Journal of Environmental Science and Technology

Year: 2012 | Volume: 5 | Issue: 3 | Page No.: 184-191
DOI: 10.3923/jest.2012.184.191

Effect of Cadmium on Abundance and Diversity of Free Living Nitrogen Fixing Azotobacter spp.

Durga Prasad, Gangavarapu Subrahmanyam and Krishna Bolla

Abstract: Soil pollution by elevated heavy metals are known to adversely effect microbial activities and their functional diversity. However, response of certain beneficial native soil bacterial population to heavy metals is poorly understood. In the present study, effect of cadmium (Cd²⁺) on abundance and diversity of free living nitrogen fixing Azotobacter spp. has been investigated by short term microcosm experiment. After 45 days, total viable counts of both heterotrophic and Azotobacter spp. have been decreased with increased Cd²⁺ concentrations. Significant negative correlation (R = -0.99, p = 0.006) was observed between abundance of Azotobacter spp. and Cd²⁺ concentration. Similar kind of result has been noticed with total viable heterotrophic bacteria. Amplified ribosomal DNA restriction analysis (ARDRA) based dendrogram revealed the Cd²⁺ induced diversity shift of Azotobacter population. Relatively decreased diversity of Azotobacter spp. was noticed at elevated concentration of Cd²⁺ (5 mg kg^-1 soil). Reduction of both abundance and diversity of Azotobacter spp. pointed apparent deleterious effect of Cd²⁺ on free living nitrogen fixation and concurrent soil health.

Keywords: Azotobacter spp., 16S rRNA gene diversity, amplified ribosomal DNA restriction analysis (ARDRA) and Cadmium toxicity

INTRODUCTION

Soil contamination with heavy metals has been considered to be an important environmental issue (Azmat et al., 2005; Benson, 2006; Kamala-Kannan and Lee, 2008; Arabani et al., 2010; Ling et al., 2010). Heavy metal induced long-term hazardous impact on soil biological processes and subsequent soil health has been reported (Perez de Mora et al., 2006; Wang et al., 2007; Bhattacharyya et al., 2008). Elevated levels of heavy metals had significant impact on the plants, microbial abundance, activity and functional diversity (Liao et al., 2005; Ahmad et al., 2005; Siham, 2007; Sengar et al., 2008; Afef et al., 2011). Generally, heavy metals have induced influence on microbial community which subsequently lead to changes in soil microbial activities including enzymes and microbial biomass C (Moreno et al., 2002; He et al., 2005; Khan et al., 2010). It was found that both short-term and long-term exposures to heavy metals resulted in the reduction of soil microbial activity and diversity (McGrath et al., 2001; Lasat, 2002; Liao et al., 2005; Khan et al., 2007, 2010). Cadmium (Cd) is one of the toxic heavy metal and released to the environment mainly due to industrial processes and phosphate fertilizers. Deleterious effect of Cd²⁺ on both plants and microbes has been established (Shaukat et al., 1999; Sandalio et al., 2001; Moreno et al., 2002; Khan et al., 2007; Benavides et al., 2005). Microbial activity and diversity have been decreased with increasing concentration of Cd²⁺ (Sandalio et al., 2001; Khan et al., 2007, 2010) Harmful effects produced by Cd might be explained by its ability to inactivate enzymes possibly through reaction with the SH-groups of proteins (Fuhrer, 1982).

Nitrogen fixation can be considered as one of the most important microbial activity as it enables the recycling of nitrogen on earth and gives a fundamental contribution to nitrogen homeostasis in the biosphere and in the geosphere (Aquilantia et al., 2004). Azotobacter is a free living diazotroph that have profound scientific and agronomic significance due to their ability to fix nitrogen. It was estimated that Azotobacter could fix at least 10 mg N per gram of carbohydrate (Becking, 1991). The ecological distribution of Azotobacter spp. was found to be intricate subject and was effected by diverse factors such as climate conditions, organic matter, moisture, C/N relation, pH and rhizosphere (Dobereiner and Pedrosa, 1987; Tejera et al., 2005). However, effect of heavy metals particularly Cd²⁺ on the distribution and diversity of these bacteria was not yet established. Moreover the negative effect of Cd²⁺ on general microbial community structure and activity was well noticed (Khan et al., 2007; Moreno et al., 2002). On the contrary, little was known about influence of Cd²⁺ on specific native soil beneficial bacterial populations. Therefore, in the present study, we have investigated specifically about response of Cd²⁺ on abundance and diversity of Azotobacter spp. by short term microcosm experiment.

