Characteristics of probes used in this study.
Abstract
The study is devoted to a very urgent and acute problem for Georgia – remediation/restoration of the arsenic (As) mining and storage sites. The approach of a given work is based on using capabilities of nature itself, which has a great adaptive potential to chemical environmental pollution. The aim of the study is to identify the bacterial strains from the endemic soil microbiota, characteristic to a specific localization of arsenic contaminated sites and able to resist to the toxicant. To determine the level of arsenic contamination, soil samples have been analyzed using Inductively Coupled Plasma - Optical Emission Spectrometry method. The distribution of arsenic in soil samples splits them into categories according to the degree of contamination, ranging from 50 ppm to 13000 ppm. The local bacteria community has been studied using conventional cultivation method along with modern method of bioindication – a biochip. The low density biochip contains the relevant probes for the identification of the bacterial consortium in soil microbiota. Chemical and microbiological analysis was based on the standards and methodologies developed by International Standards Organizations – ISO and Environmental Protection Agency – EPA. It is prospected that bioremediation can become essential part of remediation against arsenic pollution in the context of circular economy.
Keywords
- arsenic pollution
- inductively coupled plasma - optical emission spectrometry method
- biochip
- soil microbiota
- bioremediation
- PCR amplification
1. Introduction
The study deals with chemical pollution of the environment, in particular arsenic contamination of soil, which is a highly pressing problem for both Georgia and the world. Arsenic is one of the toxic metalloids that exist in more than 200 mineral forms, where 60% of them are normally arsenates, 20% are sulfosalts and sulfides and 20% are arsenite, oxides, arsenide, silicates and elemental arsenic. Arsenic mainly exists in the environment as arsine (As−3), elemental arsenic (As0), arsenite (As+3) and arsenate (As+5). Among all these forms only arsenite and arsenate are more abundant in natural environment than the other two [1]. Arsenic is naturally present in the lithosphere (earth crusts, soil, rock and sediment), hydrosphere (surface water, aquifers, deep well and oceans) and biosphere (food chain and ecosystems) [2, 3]. Arsenic is one of the top five toxic chemicals that were listed in the US Comprehensive Environmental Response, Compensation, and Liability (CERCLA) act of hazardous substances [4]. The toxicity of arsenic to living organisms is due to its functional affinity for phosphorus and its ability to form covalent bonds with sulfur. Arsenate replaces phosphate in phosphorylation reactions, and arsenite interacts with thiol groups of proteins, causing serious disturbances in cell metabolism [5]. Arsenic species are deposited in the skin, lungs, kidney, liver, etc. and cause several severe diseases by oxidative stress, altered DNA methylation, altered DNA repair, mitochondrial damage, cell proliferation, tumor promotion and co-carcinogenesis [6]. Arsenic exists both in toxic inorganic (associated with iron, cobalt, nickel coupled with sulfate minerals) and comprehensively less toxic organic (associated with carbon and hydrogen) forms. It is released into the environment by natural phenomenon (geogenic) or by anthropogenic activities. Soil texture is an important characteristic that affects arsenic chemistry. Arsenic levels in soil depend on climate, pH and redox potential. Arsenic can be chemically transformed in soils through several mechanisms. These include oxidation, reduction, adsorption, dissolution, precipitation, and volatilization. The trivalent form (As+3) is easily absorbed to ferric and aluminum oxides in the soil environment and oxidized to the pentavalent form in aerobic condition and reduced back to the trivalent form by reduction [7]. Under aerobic environment, the inorganic form of arsenic can easily bind to inorganic and organic materials in soil such as clay, iron and manganese dioxide, and exists in the