene for As(III) oxidase was detected. In order to envisage the use of a bacterial strain in the As(III) oxidation step of the arsenic removal process, Aliihoeflea aestuarii strain 2WW was tested for its As(III) oxidation activity in different conditions of temperature and pH. It completely oxidized 1 mM As(III) within 24 h at 30°C, and within 96 h at 15° C. At 5°C the complete oxidation of As(III) occurred in 350 h. Complete As(III) oxidation occurred also in the pH range 5.0-8.0. Goethite was chosen as a model arsenic adsorbing material. The experiments were conducted in Tris-HCl 5 mM (pH 7.2) containing 200 μg L-1 of arsenic, using As(III)-induced and non-induced resting cells. As(III) and As(V) adsorption capability of goethite was evaluated by using 4 g L-1 goethite and increasing arsenic concentrations (25-800 μg L-1). Finally we tested the arsenic removal efficiency of the combined 2WW-goethite system on a synthetic contaminated water and a contaminated groundwater sample. Resting cells of an As(III)-induced culture of 2WW were able to oxidize completely 200 μg L-1 of As(III) in 8 hours, while non-induced cells oxidized As(III) in 24 hours. Subsequently, results from the arsenic adsorption experiments showed that goethite removed almost the complete 200 μg L-1 of As(V) from the solution, while only 75% As(III) was adsorbed, thus leaving in solution arsenic exceeding the 10 μg L-1 threshold limit. Our results indicate that arsenic polluted groundwaters of Lombardia are reservoirs of bacterial populations able to transform arsenic, that may contribute to mobilization/immobilization of the metalloid in that environment. As(III) oxidation by Aliihoeflea aestuarii strain 2WW combined with goethite adsorption is envisaged as an efficient approach for the removal of arsenic from contaminated groundwater. Research supported by CARIPLO Foundation, project 2010-2221