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Research Paper

Characterization, Identification and Seasonal Evaluation of Microbes

in Mercury Contaminated Soils

Timothy E. Egbo1, John O. Dickson2, Carrie Miller3, Alexander Johs2, Carrie A. Sanders1,

and Boakai K. Robertson1,*

1Alabama State University, Montgomery, Alabama 36104, USA;  2Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA;  3Troy University, Troy,  Alabama 36082, USA, *Corresponding author, Boakai K. Robertson, Email: brobertson@alasu.edu

Received April 26. 2017, revised July 17, 2017, accepted July 18, 2017

Publication Date (Web): July 18, 2017

© Frontiers in Science, Technology, Engineering and Mathematics

Abstract

Mercury (Hg) contamination within the Oak Ridge Reservation has been an ongoing research. A comprehensive understanding of the potentials of predominant microorganisms in selected cleanup strategy is highly paramount. The use of sorbents has been proposed as a cleanup strategy; however, biological factors need to be evaluated as regards to the strategy. The goal of this study was to use microbes isolated from various contaminated sites and determine their effectiveness on sorbents to reduce total Hg (HgT). We isolated DNA from contaminated soils (SB 5-8, SB 14-8) and control soil (Hinds Creek) with little or no Hg contamination. Pure cultures were obtained from soil samples using broad-spectrum nutrient broth, and natural medium formulated using extracts from the different soil samples. Selected colonies and total DNA from the soils were used for 16S rDNA sequencing. Some Hg reducers (Acinetobacter rhizosphaerae and Serratia marcescens) were isolated. S. marcescens was chosen due to its role in bioremediating and transforming Hg. Studies of Hg leachability in the contaminated soils showed that the amount of leachable Hg varied in magnitude among the three soils possibly due to seasonal changes. Sorbent was added to determine control in leachability after the soils have been incubated for 14 days in diH2O. S. marcescens was inoculated into the soils along with sorbents and incubated for 48 h at 25oC. Total Hg (HgT) concentrations were determined using cold vapor atomic fluorescence in accordance with EPA method 1631. Variable amount of methylators were found in each soil type. Organisms that grew on the nutrient agar grew poorly on the natural medium with variable colonies. However, S. marcescens grew on both media. S. marcescens showed high reduction of Hg2+ compared to the sorbent; however, when inoculated in the soil, the sorbent was found to absorb more HgT in solution.

Keywords

Total mercury, Sorbents, Bioremediation, Leachability, Methylators, Aerobic microbes

Introduction

The Y-12 National Security Complex around the Oak Ridge Reservation has been identified by the US Environmental Protection Agency (EPA) to have heavily contaminated the upper andlower East Fork Poplar Creek (EFPC) ecosystem with Hg through transportation from the source areas via atmospheric deposition, sediment transport, surface water runoff, and groundwatertransport contaminating the upper and lower EFPC ecosystem (He et al 2010).

Part of the current strategy to remediate Hg from EFPC is the use of sorbents to immobilize Hg from EFPC. Tests conducted on various sorbents showed varying level of Hg affinity. Sorbents such as Thiol-SAMMS and Organoclay have been identified to have great ability to immobilize organic Hg, Hg+ and Hg+2 (Chen et al 1999; Wang and Abraham 2009) among others.

Hg is one of the most dangerous metals that freely exist in the environment. Hg pollution occurs through the release of fossil fuel globally with 90% of total Hg (HgT) released to the atmosphere due to steady combustion (Pirrone et al 2010). Hg can be found in soils, aquatic environments, and sewage. However, in soils Hg is more persistent compared to the aquatic ecosystems and sewage (Xu et al 2015). For the most part, Hg enters the food chain through soils or water thereby increasing health risks (Cui et al 2011). Hg originating from both the aquatic and terrestrial environments can seriously endanger the food chain and degrade the ecosystem thereby threatening human health. This in return increases the need to transform the Hg in the environment into its most stable and less toxic form.   

