Research Article |
Corresponding author: Galina Radeva ( galrad@abv.bg ) Academic editor: Michaela Beltcheva
© 2022 Radina Nikolova, Michaella Petkova, Nikolai Dinev, Anelia Kenarova, Silvena Boteva, Dimitar Berov, Galina Radeva.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Nikolova R, Petkova M, Dinev N, Kenarova A, Boteva S, Berov D, Radeva G (2022) Correlation between bacterial abundance, soil properties and heavy metal contamination in the area of non-ferrous metal processing plant, Southern Bulgaria. In: Chankova S, Peneva V, Metcheva R, Beltcheva M, Vassilev K, Radeva G, Danova K (Eds) Current trends of ecology. BioRisk 17: 19-30. https://doi.org/10.3897/biorisk.17.77458
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In the present study, the correlation between bacterial abundance and soil physicochemical properties along the heavy metal contamination gradient in the area of non-ferrous metal processing plant was assessed. Our results showed that bacterial abundance (number of heterotrophic bacteria and number of 16S rRNA gene copies) decreased with 45–56% (CFU) and 54–87% (16S rRNA gene) along the Zn, Pb and Cd contamination gradient. The total bacterial abundance (16S rRNA gene) increased exponentially in contrast to the abundance of heterotrophic bacteria. The reduction of bacterial abundance in heavily contaminated soil indicated that the soil properties (soil pH, total organic carbon, inorganic ions, soil texture) could modify the effects of heavy metals and the response of microorganisms to that stress in long-term contaminated soils.
Bacterial abundance, 16S rRNA gene, heavy metals, soil contamination, soil properties
Soil sustains a great abundance and diversity of microorganisms, which modify its physical and chemical environment and play an essential role in the mineralization of organic matter and nutrient recycling (Martinez-Toledo et al. 2021). Microbial communities are strongly susceptible to soil physicochemical properties and to the effect of various soil pollutants, such as heavy metals (HMs). HMs are one of the most common pollutants, which accumulate in soils of industrial and mining areas. The most common HMs found in contaminated sites are Zn, Cd and particularly Pb (
Previous studies showed that HMs severely affect soil microbial communities by reducing their diversity (
Many studies reported that soil physicochemical properties (soil pH, soil texture, organic matter, etc.) moderate HMs’ toxicity and therefore, HMs play a key role in shaping the community diversity and structure (
Taking into consideration that soil is highly heterogeneous, it is necessary to investigate the microbial communities at different sites and scales (
The study area is located in the region of a non-ferrous metal plant KCM 2000- Plovdiv, Southern Bulgaria (42°03'40.8"N, 24°48'52.0"E) (Fig.
Soil pH was measured in 0.1M CaCl2 according to
The bacterial abundance was estimated by the use of colony-forming units (CFUs) in serial dilution in R2A medium at 25 °C for 2 days. The selected dilution from each test sample was 10-4 and it was plated in triplicate. For this analysis, we used 10–100 colonies per plate.
The genomic DNA was extracted from 0.5 g soil using the E.Z.N.A DNA soil kit (Omega Bio-tek, USA) using the manufacturer’s recommended protocol. The soil DNA quality was controlled by a spectrophotometer (NanoDrop 1000, ThermoScientific, USA) and agarose gel electrophoresis.
Bacterial abundance was quantified by real-time quantitative PCR (qPCR) with bacterial universal primer pairs Eub338f (5’-ATTACCGCGGCTGCTGG-3’)/Eub518r (5’-ATTACCGCGGCTGCTGG-3’) for 16S rRNA gene (
Soil properties and HM concentrations were compared using principal component analysis (PCA). Prior to the analysis the data was normalized and checked for outliers. Linear correlations between the resulting indicator of pollution and the copies of 16S rRNA gene found in each sample were plotted and evaluated (r2 metric of plotted trendline). Additionally, the significance of the correlation between the two variables was evaluated with the Student T-test.
The PCA statistical analyses were carried out using Primer 7.0. Univariable statistical correlations were tested using STATGRAPHICS Centrion XVII software package. (
The values of studied soil properties are shown in Table
Soil physicochemical properties and concentrations of heavy metals (total and bioavailable forms) in the area of KMC-2000.
