Research Article |
Corresponding author: Galina Radeva ( galrad@abv.bg ) Academic editor: Ventsislava Petrova
© 2023 Michaella Petkova, Nadezhda Nankova, Viktoriya Kancheva, Silvena Boteva, Anelia Kenarova, 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:
Petkova M, Nankova N, Kancheva V, Boteva S, Kenarova A, Radeva G (2023) Distribution of microbial abundance in long-term copper contaminated soils from Topolnitsa-Pirdop valley, Southern Bulgaria. In: Chankova S, Danova K, Beltcheva M, Radeva G, Petrova V, Vassilev K (Eds) Actual problems of Ecology. BioRisk 20: 23-35. https://doi.org/10.3897/biorisk.20.97795
|
This study presents the distribution of bacterial and fungal abundances in long-term copper (Cu) contaminated soils in Topolnitsa-Pirdop valley – a highly industrialized zone with a number of mines and processing plants for copper and other non-ferrous metals. The bacterial (16S rRNA gene copies) and fungal (ITS rRNA gene copies) were estimated using quantitative PCR technique in five topsoils, differently Cu contaminated (ranging from 28.05 to 198.9 mg kg-1). Bacterial abundance varied in a range of 1.68 × 1011 to 3.24 × 101116S rRNA genes, whereas fungi amounted from 1.95 × 108 to 6.71 × 108 ITS rRNA genes. Fungal and bacterial abundances were significantly (fungi) and insignificantly (bacteria) influenced by Cu contamination. The fungal/bacterial ratio related negatively with soil Cu, which shifted microbial communities’ structure towards bacterial dominance. Since the ratio between bacteria and fungi are vital in explaining many soil functions, the calculated changes in this ratio indicated deterioration in soil quality, being of primary importance for plant production.
ITS rRNA gene, microbial abundance, qPCR, soil contamination, 16S rRNA gene
Heavy metals (HMs) are natural constituents of the environment, but intensive use for human purposes has altered their geochemical cycles and biochemical balance. The extraction and processing of mineral raw materials are one of the main sources of soil and water pollution with HMs and other toxic elements. In this respect, of particular interest are the open-pit mines and their generated waste materials, which cause serious environmental damage and significant changes to the landscapes of the impacted regions (
Copper is one of the most common soil contaminants (
The aim of this study was to assess the distribution of bacterial and fungal abundances in Cu long-term contaminated soils from the valley of Topolnitsa-Pirdop and its relationship with the local soil properties.
Topolnitsa River and its tributaries (Medetska, Zlatishka, Pirdopska and Bunovska Rivers) were influenced by the ore industry, agriculture activities, and domestic wastewater discharge, which led to the deterioration of its ecological status according to the
The study area is located in the Topolnitsa-Pirdop valley (Fig.
Soil pH was potentiometrically measured in 0.1 M CaCl2 soil suspension according to ISO 10390:2005. The soil organic carbon (SOC) was determined by sulfochromic oxidation according to
The genomic DNA was extracted from 0.5 g soil, using the E.Z.N.A soil DNA kit (Omega Bio-tek, USA) according to the manufacturer`s recommended protocol. The quality and quantity of the extracted DNA were subsequently assessed by Qubit 4 fluorometer (Invitrogen) and 1% agarose gel electrophoresis.
Q-PCR analyses were performed using iTaqTM Universal SYBRGreen Supermix (BioRad) on Rotor Gene 6000 (Corbett Life Science) according to the manufacturer’s protocols. Bacterial abundance was estimated using 0.4 μM of primers Eub338f (5’-ACTCCTACGGGAGGCAGCAG-3’) and Eub518r (5’-ATTACCGCGGCTGCTGG-3’) (
The fungal abundance was estimated using 0.4 μM primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCCTTATTGATATGC-3’) (
A four-point serial decimal dilution of a plasmid DNA of Uncultured Basidiomycota clone LS_Az0_D1_31 (MT785782.1) was used as a standard to generate a linear calibration curve of the threshold cycle versus a number of gene copies ranging from 102 to 105. All measurements were run in triplicates. Data were expressed as gene copy numbers per gram of dry soil. Amplification efficiency was estimated to be 92% (R2=0.99). The results were analysed via ROTOR-GENE Q SERIES, Software version 2.3.1.
The relative ratio of fungi/bacteria was calculated as the ratio of the fungal gene copy numbers to bacterial gene copy numbers (
Pearson correlation analysis was used to find the relationships between soil metrics. Hierarchical clustering (Algorithm: UPGMA; Similarity index: Bray-Curtis) was conducted to evaluate the distance between soil physical environments. Non-metric multidimensional scale (nMDS) ordination was applied to assess the similarity between abundance-weighted microbial communities. Statistical analyses were performed using the software PAST (p<0.05).
The values of studied soil properties are shown in Table
Soil physicochemical properties and Cu concentrations in the region of Topolnitsa River and its tributaries.