MATERIALS AND METHODS

Composite soil samples were collected from a healthy agricultural field located near Visakhapatnam a North Eastern coastal district of Andhra Pradesh, India. Prior to the collection of samples, the sites were cleaned by scraping the surface layer. Samples were transported to the lab as described previously (Subrahmanyam et al., 2011a). All the samples were sieved (<4 mm) and stored at 4°C. The pH of the soil was found to be 6.7 and the organic matter was estimated to be 1.1%. Total C/N ratio was 8.3. Soil texture was found to be clay loam. Heavy metal analysis was performed (Udosen et al., 2006) and no traces of Cd²⁺ were found in the field soil. In order to elucidate the effect of Cd²⁺ on Azotobacter density and diversity, metal spiked microcosms were conducted. Five different treatments of Cd²⁺ such as 0, 1, 2, 5 and 10 mg kg^-1 soil were established with spiking of aqueous solution of CdSO₄ (Khan et al., 2010). Treatment of Cd²⁺ at 0 mg kg^-1 soil was considered to be control. The experiment was completely randomized design as described elsewhere. After 45 days of incubation, total viable counts of both heterotrophic bacteria and Azotobacter spp. were performed. Treatment 1 (0 mg kg^-1) and treatment 4 (5 mg kg^-1) were chosen for diversity analysis conducted by 16S rDNA based ARDRA.

Total viable counts of heterotrophic bacteria were obtained by a procedure suggested by Massa et al. (1998) and Subrahmanyam et al. (2011b). Azotobacter spp. were isolated and enumerated by streaking of serial soil dilutions on plates containing selective Brown N free medium (Brown et al., 1962; Knowles, 1982; Aquilantia et al., 2004). After 3-7 days incubation at 27-30°C, the plates presenting growth of Azotobacter were revealed by the appearance of slimy, glistening colonies (Aquilantia et al., 2004). A total of 25 Azotobacter colonies were randomly chosen for 16S rDNA diversity studies.

Genomic DNA of the actively growing Azotobacter spp. was extracted by CTAB-SDS lysis method (Sambrook and Russell, 2001). The 16S rDNA fragment of each isolate was amplified by the universal eubacterial primers discussed elsewhere (Weisburg et al., 1991; Pramanik et al., 2003). Primers used in the study were as follows Gm3f (8-23)-5^IAGA GTT TGA TCM TGGC3^I and Gm4r (1492-1507)-5^ITAC CTT GTT ACG ACT T3^I. These two primers are considered to be specific for eubacteria and are highly conserved (Weisburg et al., 1991). The PCR reaction was performed in a 50 μL of total reaction volume, with 1.5 U of Taq DNA polymerase and the conditions for PCR were previously described. (Pramanik et al., 2003). In brief, initial denaturation at 94°C for 1 min, followed by 28 cycles of 1 min at 94°C, 1 min at 51°C and 1 min 30 sec at 72°C and a final extension step at 72°C for 10 min. Amplification products were checked on 1% agarose gel in 1xTBE buffer. The PCR products were directly subjected to restriction digestion.

16S rDNA-based phylogenetic analysis of Azotobacter spp. was revealed by ARDRA. Two tetra-cutter enzymes, viz., RsaI (New England Biolabs) and HaeIII (Genei) were used for ARDRA as described previously (Pramanik et al., 2003; Aquilantia et al., 2004). Digestions were carried out according to the manufacturer’s protocols. Digested 16S rDNA products were separated by agarose gel electrophoresis using a 3.0% agarose gel in 0.5xTris-borate-EDTA buffer. Gels were stained with ethidium bromide and visualized under UV transilluminator and photographed with gel documentation system. The two gel pictures were compared, combined and generated a single dendrogram for both the enzymes. Co-efficient based dendrogram was generated by using the band pattern of ARDRA for the isolates using NT sys P2.0 software program.

RESULTS AND DISCUSSION

Microbial interactions with metals may have several implications for the environment. Microbes would play pivotal roles in the biogeochemical cycling of toxic metals, as well as in managing the metal tainted environments. Thus, it is certainly important to have a better understanding of how microbial populations respond to elevated metal concentrations. The responses analyzed should be related to some important soil biological processes, such as C and N cycling (Martensson and Torstensson, 1996). Therefore, in the present investigation, effect of Cd²⁺ on the abundance and diversity of free living diazotrophs particularly in the emphasis of Azotobacter population has been investigated in a short term microcosm experiment.

