| Heavy metal pollution has received increasing attention in recent years mainly because of the public awareness of environmental issues as heavy metals are toxic at higher levels in both natural and man-made environment ecosystems. Heavy metals in soil above the ambient levels can adversely affect the surrounding ecology by population loss, changes in population structure, physiological activity and shifts or change in the composition of the soil microbial communities, which in turn may result in adverse effects on various parameters influencing crop quality, yield and possibly human health through food chain. Soil microbiological properties are indicator of soil pollution, but the results described in the literature are contradictory, and the greatest problem posed by the use of these properties as soil quality indicator is the lack of reference values, and regional variations in expression levels. This discrepancy may be attributed to the methods used until recently, which provide data on processes or microbial numbers and were not suitable for the analysis of soil microbial community structure or diversity. Accordingly, a study was conducted to evaluate the toxicity of lead (Pb) and cadmium (Cd) applied alone and or in combination on microbial biomass, enzymes activity, substrate utilization pattern, and structure diversity of microbial communities in soil. Here, we combined the traditional assays with modern molecular technique of polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) to evaluate their ability to detect changes in soil microbiological parameters as a result of heavy metal pollution.Soil microbial biomassThe soil microbial biomass carbon (Cmic) and nitrogen (Nmic) contents determined at 0, 15, 30, 45 and 60 days after heavy metal application (DAA) showed a significant (P<0.05) decline in the Cmic for all Pb and Cd amended soils from 15 to 30 DAA. From 30 to 60 DAA, Cmic contents changed non-significantly for all other treatments except for 600 mg kg-1 Pb and 100 mg kg-1 Cd in which it again declined significantly from 45 to 60 DAA. The Nmic contents also decreased significantly from start to 30 DAA for all other Pb and Cd treatments except for 200 mg kg-1 Pb which did not show significant difference with control. Control and 200 mg kg-1 Pb had significantly lower soil microbial biomass C: N ratio as compared with other Pb treatments from 15 to 60 DAA, however at 60 DAA, 1000 mg kg-1 Pb showed significantly higher C: N ratiocompared with other Pb treatments. On the other hand, C: N ratio for various Cd treatments in soil was higher compared with that of Pb treatments. Combining low level (200 mg kg"1) of Pb with low (20 mg kg-1) level of Cd showed no significant difference with control for CmjC up to 30 DAA, however, its combinations with high level of Cd resulted in a significant decrease in CmjC. A similar behavior was also shown when low (20 mg kg"1), medium (60 mg kg"1), and high (100 mg kg"1) levels of Cd were combined with different Pb concentrations. At 30 DAA, high level of Pb (1000 mg kg"1) combined with medium (60 mg kg"1) and high (100 mg kg"1) level of Cd application to soil resulted in more than 50 % decrease in NmjC compared to the initial value. The initial C: N ratio in the soil was 5.7 and until 60 DAA, significantly higher C: N ratio (15.7) was observed in high levels (1000-100 mg kg"1) of Pb and Cd combined treatment followed by 14.0 in 1000-20 mg kg"1 Pb-Cd treatment, respectively. Combining 200 mg kg"1 Pb with 20 mg kg"1 Cd in soil showed significantly more inhibition of CmiC compared to their individual treatments at 45 and 60 DAA. In fact, by the application of metals in combined form, the overall toxicity of metals may not be due to direct toxic effects of a particular metal applied. Instead, it may arise from the interactions among the various metals present in the system. The dynamics of CmjC and Nmic during the incubation period after the heavy metal addition was related to the dynamic of soil microbial populations. The soil microbial populations decreased upon the depletion of readily utilized carbon substrate resulted from heavy metal toxicity, and starved as the reserves were exhausted, and decreased significantly in size up to 30 DAA. The differences in CmiC and Nmic among the treatments were caused by the different concentrations of heavy metal added to the soil, which inhibited the growth of soil micro-organisms. Comparatively greater reduction of Cmic and Nmic under Cd than Pb amended soils was seen in the present experiment which might be due to the higher solubility and more direct toxicity of Cd than Pb to soil microorganisms.Soil enzymes activityEffects of Pb and Cd applied alone and or in combination on the dynamics of dehydrogenase, urease, and acid phosphatase activities in soil were studied during 60 days of incubation at 25 °C. The results showed a significant decline in the dehydrogenase activity for all Pb and Cd treated soils from 15 to 60 DAA, except for 20 mg kg"' Cd which had similar activity values compared with control from start to 45 DAA. Soil urease and acid phosphatase activities were also decreased significantly from 15 to 60 DAA for all other Pb and Cd treatments except for 200 mg kg"1 Pb which did not show significant difference from control. The declining rate of enzymesactivities was comparatively higher from start to 30 DAA than from 30 to 60 DAA, suggesting a greater toxicity in the first 30 days after which the microbial activities reached at equilibrium. Up to 60 DAA, urease activity was reduced more than 50 % from its original value for 1000 mg kg"1 Pb, and 60, 80 and 100 mg kg"1 Cd; while more than 50 % reduction in acid phosphatase activity was observed in 800 and 1000 mg kg'1 Pb, and also in 100 mg kg'1 Cd treatments. Dehydrogenase activity showed more than 50 % decline only for 1000 mg kg"1 Pb until 60 DAA. The combined application of 200 mg kg"1 Pb with 20 and 60 mg kg"1 Cd didn't show significant difference in enzymes activities compared to the individual application of 200 mg kg"1 Pb. Combined application of 60 and 100 mg kg"1 Cd with 1000 mg kg'1 Pb indicated significantly greater inhibitions of dehydrogenase activity compared to their individual treatments in soil from 15 to 60 DAA. Whereas, combining 60 and 100 mg kg"1 Cd with 200 mg kg"1 Pb in soil showed lower inhibitions compared to 60 and 100 mg kg"1 Cd individual treatments, respectively at 60 DAA. Soil urease activity seemed to be more sensitive compared to dehydrogenase in soil with Pb and Cd combined applications. Whereas, combining high level of Cd with medium and high levels of Pb resulted in more than 50 % decline in acid phosphatase activity in these treatments at 60 DAA, compared to that in the control, respectively. The