pentavalent state (arsenate AsO43−). Under anaerobic environment, anaerobic bacteria transform it into less toxic volatile forms such as dimethyl arsenic acid and monomethyl arsenic acid [8]. Oxidation and reduction of arsenic species are carried out both chemically and biologically in soil and water. The acid/base chemistry plays a major role in the types of arsenical compounds present in the soils. Naturally, arsenic can be released into the soil environment by weathering and erosion (hydrolysis and oxidation process) of primary sulfide mineral (arsenopyrite). Organic content, iron and aluminum oxides, hydroxides, and phosphate ion play an important role in the speciation as well as mobility and sorption of arsenic in sediments [9]. Arsenic retention in soil media is mainly dictated by adsorption and desorption reactions, presence of ligands and soil redox conditions [10]. Bioavailability plays an important role in uptake of arsenic from soil to plants. The widespread contamination of the inorganic form of arsenic and its level of toxicity is becoming a global problem as the metalloid does not degrade, and cannot be destroyed. It is reported that conventional methods such as oxidation or reduction, chemical precipitation, filtration, ion exchange, reverse osmosis and evaporation recovery of cleaning contaminated area are too expensive and laborious. There is need to develop eco-friendly and low cost technique to mitigate the arsenic contamination. It is well documented that bioremediation is a cost-effective and a comparatively innocuous alternative to physical methods for heavy-metal remediation [11]. Wide varieties of microorganisms are capable of growth in presence of heavy metal ions and can tolerate high concentrations [12]. Although arsenic is generally toxic for life, microorganisms can use arsenic compounds as electron donors, electron acceptors or show arsenic detoxification mechanisms [13]. Furthermore, suggestions have been made about the existence of microorganisms in which arsenic can play the role of phosphorus. These microorganisms are able to live and reproduce in conditions of phosphorus deficiency, replacing phosphorus in DNA with arsenic toxic to other life forms [14]. Various bacteria such as
2. Biochip applications
The establishment of the structure of the collective genomes (metagenomes) of environmental microbial communities (microbiomes) has a decisive influence on the development of earth sciences of the 21st century. However, it soon has become apparent that gene sequence data alone is of relatively little use unless it was directly linked to agriculture, alternative energy production, industrial processes, and environmental cleanup relevance. Monitoring of bacterial consortium in contaminated regions will enable to assess in advance the extent of native bioremediation and accordingly suggest a strategy of external bioremediation (detoxification). Diagnostic biochip is able to enumerate bacteria, involved in bioremediation. A biochip is a platform for screening a single sample for multiple purposes. The constructed and applied in the presented study low density DNA-biochip is a small solid matrix with multiple dot markers (probes). Each marker contains genetic DNA-sensors (pico-amounts of oligonucleotides), which is designed according to the genetic map of the studied objects.
2.1 Biochip design: DNA probes and biochip matrix
Effect of toxic metals in natural conditions is rarely revealed by the action of one of them, mainly it is a complex action of several metals (sometimes even non-toxic), and the same concerns
N | Probe name | Target species | Gene name | Sequence 5′ → 3′ | Ref. |
---|---|---|---|---|---|
1 | Archaea1 | 16S rRNA | GTG CTC CCC CGC CAA TTC AT | [21] | |
2 | GeoS | 16S rRNA | TTC GGG CCT CCT GTC TTT C | [22] | |
3 | GeoM | 16S rRNA | TTC GGG CCT TTT GTC ACC | [22] | |
4 | BacilPhosL | Phospholipase PL | CTA CTG CCG CTC CAT GAA TCC | [23] | |
5 | Pseudomonas | Polyketide synthase phlD | GAG GAC GTC GAA GAC CAC CA | [24] | |
6 | napA | nitrate reductase (napA) | CCG CGG CTA TGT GGG TCG AAA AAG | [22] | |
7 | nirS | nitrite reductase (nirS) | CGC TGT TCG TCA AGA CCC ATC CG | [22] | |