Hg exists in three (organic, inorganic and elemental Hg) forms. Organic Hg results from Hg combining with carbon and other elements. The most common and toxic form of organic Hg is methylmercury (MeHg) (Reinfeldera et al 1998). Bacteria methylate Hg by the excretion of methylcobalamin (Robinson and Tuovinen 1984). The production of MeHg is predominant among microorganisms especially anaerobes (Hsu-Kim et al 2013; Gilmour et al 2013; Podar et al 2015). Moreover, obligate anaerobes are mostly associated with Hg methylation and they include diverse groups such as sulfate reducers, iron reducers, methanogens, syntrophs, acetogens (Gilmour et al 2013; Lee et al 2016; Jeremiason et al 2006; Marvin-DiPasquale et al 2014; Gilmour et al 2011; Yu et al 2013; Yu et al 2012). Inorganic Hg is more soluble and more reactive than the other forms of Hg. Inorganic compounds exist as mercuric sulfide (HgS), mercuric oxide (HgO) and mercuric chloride (HgCl2). Several bacteria strains possess reductase genes that are encoded by mer operon that aids in reducing Hg2+ to Hg0 (Barkay et al 2003; Lin et al 2012; Tottey et al 2007).

On the other hand, organisms need some heavy metals (nickel, iron, zinc and copper) in small amounts for their metabolic activity; other metals (Hg, silver and cadmium) have no role in microbial metabolism. However, at low concentration they can be metabolized (Ehrlich 1997). Factors such as pH, metal concentration, speciation and organic matter have an influence on microorganisms (Nwuche and Ugoji 2008; Sterritt and Lester 1980). Studies have also shown that the presence of these factors in Hg contaminated environments can create a huge impact on the ecosystems (Sobolev and Begonia 2008).

In this study, we characterized the soil properties to better understand its biotic and abiotic characteristics. To understand the long-term stability of total Mercury (HgT) in the soils during various seasons, a study was performed to determine the leachability of HgT through wet and dry analyses. We also developed an easy and bio-friendly approach to isolate aerobic Hg resistant microorganisms that can transform Hg either via metabolically dependent or independent processes, or as a potential target for bioremediation. We were also able to develop a more quantitative and qualitative method to isolate genomic DNA (gDNA) from the soil samples.

Experimental Design

Soil collection

      Bank soils contaminated with Hg from EFPC was collected from two sampling sites with soil profiles based on soil color. For soil profile, the samples were collected from twodistinct soil layers (Figure 1), the top lighter-colored soil labeled SB 14-8 downstream of the creek and the bottom dark-colored soil upstream of the creek labeled SB 5-8.  Soil samples fromthe uncontaminated Hinds Creek (45 km away from EFPC) with similar general chemistry, hydrology and underlying geology as the contaminated sites were included as control. Sampleswere collected in summer 2015, fall 2016 and winter 2017. The samples were transported on dry ice to Alabama State University within 24 hours of sampling and kept at -20°C until analysis.

Figure 1. East Fork Popular Creek where the soil was sampled from the bank of the creek.

Determination of the physical characteristics of soils and stream water

Hg in the soils was analyzed according to EPA method 1631 using a Brooks Rand MERX Cold Vapor Atomic Fluorescence Spectrophotometer (CVAFS) system. MeHg was analyzed usingmodified EPA method 1630 on a Brooks Rand MERX MeHg instrument coupled with a Perkin Elmer Elan-DRC ICP-MS. The pH was determined with a glass electrode according to standardmethods. Total carbon (C), sulfurs, and nitrogen (N) were determined by the dry combustion method using a Leco 2000 CNS analyzer equipped with thermal conductivity detector. Major cations were analyzed in accordance to standard procedure using sodium peroxide fusion method coupled with Varian 735ES ICP-OES detection. A multi-parameter sonde (TROLL 9500series) was used to record pH, specific conductivity, oxidation-reduction potential and temperature. Particle size distribution (PSD), Atterberg limits and bulk density of samples were determinedin accordance with the American Society of Testing and Materials (ASTM) standard test methods D422, F1632, and D2937 respectively.