observed decreases in the enzymes activities under Pb and Cd could be due to the direct inhibition of soil enzymes by binding of metal with the functional group of enzyme and indirect effect by declining the size of microbial communities.Functional diversity of soil microbial communitiesA functional approach in community analysis can provide an ecologically meaningful measure of heavy metal toxicity to soil microbial communities; since the functional abilities of the microbial community are related to the essential processes in the ecosystem Functional diversity of soil microbial communities under heavy metal application was determined by inoculating Biolog ECO plates. The results indicated that various levels of Pb and Cd addition markedly inhibited the functional activity of soil microbial communities as indicated by the intensity of average well color development (AWCD) during 168 hours of incubation. Multivariate analysis of sole carbon source utilization pattern for 600, 800 and 1000 mg kg"1 Pb, and 80 and 100 mg kg"1 Cd applications in soil showed significantly lower AWCD values compared with that of control and had different substrate utilization pattern of soil microbial communities. Correlation and analysis of the loadings of the most influential C sources byPrinciple components analyses indicated that microbial communities of Pb- amended soils had more utilization of L- phenylalanine, D-mannitoI, D-malic acid and a- ketobutyric acid, while less utilization of tween 40, pyruvic acid methyl-ester, hyrdoxy butyric acid and itaconic acid. On the other hand, the results of Cd treated soils showed higher utilization of L- asparagine, L-phenylalanine, pyruvic acid methyl ester acid and L- threonine and lower utilization of N-Acetyl-D-glucosamine, D-galacturonic acid, ot-cyclodextrin and D-xylose. Pb and Cd combined application in soil showed more inhibition of AWCD values compared with that of their individual applications. Multivariate analysis of sole carbon source utilization pattern for over all Pb and Cd combined treatments showed more utilization of B-methyl-D-glycoside, pyruvic acid methyl-ester, D-xylose, L-phenylalanine, N-acetyl-D-glucosamine, glycogen and D-glucosamine acid, while less utilization of tween 40, tween 80, D-mannitol and D-cellobiose. Diversity indices indicated that a significant decrease of microbial diversity in combined treatments compared to that in control resulted only when high level of Pb and Cd was combined in soil. However, no significant decrease in species richness was seen in combined treatments compared to control, except for high level of Cd combined with medium level of Pb treatment in soil.Structural diversity of soil microbial communitiesMethod of PCR-DGGE was used to investigate the change in the structural diversity of soil microbial communities under heavy metal pollution. The results showed that the size of DNA extracted from soil decreased with increasing Pb and or Cd concentrations in the soil, however, PCR results indicated that DNA template for all treatments was equally amplified by using primers and PCR cycles. DGGE profiles generated from the universal bacterial primers (F968 and R1401) revealed the structural composition of communities for different heavy metal treatments. Each of the distinguishable bands in the separation pattern is represented as an individual bacterial species. DGGE patterns in the soil treated with Pb and Cd simplified with increasing their concentration, and all Pb and Cd treatments except 200 mg kg'1 Pb showed significantly less number of visible bands compared with that for control. Phylogenetic clustering of DGGE profiles for various Cd treatments revealed that they had more than 50 % difference with that of control. Several similarities in banding positions were also found among the treatments receiving different levels of heavy metals, indicating that many common microbial members were still present in each treatment regardless of the heavy metal rates. The structuraldiversity indices generated for soil with various levels of Pb application indicated that all Pb treatments except 200 mg kg"1 had significantly lower diversity; however, no significant difference for species evenness was detected. On the other hand, combining Pb and Cd at medium and higher levels resulted in a significant decrease in species richness, but no significant change in diversity was observed at any of the Pb and Cd combined applications in soil except for 1000 mg kg'1 Pb combined with 100 mg kg'1 Cd treatment. It appeared that with increasing rate of heavy metal application in soil, many bacteria with low resistance or without resistance to heavy metal couldn't survive under the high selective pressure of Pb and Cd, and caused the bacterial diversity to change.Relationship among soil microbiological indicesSoil microbiological and biochemical properties are indicator of soil quality and these properties have been used individually, as simple indices, or in combination using complex equations derived from mathematical combinations or the application of statistical programs. These properties exhibited differential relationships among each other under present investigation. Soil enzymes activity showed more strong positive correlation with microbial biomass than functional and structural diversity of soil microbial communities. Microbial biomass N was more closely related to C: N ratio than microbial biomass C. Out of all the three enzymes, dehydrogenase activity showed more close relation to microbial biomass C and N than that of urease and acid phosphatase under Pb pollution in soil, however, soil urease activity was more strongly related to C: N ratio. Average well color development (AWCD) values indicated more close relation with microbial biomass and enzymes activities compared to average band intensity (ABI). Among different Pb and Cd treatments, higher levels of Pb showed more similar effects with Cd at medium levels. Regressions lines for various microbiological properties indicated that soil microbial biomass N and dehydrogenase activity decreased more sharply by increasing Pb concentrations in soil, while microbial biomass C and acid phosphatase activity decreased more sharply by increasing Cd levels in soil. Comparing Biolog ECO plate and PCR-DGGE data results by principle components analyses and diversity indices showed similar trends for various heavy metal treatments in general, but specifically, values for individual treatments were less comparable. So it can be assumed that the responses of soil microorganisms to soil treatments depend not only on the type and rate of treatment applications but also the methods used to investigate the response.
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