8 | Clostridia | Fe-hydrogenase hydA | CGG CGA GCA TGA TCC AGC AAT | [22] | |
9 | Shew | Ni,Fe-hydrogenase hydA | ACA ACT GCC CAA CCG AGC G | [22] | |
10 | FTHFS | formyltetrahydrofolate synthetase (fthfs) | TGC ATG GCC AAG ACC CAA TAC AGC | [22] | |
11 | a/bssA | alkylsuccinate synthase and benzylsuccinate synthase alpha subunits (assA/bssA) | TCG TCA TTG CCC CAT TTG GGG GC | [22] | |
12 | SRB | adenosine 5′-phosphosulfate-reductase APR | CCA GGG CCT GTC CGC CAT CAA TAC | [25] | |
13 | Dsmicrobium1 | hydA (Ni,Fe-hydrogenase) | CCA CAA CCT GGC CAT CCC GGA AAT | [22] | |
14 | Dtomaculum2 | hydA (Fe-hydrogenase) | CAC GCA TCG GGG AGA GGG TGG | [22] | |
15 | Dbulbus | hydA (Ni,Fe-hydrogenase) | GCG CCA CCC TGC CGT TCA AC | [22] | |
16 | Dbacterium1 | hynB (periplasmic Ni,Fe hydrogenase) | CAC TGG AAC AGG CGA TCA AG | [22] |
2.2 Analysis of bacterial composition using a biochip
The intensive study of heavy metal contaminated areas has revealed special property of bacteria living in these areas. It is their selective metallophilic property. That is largely due to the activity of these organisms to change the oxidation state of soluble heavy metal oxyanions by transformation them to an insoluble form. Metal-reducing bacteria may receive metal ions as soluble oxyanions via the anionic transport system. Some metal-reducing bacteria can play a key role in the mobilization of arsenic in sediments collected from a contaminated aquifer [27].
To evaluate and compare bacterial composition of arsenic contaminated area soil profiles were sampled at different points. Three sampling points were selected for a biochip analysis. Samples from the arsenic ore collector area were selecting based on the location: soil from arsenic processing plant (
2.2.1 Comparison of bacterial functional assemblages
The Figure 1A represents the hybridization signal intensities estimated as signal to noise (S/N) ratio for the probes targeting key genes encoding enzymes involved in metabolic processes. Functional gene array is the approach to assess diversity and functional activities of microbial communities in natural environments. The Figure 1A expresses the proportion of the issued bacterial species in the samples
The hybridization signals from the probes targeting 16S rRNA genes are presented in Figure 1B. The presence
2.2.2 Comparison of sulfate reducing bacteria functional assemblages
Sulfate reducing bacteria take the special attention, as SRB along with methanogens are involved in arsenic methylation and demethylation in soils [29], and microbial arsenic methylation and demethylation are important components of the As biogeochemical cycle. Only a limited number of organisms are well known for their narrowly defined metabolic capabilities, and SRB is such group. The detection of SRB in the bacterial consortium could be carried through functional genes as
PCR primers are listed in Table 2. Individual gene-specific PCR primers were designed using Primer3Plus software. The PCR mixture (20 μl) contained 1xiProof High-Fidelity Master Mix (Bio-Rad Laboratories, USA) with 1.5 mM MgCl2, 200 μM (each) deoxynucleoside thriphosphate, 500 nM each primer, 20 ng of DNA template. PCR amplification was conducted using the following conditions identical for each primer pair: an initial denaturation step (30 s, 98°C) was followed by 30 cycles of denaturation (10 s, 98°C), annealing (20 s, 60°C), and extension (15 s, 72°C) and one terminal extension step (10 min, 72°C).
Positive control DNA | Target gene GenBank accession no. | PCR Primers name | ||
---|---|---|---|---|
For2 | 150 | |||
Rev2 | ||||
CP002364 | For1 | 165 | ||
Rev1 | ||||
CP001629 | For1 | 109 | ||
Rev1 | ||||
CP002780 | For2 | 170 | ||
Rev2 |
As it follows from the analysis of the SRB assemblages, the use of nothing but the wide spectra probe (SRB) on a biochip does not differentiate the content of SRB in the studied samples. The expansion of the SRB allows distinguishing SRB composition between the samples. Specifically,
Taking into consideration biochip data about bacterial composition assessment, it can be said that a biochip is not only diagnostic, but also a predictive instrument.
