Isolation and identification of the native population bacteria for bioremediation of high levels of arsenic from water resources

Highlights

• We isolated arsenic-resistant bacteria from arsenic-contaminated spring water.

• Identification of 14 isolated bacteria with resistance to some heavy metals was performed.

• Two strains of bacteria: Pseudomonas strain As-11 and Bacillus strain As-12, were identified as arsenic transformer.

• Introduction of Silver Diethyldithiocarbamate method for the first time to measure the concentrations of arsenic species in the biological samples.

Abstract

Health of millions of people is threatened by the risk of drinking arsenic-contaminated water worldwide. Arsenic naturally conflicts with the concept of life, but recent studies showed that some microorganisms use toxic minerals as the source of energy. Hence, the researchers should consider the development of cost-effective and highly productive procedures to remove arsenic. The current study was conducted on a native bacterial population of Seyed-Jalaleddin Spring Kurdistan, Iran. Accordingly, the arsenic amount in water samples was measured >500 μg/L by the two field and in vitro methods. Water samples were transferred to laboratory and cultured on chemically defined medium (CDM) with arsenic salts. A total of 14 native arsenic-resistant bacterial strains were isolated and after providing pure culture and performing biochemical tests, the isolates were identified using polymerase chain reaction (PCR) and 16s rRNA genomic sequencing. The potential of bacterial strains for the biotransformation of arsenic was assessed by the qualitative assessment of AgNO₃ method and efficiency of arsenic speciation was determined for the first time by silver diethyldithiocarbamate (SDDC) method with an error of less than 5%. Among the isolated strains, only strain As-11 and strain As-12 showed arsenic transformation characteristics and were registered in NCBI database by the access numbers KY119262 and KY119261, respectively. Results of the current study indicated that strain As-11 had the potential of biotransformation of As(V) to As(III) and vice versa with the efficiency of 78% and 48%, respectively. On the other hand, strain As-12 had the potential for biotransformation of As(V) to As(III) and vice versa with the efficiency of 28% and 45%, respectively.

Introduction

Arsenic is a rare metal ranked the 20th regarding its frequency in the earth crust (Jolly, 1966), and is found combined with igneous and sedimentary rocks (Merian et al., 2004). Many springs and water running resources are naturally contaminated with arsenic. The regular consumption of such water resources by human beings gradually causes diseases such as melanosis, gangrene, cancer, and finally, death (Hopenhayn, 2006, Maleki et al., 2015). Geothermal areas usually generate springs with high concentration of arsenic (Webster and Nordstrom, 2003). Concentrations more than 50 μg/L of arsenic in drinking water resources increases the risk of bladder and lung cancers and total amounts less than 50 μg/L in drinking water may cause skin cancer. Hence, arsenic in water is a high risk for human health (Jebelli et al., 2017, Lizama et al., 2011). Since the toxic effects of arsenic results from its oxidation state (Pongratz, 1998), identification of all arsenic-resistant strains and the evaluation of factors influencing the speciation are important issues to remove arsenic from the environment. The most frequent forms of arsenic in the environment are its inorganic compound known as arsenite As(III), followed by arsenate As(V) (Balasoiu et al., 2001). The toxicity, bioavailability, and mobility of arsenic in the environment are highly associated with its binding capacity (Bissen and Frimmel, 2003, Inskeep et al., 2001). The mobility of As(III) is more than that of As(V) and accordingly, it is more toxic (Mahimairaja et al., 2005). The main factors controlling the speciation of arsenic are the oxidation state and pH (Masscheleyn et al., 1991).

Arsenic remediation process from drinking water was introduced by the United States Environmental Protection Agency (EPA) includes sedimentation, absorption, ion exchange, and membrane processes. In addition to rather fine removal efficiency for the above mentioned processes (80%–95%), they also had disadvantages such as high cost of administration, high efficiency just in small scales, necessity for secondary refining in most of the cases, sludge production with the high concentration of arsenic, sludge disposal, water loss 10%–15% (based on water quality and the employed process known as a main problem in low-water areas), and the selective performance of some processes during the exposure to different ions concentration (Pirnie, 1999). Most of such techniques are used to remove As(V) and are not efficient enough to remove As(III). Hence, a pre-oxidation stage is required to transform As(III) to As(V) (Katsoyiannis et al., 2004). As(III) oxidation is usually done by adding chemical compounds such as ozone, oxygen peroxide, chlorine, or potassium permanganate (Jekel, 1994, Kim and Nriagu, 2000). Although such compounds affect the oxidation of As(III), due to the formation of hazardous byproducts resulted from their consumption, the costs of coping with the byproducts dramatically increases. In the recent years, biological remediation of arsenic was introduced as a cost-effective method. Hence, microbial oxidation of As(III), as a suitable alternative for the chemical oxidation, can be employed (Duarte et al., 2009).

Microorganisms play important roles in the bioenvironmental state of arsenic, by different effective mechanisms to transform soluble to insoluble species of arsenic as well as toxic to nontoxic ones (Jebeli et al., 2017). There are 4 different microbial mechanisms in the transformation of arsenic as methylation, demethylation, oxidation, and reduction (Gihring et al., 2001, Ilyaletdinov and Abdrashitova, 1981, Oremland et al., 2000, Ridley et al., 1977, Sohrin et al., 1998, Stolz and Oremland, 1999). Therefore, in order to determine the efficiency of arsenic microbial oxidation, various analyses on different species of arsenic in biological samples are required. In recent years, different methods have been introduced to measure the concentration of arsenic species. However, there is still no common method of measurement in this subject. (Bednar et al., 2004, Matera et al., 2003, Rasmussen et al., 2002). The current study aimed at 1) determining the native arsenic-resistant population of bacteria in Emamzadeh Seyed-Jalaleddin travertine spring water, 2) identifying the bioprocess of the spring and 3) measuring the concentrations of arsenic species in the biological samples for the first time by the in vitro method of SDDC, based on the accuracy and validity of the tests, availability, and ease of use. SDDC is a spectrophotometric-based method; it benefits from the possibility to measure different species of arsenic separately and without the need for expensive instruments.