Leachability of Hg under wet and dry conditions

To examine the amount of leachable Hg from the soil samples (SB 5-8, SB 14-8 and Hinds Creek) under wet or dry conditions, soils were leached with artificial creek water (ACW).  The ACW is composed of deionized water with 1.93 mg/L Ca(NO3)2 to simulate the ionic strength and the major anion and cation composition present in EFPC. A preliminary experiment was conducted in which the concentration of leachable Hg was measured from naturally wet soils and soils dried at 60°C for 3 days. The wet conditions were conducted on the soil samples, while the dry conditions were conducted on the SB 5-8 and Hinds Creek soils.  The leachability of dry HgS mixed with sand was also tested. The ACW was added to the samples and shaken for 24 hours, the samples subsequently filtered (GFF filter) and mercury content in the aqueous phase determined. Soils samples were either dried at 30°C for 5 days or left under wet conditions for 5 days.  ACW was added to all samples on day 6 and shaken for 24 hours and then filtered on day 7.  This process was repeated until the amount of Hg leached from the samples reached a steady state.

Isolation of microorganisms

The soils were cultured in a non-organism specific nutrient broth used for pure culture isolation. Approximately, 1 g of the soil was transferred to 99 mL of nutrient broth and incubated overnight, ≤18 h at 25°C on a rotary shaker and about 1 mL of the overnight culture grown to 10-6 was plated on both selective and non-selective solid media (Remel INC, San Diego, CA and WARD’s science, Rochester, NY) respectively, and incubated at 25°C for 24 h. Pure bacteria colony was repeatedly transferred to fresh solid media until single colonies were isolated. The isolateswere suspended in sterile saline solution until further testing. Morphological studies of the isolates were performed using a Nikon compound high power microscope (1000 x), followed by Gramstaining. Chemical characterizations of the isolates were determined based on the Bergey’s Manual of Determinative Bacteriology.

Bacterial Enumeration

Each soil extract was formulated using 500 g of soil added to 1 L of 50 mM NaOH solution and incubated at room temperature overnight. The supernatant was separated through decantation and centrifuged for 1.5 hours at 14,000 rpm. Approximately 7.5 g/L of agar was added in 250 mL of soil extract and sterilized by autoclaving. The culture was serially diluted to 10-6 in 0.85% (w/v) NaCl. The 0.1 mL dilution was plated on the formulated agar and incubated at 25°C for 72 h. Colony formation was recorded after 24, 48 and 72 h.

DNA Extraction

Soils from contaminated and reference sites were used for total community genomic gDNA extraction at ~1 g of each sample (wet). The soils were homogenized prior to subsequent studies. A survey of the total DNA in the soil was performed using gDNA extracted from soil using PowerSoilTM DNA Isolation Kit (QIAGEN, Carlsbad, CA), with the addition of a few optimizations, and quantification using N200 spectrophotometer for soil (MP Biomedicals) in accordance to the manufacturer’s instructions. The gDNA was concentrated and further purified by precipitation with ethanol. The quality and concentration of the generated DNA was evaluated using NanoDrop N200 spectrophotometer (Thermoscientific, Wilmington, DE). The isolated DNA was stored at -20°Cfor further analysis using 125-nucleotide paired-end multiplex sequencing on the Illumina platform.

Mercury Biosorption using Sorbents and Microorganism

All glass wares were subjected to 10% HCl and 25% HNO3 overnight washing before sterilization to purge any traces of Hg. Microorganisms were grown between OD600 0.4 to 0.6 before inoculation into the culture. About 50 ng/L of elemental Hg was used to analyze the amount of Hg that can be absorbed by either the microorganisms or the sorbents (Thiol-SAMMS™ and OrganoclayPM-199) (Oak Ridge National Lab. Oak Ridge, TN) (Table 1). The culture was incubated at 25°C for 48 h on a rotary incubator, spun down at 4,000 rpm for 15 mins, collecting the supernatant and preserving with 0.1% metal grade HCl until further analysis.

Table 1. Overview of sorbent materials evaluated.