Soil parameter | Soils | ||||
---|---|---|---|---|---|
KCM_1 | KCM_2 | KCM_3 | KCM_4 | KCM_5 | |
pH | 7 | 7.1 | 7.2 | 6.7 | 6.8 |
Sand (%) | 17.6 | 53.6 | 47.1 | 49.0 | 51.7 |
Silt (%) | 39.2 | 30.9 | 31 | 30.8 | 36.2 |
Clay (%) | 43.3 | 15.5 | 21.9 | 20.2 | 12.1 |
Soil moisture (SM) (%) | 16.7 | 12.3 | 9.3 | 14.7 | 22.7 |
TOC (g kg-1) | 9.65 | 14.07 | 6.45 | 12.33 | 7.035 |
NO3-N (mg g-1) | 43.38 | 16.32 | 3.01 | 5.13 | †ND |
NH4-N (mg g-1) | 6.62 | 5.13 | 3.26 | 2.25 | 2.22 |
P2O5 (mg kg-1) | 5.69 | 24.02 | 7.42 | 6.89 | †ND |
Zn (mg kg-1) | 9452 | 1558.2 | 216.2 | 6872 | 740 |
Pb (mg kg-1) | 11569 | 1370.1 | 135.6 | 5723 | 335 |
Cd (mg kg-1) | 184.9 | 15.7 | 3.9 | 86.2 | 9.3 |
Znb‡ (mg kg-1) | 8.2 | 0.1 | 0.1 | 3.3 | 0.3 |
Pbb‡ (mg kg-1) | 2.6 | 0.2 | 0.9 | 0.2 | 0.8 |
Cdb‡ (mg kg-1) | 9 | 0.2 | 0.5 | 1.1 | 0.4 |
NPI§ | 73.27 | 8.74 | 1.00 | 37.46 | 3.11 |
The HM concentrations at KCM_3 were under (Zn) and slightly higher (Pb and Cd) than the maximum permissible concentration (MPC) allowed under Bulgarian Regulation 3/2008 (http://eea.governement.bg/bg/legislation/soil), and this sample was considered as a control in our study. Pb was the most serious soil pollutant, and its concentration was over 100 (KCM_1), 57 (KCM_4), 13.7 (KCM_2) and 3 (KCM_5) times higher than the guideline limit. Cd was the other most serious soil pollutant and its concentrations exceeded the MPC in the following order: KCM_1 (92.45 times) >KCM_4 (43 times) >KCM_2 (7.85 times) >KCM_5 (4.0 times). The order of Zn soil contamination comparing to MPC was: KCM_1 (29.5 times) >KCM_4 (21.0 times) >KCM_2 (4.8 times) >KCM_5 (2.0 times). Nemerow’s Pollution index (NPI) assessed the overall level of soil contamination (
The results from the bacterial abundance of heavily contaminated soils were compared as a percent to KCM_3, which was a control soil in the experiment (Fig.
Soil properties (total organic content, inorganic ions, soil particles of silt, clay and sand) and HM concentrations in soils were compared using principal components analysis (PCA) and the results are presented in Fig.