Soil sample | Soil physicochemical properties | ||||||
---|---|---|---|---|---|---|---|
pH | WtC (%) | SOC g kg-1 | NO3-N mg kg-1 | NH4-N mg kg-1 | P2O5 mg kg-1 | Cu mg kg-1 | |
S1 | 5.57 | 13.33 | 2.56 | 11.44 | 7.82 | 2.93 | 198.90 |
S3 | 5.60 | 11.67 | 1.22 | 0.65 | 8.32 | 2.37 | 110.00 |
S4 | 5.76 | 12.33 | 2.16 | 8.84 | 5.74 | 3.40 | 82.40 |
S2 | 5.57 | 12.33 | 2.67 | 11.12 | 3.07 | 3.47 | 67.40 |
S5 | 5.61 | 10.00 | 2.05 | †ND | 10.74 | 2.78 | 28.50 |
Cu concentrations varied among the soils as follows: the concentrations in S1 and S3 exceeded the maximum permissible concentration (MPC) of 80 mg kg-1 under
Pearson correlation analysis indicated a linear relationship (-0.61; p=0.015) between the level of soil Cu contamination and the distance to the source of pollution.
A cluster analysis of soil abiotic factors was conducted to visualize the similarity among the tested soils (Fig.
Two main clusters were demonstrated: cluster I containing soil S5 with the lowest Cu concentration and cluster II, consisting of other soils sub-clustered according to the Cu threshold concentration of 100 mg kg-1. This pattern of soil clustering identified the major role of Cu in structuring soil environments.
Bacterial abundance varied in a relatively narrow range. The highest number was observed in S4 (3.24 × 1011), and the lowest one in S5 (1.68 × 1011) (Fig.
The highest fungal abundance was detected in uncontaminated soil S5 (6.71 × 108) and the lowest one in S3 (1.95 × 108) (Fig.
An abundance of ITS rRNA gene. Error bars are the standard deviations of the mean for the three replicates.
Pearson correlation analysis showed a significant negative correlation (-0.76; p=0.001) between fungal abundance and soil Cu contamination.
The highest relative fungal to bacterial abundance was observed in the uncontaminated soil S5 (3.98 × 10-3), and the lowest ratio was recorded in the highly contaminated soil S3 (6.80 × 10-4) (Fig.
Statistical non-metric multidimensional scaling (nMDS) was performed on patterns of fungal and bacterial abundances to assess soil similarities with respect to their microbiology (Fig.
Microbial abundances in soils of Topolnitsa-Pirdop valley as indicated by non-metric multi-dimensional scaling plots.
Plots separated the soils into four distinct abundance-weighted microbial communities The soils S2 and S4 grouped closely, indicating similar microbial abundances. The other soils were placed individually both away from the other and from the group S2 and S4. This distribution was probably due to differences in microbial abundance-weighted communities. Our results showed that the important factors influencing soil microbial abundance were soil Cu concentration and water content in S1, and soil organic carbon and phosphates concentrations in S2 and S4 (Fig.
The present study was focused on the distribution of microbial abundance as a response to the influence of Cu contamination in the area of Topolnitsa-Pirdop valley – a highly industrialized zone with a number of mines and processing plants for Cu and other non-ferrous metals.
The soil concentrations of the main contaminant Cu varied from 28.5 mg kg-1 to 189.9 mg kg-1, being above MPC in three (S1, S3, and S4) of five tested soils. The mode of clustering highlighted the significant effects of Cu on soil environments, grouping soils in clusters or sub-clusters depending on the level of contamination. Additionally, soil pH (acidic) may influence soil microbial communities by primarily suppressing the bacterial diversity (
The results showed high bacterial abundance in soils, ranging from 1.68 × 1011 to 3.24 × 1011 16S rRNA gene copy numbers, being one-fold higher compared to the abundances estimated in our previous studies on mining activities in the area, where the soil Cu concentrations ranged from 53 mg kg-1 to 860 mg kg-1 (
Similar to other authors (
The higher exerted Cu toxicity on fungi reflected also on the fungal/bacterial ratio, which decreased with increasing soil Cu contamination. According to some authors (
Although microbial communities were differently abundance-weighted (except that of S2 and S4) according to the ordination procedure nMDS, it demonstrated that only the highest soil Cu concentration (189.9 mg kg-1) influenced significantly soil microbial communities (S1). The abundances of soil microorganisms from the other sampling sites were under the influence of local soil abiotic factors.
The present study showed that microbial abundance, especially fungal, was significantly affected by long-term Cu contamination of Fluvisols. The Cu shifted microbial communities’ structure towards bacteria, suggesting that in this case, bacteria could be better at developing Cu resistance than fungi. Further studies should be implemented to clarify microbial functional responses to Cu.
This study was supported by the National Research Fund of the Bulgarian Ministry of Education and Science under Grant [KP-06-М41/3].