Eco-toxicity and bioremediation of mercury in terrestrial environments

Research Doctorate - Doctor of Philosophy (PhD)The heavy metal mercury (Hg) poses serious risk to environmental and human health. The toxicity and biogeochemistry of mercury have both been well studied in aquatic systems which has led to the development of successful bioremediation technologies to treat Hg contaminated waters. On the other hand, the eco-toxicological role of Hg in soil systems is poorly understood. Moreover, there is no evidence of the utilization of bioremediation principles to remediate Hg contaminated soils. The present study was carried out to investigate the toxicity of the inorganic form of Hg to soil micro and macro inhabitants, and to exploit bacterial volatilization potential to remediate inorganic soil bound Hg. Furthermore, a microbial whole cell biosensor was designed to detect low levels of inorganic Hg in solution. The isolation of Hg resistant bacteria was carried out from contaminated soils by an enrichment technique using Hg supplemented liquid media. The strains were identified based on their 16s rRNA sequences which are considered one of the most conserved regions in the bacterial genome. The isolates were subjected to growth experiments under the stress of Hg gradients to elucidate effective concentrations of Hg that cause 50% inhibition to their growth (EC₅₀). The growth experiments were carried out in both nutrient rich media (nutrient broth) and less nutrient containing chemically defined media (low phosphate broth) to understand the effect of complex ingredients in nutrient media on the toxicity tests. After determining their Hg resistance patterns, the strains were tested (both qualitative and quantitative) for their capability to remediate inorganic Hg from solution by the process of bacterial volatilisation. Electron microscopy was employed to visualise any cellular accumulation. Then the activity of the key enzyme for Hg volatilisation, mercuric reductase was determined in crude cell extracts. The presence of three important genes namely <i>merA</i>, <i>merR</i> and <i>merT</i> in the <i>mer</i> operon was determined in bacterial isolates by polymerase chain reaction (PCR) method and the isolated genes were sequenced. Eco-toxicological investigations of Hg were carried out using a range of bio markers in soil, such as soil microbial enzymes, soil microbial community structure, soil invertebrate and native Australian terrestrial plants. To carry out these experiments, three different soils with varying pHs and organic profiles were spiked with different concentrations of inorganic Hg. Following Hg supplementation, the soils were kept for periods of 60 – 120 days to allow the Hg to react with soil particles. After aging period, soils were analysed for total and water extractable Hg. Two important soil microbial activity indicators – dehydrogenase enzyme activity (DHA) and nitrification rate were monitored after Hg supplementation using standard methods. A dose – response correlation was established between Hg doses and microbial activities. To understand the changes in soil bacterial diversity and community structure, DNA from Hg spiked soil samples was extracted and subjected to Illumina Miseq based sequencing which revealed the changes in bacterial taxa with Hg gradients. The effect of soil physicochemical parameters and Hg stress on various bacterial taxa and diversity matrices was established by Canonical Correspondence Analysis and non-linear regression analysis respectively. The effect of inorganic soil bound Hg to the soil invertebrate <i>Eisenia fetida</i> was investigated by observing their mortality, weight loss and bioaccumulation. Dose – response correlation was established between Hg gradients and their mortality in different soils. Finally, three native Australian grass species were tested for their Hg sensitivity by observing root growth while grown in different soils. To test the efficiency of the isolated Hg resistant bacterial strains to be used in bioremediation, they were subjected to growth under the stress of four divalent heavy metals namely cadmium (Cd), lead (Pb), zinc (Zn) and copper (Cu). The best resistant strain (to Hg and other metals) was used as bio-agent to volatilise soil bound inorganic Hg from field contaminated and spiked soils. Cucumber and lettuce plants were grown in remediated and contaminated soils to determine the efficiency of our strategy. Finally, a whole cell bacterial biosensor was developed employing green fluorescence protein (GFP) as reporter element to detect inorganic Hg from solution. The bio-sensing cassette was integrated into chromosomal DNA of one of the isolates following standard cloning procedures. The sensing time and detection range of the biosensor were determined. Three Hg resistant bacterial strains were isolated from contaminated soils which were identified as <i>Sphingobium</i> sp. SA2, <i>Sphingopyxis</i> sp. SE2 and <i>Pseudoxanthomonas</i> sp. SE1. These strains showed high resistance to Hg²⁺ while grown in a low phosphate