Variable | PC1 | PC2 | PC3 | PC4 |
---|---|---|---|---|
Variation explained (%) | 65.7 | 17.7 | 13.4 | 3.2 |
pH | -0.001 | -0.308 | -0.618 | -0.327 |
TOC | -0.011 | -0.501 | 0.437 | 0.205 |
Sand | 0.324 | -0.047 | 0.119 | 0.110 |
Silt | -0.248 | 0.266 | -0.137 | 0.708 |
Clay | -0.316 | -0.027 | -0.102 | -0.358 |
Zn | -0.284 | -0.019 | 0.357 | -0.211 |
Pb | -0.313 | -0.028 | 0.219 | 0.127 |
Cd | -0.316 | 0.000 | 0.197 | -0.117 |
Znb | -0.321 | 0.032 | 0.156 | -0.119 |
Pbb | -0.296 | 0.142 | -0.276 | 0.067 |
Cdb | -0.328 | 0.019 | -0.057 | 0.037 |
NO3-N | -0.305 | -0.216 | -0.080 | 0.199 |
NH4-N | -0.244 | -0.357 | -0.240 | 0.251 |
P2O5 | 0.068 | -0.620 | -0.022 | 0.116 |
The soils had a high abundance of bacteria, whose number varied from 17.80×1010 (KCM_2) to 1.49×1010 (KCM_1) 16S rRNA gene copies (Table
Sample | PC1 score | 16S rRNA gene copies ×1010 |
---|---|---|
KCM_1 | -5.35 | 1.49 |
KCM_2 | 1.57 | 17.80 |
KCM_3 | 1.6 | 11.50 |
KCM_4 | 0.486 | 4.17 |
KCM_5 | 1.7 | 9.57 |
The present study focused on the correlation between bacterial abundance and soil properties in long-term contaminated soils in the area of non-ferrous metal processing plant KCM-2000. The gradient of Zn, Pb, and Cd concentrations in the soil from KCM_3 to KCM_1 provided a good soil pattern for estimating the changes that occurred in soil bacterial abundance under the power of long-term HM contamination. The soil of KCM_3 was determined in this study as a control (NPI=1.00). In KCM_3, the concentration of Pb was slightly higher than the MPC, and Pb bioavailable forms were equal to that of KCM_5, exceeded by 2.5 times that of KCM_2 and KCM_4, and was by 3.0 times lower than Pbb of KCM_1.
The distribution of bacterial abundance of unculturable and cultivable bacteria along a gradient of contamination was estimated through quantitative PCR of 16S rRNA gene and numbers of colony-forming units (CFU). In general, the soils of the site of interest showed a high bacterial abundance – 1.49×1010-17.80×1010 16S rRNA gene copies (total bacterial abundance) and 1.30×106 -3.70×106 CFU (abundance of heterotrophic bacteria).
Although, the HM gradient of soil contamination determined a gradient of soil bacterial distribution, only in the case of KCM_2 bacterial abundance was higher (around 55% for 16S rRNA and 27% for CFU) compared to that of the control. We suggested that this inconsistency with the model of general bacterial distribution could be due to the toxicity of the much higher Pbb concentrations in KCM_3, or attributed to the modulating effects of higher concentrations of TOC, NO3-N and P2O5 in KCM_2 compared to KCM_3 soil. Bacterial reduction in HM contaminated soils varied between 45–56% (CFU) and 54–87% (16S rRNA), except for the relatively low decrease in 16S rRNA gene copies in KCM_5 (17% compared to KCM_3). This fact could be explained by the relatively low level of soil contamination compared to the other studied sites (NPI=3.11). The obtained results were consistent with our previous study, where bacterial abundance (CFU and 16S rRNA gene copies) decreased in long-term contaminated with Cu, Zn and Pb soils in the area of copper mine and smelter (
To elucidate the effects of HMs and soil properties on the distribution of bacteria, an exponential correlation between the values of PC1 scores, and both 16S rRNA gene copies and CFU was conducted. There was found to be a good correlation between soil variables and 16S rRNA gene copies, but a low relationship between soil characteristics and CFU. We assumed that the lack of significant correlation between soil variables and heterotrophic bacteria (CFU) might have resulted from the limitation of cultivation technique or the high ecological tolerance of cultivable representatives of bacterial communities. Some authors (
The total bacterial abundance (estimated by the quantified 16S rRNA genes) increased exponentially in contrast to the abundance of heterotrophic bacteria, which could be explained by the limitations of the cultivable method. Regarding the statistical analysis, the bacterial abundance, expressed by the 16S rRNA gene copies, was considered as a more valuable indicator of the HM contamination effects on the soil inhabitants in comparison to the heterotrophic bacterial abundance. The reduction of bacterial abundance in heavily contaminated soil indicated that the soil properties (soil pH, total organic carbon, inorganic ions, soil texture) could modify the effects of heavy metals and the response of microorganisms to that stress in long-term contaminated soils. Further studies are needed for investigating the shifts in bacterial community structure in this area in response to the HM contamination gradient.
This study was financially supported by the National Research Fund of the Bulgarian Ministry of Education and Science (Grant-DN11/4-Dec.2017).