Results and Discussion

Determination of chemical and physical characteristics of soil and stream water

In our initial experiments, we determined the chemical and physical characteristics of the soils. The concentration of HgT in soils was slightly greater in SB 5-8 sample (14.268 mg/gdw)compared to SB 14-8 (13.468 mg/gdw) (Figure 6).  However, the MeHgT in the SB 14-8 was twice the amount in SB 5-8. The pH of the soils and stream water was slightly basic (Table 3a). This indicates that the formation of MeHg can occur downstream regardless of the amount of HgT if a methylating microorganism is present. Elemental analysis of the soil showed that most of themajor elements in the soils were less than 1% except for K, Al, Fe, and Si with varying concentrations (Table 4a, 4b, 4c, and 4d). Total carbon content was much greater in SB 5-8 (13.8%)compared to SB 14-8 (2.81%) and similarly, twice the amount of organic carbon in SB 5-8 (4.2%) versus 2.2% in SB 14-8 (Table 3c).

Particle size analysis showed that the soils are basically silty clay loam. Results of detailed characterization of the stream water are presented in Table 3a. Other measurements such asgeographical location, Atterberg limits, sample collection time, and depths are shown in Table 5a, 5b, and 5c respectively. 

Mercury Leachability under wet and dry conditions

Experimental results indicate that the amount of leachable Hg varies by orders of magnitude in the three soil samples.  Soils from Hinds Creek, control site, leached 10 ng/L when the soils were held under wet conditions.  The wet soils from EFPC sites SB 14-8 and SB 5-8 leached 1.0 x 103 ng/L and 1.0 x 104 ng/L respectively (Figure 7). The concentration of Hg in the soils from SB5-8 is approximately 100 times higher than those from SB 14-8. The results showed how the “leachable” Hg fraction (defined as the fraction of Hg in the soil which can be mobilized into water) changes when soils undergo wetting and drying cycles, which are common in nature.

Table 2. (A) Isolation of bacteria on non-selective nutrient media and (B) Soil extracts or natural media. “+” is growth; “-“ is no growth

(B)

(C)

We have isolated and identified different microbes that can thrive in Hg concentrations; some with different pigmentations based on microscopy (Table 2a and Figure 3), physiological and chemical characteristics. Potential organisms able to survive in Hg contaminated soils were isolated on both non-selective commercially prepared media and natural media prepared from the soil extracts. The organisms were also cultured on various selective media (Table 2a). Several studies have evaluated Hg resistance in different microorganisms by inoculating various amounts of Hg to the culture medium followed by incubation (Száková et al 2016; François et al 2012). When S. marcescens was directly introduced to soils with the sorbent, the sorbent absorbed more Hg from the soil compared to culture solution without soil (Figures 5 and 8).

A clear understanding that not all bacteria from a given environment will grow on all laboratory media (Xu et al 2015) is very important to further characterize microbial diversity in an environment. Using the Hg contaminated soils containing known amounts of Hg to develop a formulated agar for microbial growth, we observed that only the pigmented organism grew on the formulated agar after 72 h compared to when grown on the nutrient agar (growth rate of 24 h). From the results, the growth rate of the organisms on the different medium varied. After 24 hours of incubation, robust colonies were found on the rich nutrient agar: SB5-8 = 9.0 x 107, SB14-8 = 9.4 x 107 and HC = 8.9 x 107, but not on the agar containing the soil extract. However, colony formation on the plate containing soil extracts became more detectible after 72 hours. Organisms that were transferred from nutrient agar to the natural medium grew poorly with little or no colonies. This could be due to the Hg concentration, which did not vary in the soil samples. Also, the availability of nutrient was strictly the same as in the sampling environment with minimal to no modification.

Table 4. Soils chemical compositions. 

(a)

(b)

(c)

(d)

Table 5. (A) Soils physical characteristics measured; (B) Geographical location where soils were collected; (C) Information on soil collection time, depths and acidity level. Depths are in inches below ground surface; DIW is distilled water.

 (A)

(B)

(C)

Figure 2. Graph showing Soil DNA concentration 

Figure 3. Electron micrograph showing the morphology of S. marcescens gram staining.

Figure 5. MCO: S. marcescens; T: Thiol SAMMS; O: OrganoclayPM-199; TMCO: Thiol-SAMMS and S. marcescens; OMCO: OrganoclayPM-199 and S. marcescens; M: Nonselective nutrient broth; TMCOM: Thiol-SAMMS, S. marcescens and nonselective nutrient broth; OMCOM: OrganoclayPM-199, S. marcescens and nonselective nutrient broth. Comparing the amount of Hg that can be sorbed by S. marcescens compared to sorbents or combined.