Total viable counts of heterotrohpic and Azotobacter sp. were shown in the Table 1. Obvious effect of Cd²⁺ on both the bacterial counts has been noticed. Heterotrophic bacterial counts were significantly (p = 0.05) decreased with increased Cd²⁺ concentration. Relatively Cd²⁺ at 10 mg kg^-1 was found to be most deleterious to hetrotrophic bacteria where the bacterial population reduced up to 5.4x10⁴ times compared to control (0 mg kg^-1). These findings were supportive to earlier studies (Khan et al., 2010; Ahmad et al., 2005) and pointed that the cultivable heterotrophic bacteria were sensitive to heavy metal pollution. After 45 days of incubation, significant negative correlation (R = -0.99, p = 0.001) was observed between Cd²⁺ concentrations and total hetrotrophic bacterial counts (Fig. 1a). Upon exposure to a high concentration of heavy metals, nontolerant bacterial species could be diminished, on the other hand tolerant species survived and increased in their abundance. Typically, this enhancement in abundance would be due to physiological adaptation and genetic modifications exhibited by metal tolerant bacterial species which may lead to replacement of more sensitive species (Bruins et al., 2000).

Relatively, significant decrease in the bacterial counts of Azotobacter sp. was noticed at elevated Cd²⁺ concentrations (Table 1). When compared to control (0 mg kg^-1), a reduction up to 1.2x10² times has been noticed with Azotobacter spp. counts at 5 mg kg^-1 of Cd. No colonies were found at a concentration of 10 mg kg^-1 of Cd²⁺. Apparent negative correlation (R = 0.99) was existed between Cd²⁺ concentrations and abundance of Azotobacter sp. and the results were found to be significant at p = 0.04 (Fig. 1b).

Results obtained from viable counts of Azotobacter spp. were pointed the toxicity of Cd²⁺ on free living diazotrophs. Similar kinds of observations were made by Moreira et al. (2008). Substantial decreases of Azospirillum spp. in Cd²⁺ polluted soils were observed (Moreira et al., 2008). Moreover, in the present study it was found that Azotobacter spp. were found to be more sensitive to Cd²⁺ than normal heterotrophic bacteria. Related findings were obtained by Ahmad et al. (2005) during their study on heavy metal effect on survival of certain indigenous microbial populations. In their findings, relatively diazotrophic bacteria showed higher sensitivity to metal groups like Cd, Pb, Hg followed by Cu, Cr, Mn, Ni and Zn than any other observed bacterial populations. However their studies were restricted to symbiotic nitrogen fixers. In other study conducted by Oliveira et al. (2009), it was noticed that contaminated soil showed a decrease in the diazotrophic population size of about 80%, confirming the greater sensitivity of this group to heavy metals. It was pronounced that Azotobacter sp. were one of the key stone species in free living nitrogen fixation and estimated to be fix 10-20 kg N ha^-1 (Aquilantia et al., 2004). Hence we infer that the decreased density of Azotobacter sp. associates with reduced free living nitrogen fixation in Cd²⁺ spiked soil. Earlier studies also pointed that cycling of nitrogen particularly N₂ fixation, are sensitive to metal additions and also dependent on soil properties (Aticho et al., 2011). For example, the N₂-fixing potential of heterotrophic diazotrophs was found to be sensitive to small concentrations of heavy metals (Martensson, 1993; Martensson and Torstensson, 1996).

Diversity of Azotobacter spp. was estimated by ARDRA based dendrogram (Fig. 2, 3) and obvious effect of Cd²⁺ on diversity of Azotobacter spp. was noticed. In the control soil, isolates were distributed in 8 distinct clades at a coefficient of 0.88 (Fig. 2).

Majority of the isolates were grouped in two 6 different clusters (C1-C6). Whereas, in Cd spiked soil (5 mg kg^-1) isolates were distributed in 4 distinct clades at a coefficient of 0.88 (Fig. 3) and majority of the isolates were grouped into 3 different clusters (C1-C3). Relatively, about 50% reduction in the diversity of Azotobacter spp. was observed at a concentration of Cd²⁺ 5 mg kg^-1 soil. Substantial information of Cd²⁺ toxicity on the microbial activity and diversity was existed (Khan et al., 2007, 2010; Moreno et al., 2002). However, limited information is available with diazotrophs. For example, decreased diversity of free-leaving Rhizobium population was noticed in soils contaminated with Cu, Zn and Pb (Stan et al., 2011). Similarly, symbiotic efficiency of the Rhizobium was reduced by elevated Al (Paudyal et al., 2007). As for our knowledge, no information is available in the perspective Cd toxicity on Azotobacter spp.

CONCLUSION

Azotobacter spp. are found to be more sensitive to Cd²⁺ and 10 mg Cd kg^-1 soil, is highly deleterious to Azotobacter spp. Elevated concentration of Cd²⁺ reduced both diversity and abundance of Azotobacter spp. Therefore, we could infer that these decreased density and diversity of Azotobacter spp. may reduce free living diazotrophy in soil. Further characterization of the isolates such as phylogenetic identification and nitrogen fixing efficiency are presently under investigation.

REFERENCES