The detailed analysis of total As content in the contaminated area is presented in the next Section.
3. Total As content analysis
Arsenic contamination in Georgia has many natural and anthropogenic sources. Arsenic ore extraction, processing and production of arsenic-containing preparations have been carried out on the territory of Georgia for decades. The processing of the ore, which is located in Ambrolauri region, started in 1937. Main products of processing were metallic arsenic of high purity, As2O5, As2S5 and tin arsenate. Technological cycle in factory was very simple and included thermal treatment of ore. Amount of waste is about 60 tons, which contains up to 1% As2O5. Waste was not used and it was stored at Kajiani territory in special hydrotechnical building, called tailings. The mining of ore was carried out at Lukhuni ore, processing was performed in the factory nearby village Uravi. Chemical mining factory in village Tsana started working is 1938. The main products were metallic arsenic and refined “white arsenic” (As2O3) (I grade – 99.9%, II grade – 99.5%). This substance is poorly soluble in water and permanently can have great negative influence on environment. The factory in Tsana, administrative buildings and warehouse farming are fully destroyed and collapsed. There is no fence around the territory. Arsenic kilns and containers are taken from the ground. Thus the problem is arsenic containing waste and soils. Until today the great amount of toxic waste of arsenic production is stored in villages Uravi and Tsana, near the territory of the factories (more than 120 000 tons waste, containing 4–9% of white arsenic), which is not located safely and there is a high risk of ecological disaster in rivers and soils, especially risks of natural disasters (floods, rockslide, erosion and etc.). It should be mentioned that LEPL National Environmental Agency is conducting permanent monitoring in order to determine arsenic content. The data are not encouraging [31]. Based on this it is necessary to determine arsenic content in ecosystems (soil, water) of this region. The fifty four soil samples encompassing two hectares have been analyzed using Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES) method. Preparation (drying, loosening, sieving, extraction, etc.) of soil samples for pre-treatment and chemical analysis was carried out - ISO-11464; EPA 3050; According to EPA-TCLP-1111 ST methodologies. The results are presented in Figure 3. According to Figure 3, As content ranges from 47 mg/kg soil to 13000 mg/kg soil.
4. Characteristics of bacterial isolates from As highly contaminated area
4.1 Conditions of soil samples treatment for bacterial isolates recovery
Soil samples for bacterial isolates recovery were taken from the arsenic contaminated area and were selecting based on the location: soil sample from arsenic storage area (
4.2 Microorganisms properties
Bacterial isolates that were motile, had oxidative metabolisms, were oxidase and catalase positive, ornithine decarboxylase positive, and DNase positive, and produced H2S on triple sugar iron slants within 72 h of incubation were identified as belonging to the phenospecies, respectively based on acid production from sucrose and maltose, growth on SS agar, and growth in the presence of 6.5% NaCl. All tests were performed according to the manufacturer′s instructions. Routine biochemical test results were read daily for 72 h; oxidation of various carbohydrates was assessed by Liofilchem® Microbial Identification system after 7 days of incubation. Enzymatic plate assay results were read daily for 7 days; appropriate positive and negative control strains were included for each assay. Some additional enzymatic activities (2 h) and gelatinase activity (3 days) were estimated using Wee-Tab tablets or gelatin strips (Key Scientific Products, Roundrock, Tex.). Microorganisms have been studied for intracellular and extracellular enzymatic activities such as Oxidase test, Catalase test, Carbohydrate utilization test, Urease test, Indole test, Esculine test, Nitrate reduction test, Citrate utilization test, Gelatin hydrolysis test and Motility test.