Mercury Biosorption using Sorbents and Microorganism: 

Organoclay complex was shown to have a great affinity with Hg in solution (Wang and Abraham 2009). In our analysis, organoclay did not show any significant absorption of Hg compared to S. marcescens alone (Figure 8) or neither when both were combined. In the presence of nutrient media used for culturing there was no increase of HgT. Subsequently, S. marcescens showed more reduction of HgT.

Figure 6. Total mercury concentration in soils. 

Figure 7. Leachable Hg in soils under wet and dry conditions.

Total DNA

The results after extraction showed that SB 5-8 gave more highly concentrated DNA than that of Hind-1 or SB 14-8 (Figure 2). Ethanol precipitation increased the quality yield of the gDNA and further aided in removing any bound organic matter that might inhibit chances for optimal DNA sequencing or binding during molecular studies like PCR. 

Figure 8. Recovery of Hg2+ after incubating S. marcescens with culture media and sorbents. MCO: S. marcescens; Sorbent: Thiol SAMMS. Comparing the amount of Hg that can be sorbed by S. marcescens compared to sorbents alone or combined. There was a significant difference in the amount of Hg sorbed by S. marcescens compared to the sorbent or combined means. P value 0.0012**. Error bars represents the standard deviation of triplicates.

Conclusions 

Isolation and identification of environmental microorganisms is very challenging due to the heterogonous nature of soils (Kirk et al 2004). Various strategies have been employed to isolate and understand the behavior of individual microorganism in the presence of increased soil Hg contents (Száková et al 2016; Frentiu et al 2013). Growing organisms in their natural environment provides information in the selection of suitable organisms capable of surviving using bioavailable nutrients. In addition, pH also plays a very crucial role for the optimum growth of bacteria.

S. marcescens has been identified to significantly reduce HgT (Cui et al 2011); however, little is known on its role in Hg speciation. It does contribute to the conversion of organomercuric and mercuric Hg to volatile Hg through intracellular transformation linked to the expression of mercury resistance (mer)S genes (Reinfeldera et al 1998). The pathway of Hg methylation in this microorganism has not been fully understood nor the specific mechanism that S. marcescens uses to reduce Hg.

On the other hand, Hg transformation in S. marcescens could be associated with substrate specificity of its enzymes (Gilmour et al 2013). Our result using spiked Hg showed that S. marcescenscan absorb 50% of Hg2+ (Figure 8). However, when inoculated in the contaminated soil, sorbent (OrganoclayPM-199) showed more HgT reduction compared to S. marcescens alone (Figure 5). However, their performance in an environmental condition with other organisms needs to be evaluated. Unlike other S. marcescens isolates used in the previous Hg studies, ours was a pure culture isolated from a Hg contaminated site that is currently being studied. This strain has been shown to withstand rigorous environmental conditions and can easily be cultured in laboratory medium.

Sorbent Organoclay complexes have been tested to effectively reduce HgT in solution, but have not been evaluated for its efficacy in the presence of microorganisms. Consequently, we performed a study to examine the influence of microorganisms on the efficacy of HgT reduction by the sorbents. Our results did not show any significant influence of S. marcescens on the sorbents compared to S. marcescens alone, even though S. marcescens was isolated from the contaminated sites. Therefore, a clear understanding of its role or influence in reducing HgT in contaminated soils in the presence of sorbents and other microbes needs to be further evaluated because S. marcescens can form biofilms in the presence of other microbes.

Acknowledgements

This research is funded by the U.S. Department of Energy (DOE) and Savannah River Nuclear Solution (SRNS) subcontract grant no. 0000217390. Oak Ridge National Laboratory (ORNL). The National Science Foundation's Alliances for Graduate Education and the Professoriate (AGEP) Program, Grant No. 1432991. We are grateful to Rajnish Sahu for the EM image.

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Citation:

Timothy E. Egbo, John O. Dickson, Carrie Miller, Alexander Johs, Carrie A. Sanders, and Boakai K. Robertson (2017) Characterization, identification and seasonal evaluation of microbes in mercury contaminated soils, Frontiers in Science, Technology, Engineering and Mathematics, Volume 1, Issue 1, 15-26