زيست¬پالايي كادميم با روش رسوب¬گذاري زيستي كربنات كلسيم در خاك شني

Bioremediation of Cadmium in Contaminated Sandy Soil by Microbially Induced Calcite Precipitation

آلودگی زیستگاه¬های طبیعی به فلزهای سنگین به سبب سمیت بالا سلامت موجودات زنده و محیط زیست را تحت تأثیر قرار می¬دهد. معدنی شدن زیستی بر اساس تشكیل رسوب كربنات كلسیم زیستی روشی نویدبخش برای پالایش فلزهای سمی در خاك¬های آلوده می¬باشد. این پژوهش به منظور بررسی حذف كادمیم به روش رسوب¬گذاری زیستی كربنات كلسیم از مسیر هیدرولیز اوره با باكتری اسپورسارسینا پاستئوری انجام شد. آزمایش ابتدا در محلول دارای كادمیم (در سه سطح 5/0، 1 و ۲ میلی¬مولار كادمیم) و سپس در یك خاك شنی آلوده شده با كادمیم (در پنج سطح صفر، 10، 20، 40 و 50 میلی¬گرم بر كیلوگرم) در قالب طرح كاملا تصادفی و در 3 تكرار اجرا شد. آزمایش¬ها در دو سطح با باكتری و بدون باكتری بودند. رسوب كادمیم در محلول آلوده در سه غلظت 5/0، 1 و ۲ میلی¬مولار كادمیم در حضور باكتری به ترتیب 6/99، 8/99 و 8/99 درصد بود. نتایج این پژوهش نشان داد كه در تیمارهای دارای باكتری در مقایسه با تیمارهای بدون باكتری مقدار كادمیم در بخش محلول+ تبادلی كاهش یافته و در بخش كربناتی افزایش آماری چشم¬گیری (P≤0.05) پیدا كرد. به طوری‌كه مقدار كادمیم در بخش محلول+ تبادلی خاك در 4 غلظت 10، 20، 40 و 50 میلی¬گرم بر كیلوگرم كادمیم در حضور باكتری به ترتیب 9/85، 1/61، 3/74 و 3/80 درصد كاهش نشان داد. بنابراین، نتایج این پژوهش حاكی از آن است كه جداسازی فلزهای سنگین بر مبنای روش رسوب¬گذاری زیستی كربنات كلسیم از طریق رسوب همزمان با كلسیت می¬تواند برای پالایش زیستی فلزهای سنگین سودمند باشد.

Introduction: Cadmium is considered to be one of the heavy metals with the highest toxicity, because it has high activity and a relative high dissolution rate in water and in living tissues. In recent years, due to the high volume of natural resources pollution and the inefficiency of conventional physicochemical methods for refining these resources and the occurrence of environmental crisis, bioremediation process has been at the forefront. Microbially induced calcite precipitation (MICCP) has been considered as a novel solution for these problems, and several bacterial species have been already utilized for MICCP. MICCP based degradation of urea occurs through the ureolytic pathway. Urease (urea amidohydrolase) is an enzyme that hydrolyzes urea into one mole of carbonate and two moles of ammonia per mole of urea. In this aspect, microbial mineral precipitation products such as calcite can strongly adsorb heavy metals on their surfaces and incorporate heavy metal ions into their crystal structure. Some studies have reported MICCP-based sequestration of soluble Cd via coprecipitation with calcite was useful for Cd bioremediation. Several bacterial species have been utilized for MICCP. The endospore forming bacteria Sporosarcina pasteurii have been shown to produce high levels of urease and have therefore been extensively studied. Sporosarcina pasteurii has attracted significant attention for its unique feature of calcium carbonate precipitation, which can be easily controlled. So, In the present study the ability of Sporosarcina pasteurii bacterium has been investigated in the remediation of Cd(II) in Cd-contaminated sandy soil based on MICCP method. Materials and Methods: Sporosarcina pasteurii (PTCC 1645) was procured from Microbial Bank of Iran (Central Collection of Industrial Fungi and Bacteria, Karaj, Iran). The bacterial strain was inoculated into NB (nutrient broth) media containing 2% urea and 25 mM CaCl2 (NBU media) and then incubated at 37◦C for 6 days. The urease activity was determined at regular time intervals by measuring the amount of ammonia released from urea according to the phenol-hypochlorite assay. Minimum inhibitory concentration (MIC) test was performed to determine the lowest concentration of cadmium chloride, which prevents the growth of bacteria. Sporosarcina pasteuriiwas inoculated into NBU media supplemented with 0.5, 1,2,4,8 and 10 mmol l-1 Cd and incubated at 37◦C, 130 rpm for 50 hours. Control flasks without adding Cd were also incubated. Bacterial growth was determined in terms of optical density (OD) by measuring absorbance at a wavelength of 600 nm at regular time intervals (0, 10, 20, 30, 40 and 50 hours) and colony-forming units (CFU) were also counted. The cadmium removal in 0.5, 1 and 2 mM cadmium solutions (based on MIC) was measured.A sandy soil from a depth of 0 to 30 cm was sampled. The soil was polluted with 10, 20, 40 and 50 mg/kg of cadmium and incubated in room temperature for two weeks. After incubation time, the cadmium remediation studies were performed at 30◦C in the beakers containing 100 g of sterilized dried contaminated soils and 200 mL of overnight grown of Sporosarcina pasteurii (~ to 107cfu ml−1) in NBU media. For each treatment corresponding control were included with the same condition but without bacteria.After 7 days of incubation, urease and dehydrogenase enzymes activity and concentration of cadmium in soluble + exchangeable and carbonate fractions were measured. The concentration of cadmium in iron-manganese oxides, organic matter and residual fractions in concentration of 50 mg/kg cadmium was also determined according to the continual extraction procedure of Tessier et al. (1979). Results and Discussion: The amount of released ammonia by ureolytic activity of Sporosarcina pasteurii increased up to fourth day and then became almost constant.Optical density in different concentrations of cadmium decreased in comparison to control treatment after 48 hours. The minimum inhibitory concentration of cadmium for bacteria growth was 2 mM as determined by colony counting after 48 hours of incubation. Cadmium removal efficacy from solutions containing 0.5, 1 and 2 mM of cadmium was 99.6, 99.8 and 99.8%, respectively. The amounts of urease and dehydrogenase activities in the presence of bacterium were significantly higher than control treatments (P≤0.05). The results of the fractionation of contaminated soils in the absence of Sporosarcina pasteurii showed the distribution of cadmium as organic matter