4.3 Bacterial isolates identification and characterization
The screening of the bacterial composition using a biochip (Section 2) showed that sulfate-reducing bacteria, along with the representatives of
The biochemical characteristics of selected
Soil sample | Determined microorganism | Strain | Catalase test | Oxidase test | Mannitol fermentation | Starch hydrolysis | Nitrate reduction test | Urease test | Indole test | Citrate utilization test | TSI agar test | Gram staining |
---|---|---|---|---|---|---|---|---|---|---|---|---|
#01 | p | p | p | p | n | n | n | p | alk/acid/alk | p | ||
#02 | p | p | p | p | p | n | n | p | alk/acid | p | ||
#03 | p | p | p | p | n | p | n | p | alk/alk/H2S | p | ||
#04 | p | p | p | n/d | n | p | n | n/d | alk/alk/H2S | p | ||
#05 | p | p | p | p | n | n | n | n/d | alk/alk/H2S | p | ||
#06 | p | p | p | p | p | n | n | n | alk/acid | p | ||
#07 | p | p | p | n/d | n | n | n | n | alk/acid | p | ||
#08 | p | p | n/d | p | p | p | n | p | alk/acid | p | ||
#09 | p | p | p | p | p | n | n | p | alk/acid | p | ||
#10 | p | p | p | p | p | n | n | n | alk/acid | p | ||
#11 | p | p | p | p | n | n | n | n | alk/alk/H2S | p |
The biochemical characteristics of
Soil sample | Determined microorganism | Strain | Catalase test | Oxidase test | Mannitol fermentation | Starch hydrolysis | Nitrate reduction test | Urease test | Indole test | Citrate utilization test | TSI agar test | Gram staining |
---|---|---|---|---|---|---|---|---|---|---|---|---|
SH01 | p | p | p | n/d | p | n | p | p | alk/alk/ H2S | n | ||
SH04 | p | p | n/d | n/d | n | n | n | n | alk/acid/ | n | ||
X01 | n | p | n/d | n/d | n | n | n | p | alk/alk | n | ||
X02 | n | p | n/d | n/d | n | n | n | p | akl/alk | n | ||
X06 | p | p | n/d | n/d | p | n | n | n | alk/alk/ H2S | p | ||
X07 | p | p | n/d | n/d | p | n | n | n | alk/alk/ H2S | n |
Soil sample | Determined microorganism | Strain | Arsenic reduction | Arsenic oxidation | ||
---|---|---|---|---|---|---|
M2 | M6 | M2 | M6 | |||
SH01 | n | n | p | n | ||
SH04 | n | n | p | n | ||
X01 | n | n | p | n | ||
X02 | n | n | p | p | ||
X06 | n | n | p | n | ||
X07 | n | n | p | n |
As follows from Table 5, the
5. Conclusions
Evaluation of arsenic pollution and health risks, prevention of natural soils and waters from pollution in order to provide ecosystem enhancement and population safety is extremely important for the reality of Georgia. Arsenic as the most heavy metals are scattered and unevenly located in the earth’s crust. Under natural conditions, in sufficiently high concentrations, it is found in areas of ore deposits. Arsenic toxicity is governed by its high affinity to sulfur and phosphorus, what is extremely dangerous for living organisms. In modern ecological biotechnologies aimed at cleaning up a chemically polluted environment, both from an economic and an ecological point of view, phytoremediation is considered the most effective, which consists in the purposeful planting of specially selected plants in contaminated areas. To use phytoremediation, it is necessary to study not only the level of pollution, but also the physicochemical properties of the soil and, mostly important, the soil microbiota. This is due to that a certain consortium of microorganisms has already formed in the contaminated soil, which, on the one hand, is accustomed to being present in a polluted environment, on the other hand, determines the conditions for plant growth on this soil and, in some cases, leads to the transformation of pollutants. Therefore, the screening of the indigenous bacteria was the first key step on the way of the bioremediation of arsenic contaminated area. Diagnostic biochip is used for the monitoring of the indigenous bacteria. The bacterial composition revealed the prevalence of metal-tolerant species as
Acknowledgments
This work was supported by grant # CARYS-19-179 from Shota Rustaveli National Science Foundation (SRNSF).
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