containing LP media with estimated EC₅₀ values 4.5, 6 and 1.5 mg/L respectively. In nutrient rich media, these values were almost 5 to 20 times higher which indicates the inappropriateness of using nutrient media for microbial metal resistance analyses. All these three strains volatilised inorganic Hg from liquid media with varying degrees. Strain SA2 achieved the best result with the removal of 80% of initially added Hg in 6 h. A considerable amount of Hg was also detected in the cell pellets of both live and dead cultures. All three strains produced active mercuric reductase enzyme while the responsible gene <i>merA</i> was detected, amplified and sequenced. Other Hg resistant genes <i>merR</i> and <i>merT</i> were detected in strains SA2 and SE2 using designed primers. Eco-toxicological investigations revealed that the bioavailability of Hg in our experimental soils was as low as ~1% of the total amount of added Hg and the degree of bioavailability varied depending on soil properties. Acidic pH with higher organic carbon led to less solubility of Hg in soil which subsequently resulted in less toxicity. The EC₅₀ values obtained from dose – response analyses for the inhibition of soil DHA and nitrification rates were estimated to be 0.02 to 13.2 mg/kg Hg in different soils. The study of bacterial diversity changes in response to Hg stress revealed that the unclassified group of bacteria became dominant with Hg stress which was followed by <i>Proteobacteria</i>. Mercury had a considerable negative impact on key soil functional bacteria such as ammonium oxidizers and nitrifiers. Canonical Correspondence Analysis (CCA) indicated that among the measured soil properties, Hg had a major influence on bacterial community structure. Furthermore, nonlinear regression analysis confirmed that Hg significantly inhibited soil bacterial alpha diversity in lower organic carbon containing neutral and alkaline soils, whereas in acidic soil with higher organic carbon there was no significant impact. EC₅₀ values obtained by a non-linear regression analysis indicated that a small amount of bioavailable Hg (0.038 to 0.228 mg/kg water soluble Hg) significantly inhibited bacterial diversity in experimental soils. Similarly, The toxicity studies using <i>Eisenia fetida</i> revealed that Hg exerted less lethal effect on earthworms in acidic soil with higher organic carbon where water soluble Hg recovery was very low compared to the water soluble Hg fractions in soils with less organic carbon and higher pH. The concentrations of total Hg that caused 50% lethality to <i>E. fetida</i> (LC₅₀) after 28 days of exposure in experimental soils ranged from 152 - 367 mg/kg. Whereas the highest bioaccumulation took place when <i>Eisenia fetida</i> were cultured in acidic higher organic matter containing soil. In plant experiments with three native Australian grass species namely <i>Iseilema membranaceum</i> (Barcoo), <i>Dichanthium sericeum</i> (Queensland Blue) and <i>Sporobolus africanus</i> (Tussock), the 28 days root elongation data showed that in most of the cases Hg is less toxic to the plants grown in acidic higher organic carbon containing soil. The calculated EC₅₀ values ranged from 10 – 224 mg/kg total Hg in soil. Considering their tolerance to soil Hg, these grass species may have the potential for their use in rehabilitation of Hg contaminated sites. All three bacterial isolates, SA2, SE2 and SE1 were tested to tolerate four divalent heavy metals other than Hg. It was confirmed that the Hg resistant strain SA2 could be the best bio agent for remediation when it showed simultaneous resistance to other divalent heavy metals namely Cd, Pb, Cu and Zn with estimated EC₅₀ values 13, 28, 3 and 63 mg/L respectively. This strain removed 60% of Hg from a field contaminated soil with ~280 mg/kg Hg in only 7 d and the removal rate improved when the soil was supplemented with nutrients. Whereas in artificially spiked soils with ~100 mg/kg Hg, the removals due to bio augmentation were 33 to 48% in 14 days. In the field contaminated soil, nutrient amendment alone without bio augmentation removed 50% of Hg in 28 days. Higher root lengths of lettuce and cucumber grown in the remediated soils confirmed that the soil quality improved after bioremediation. Finally, a GFP based whole cell bacterial biosensor was developed using SA2 as host strain. The transformant could emit fluorescence in the presence of nano – molar concentration of Hg in liquid media. The sensing time was 2 – 5 h. The detection range was determined to be 3 – 40 nm based on a linear regression. This thesis provides new information in the eco-toxicity of Hg in terrestrial systems, bacterial mercury resistance and utilisation of resistant bacteria in the bioremediation of Hg from contaminated soils. Furthermore, an attempt was made to design a whole cell Hg biosensor where the sensing genetic element was integrated into the chromosomal DNA, rather than conventional plasmids, to potentially prevent sudden loss and transfer of the genetic element to other environmental bacteria