Impact of heavy metals on bacterial communities from ...

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Impact of heavy metals on bacterial communities from mangrove soils of the Mahanadi Delta (India) a

b

c

d

B. C. Behera , R. R. Mishra , J. K. Patra , K. Sarangi , S. K. e

Dutta & H. N. Thatoi

c

a

Department of Biotechnology , North Orissa University , Baripada , India b

Department of Biotechnology , MITS School of Biotechnology , Bhubaneswar Phone: nIndia c

Department of Biotechnology , College of Engineering and Technology, Biju Pattnaik University of Technology , Bhubaneswar , India d

Institute of Minerals and Materials Technology , Bhubaneswar , India e

Centre for Ecological Sciences, Indian Institute of Science , Bangalore , 560012 Published online: 10 Jul 2013.

To cite this article: Chemistry and Ecology (2013): Impact of heavy metals on bacterial communities from mangrove soils of the Mahanadi Delta (India), Chemistry and Ecology, DOI: 10.1080/02757540.2013.810719 To link to this article: http://dx.doi.org/10.1080/02757540.2013.810719

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Chemistry and Ecology, 2013 http://dx.doi.org/10.1080/02757540.2013.810719

Impact of heavy metals on bacterial communities from mangrove soils of the Mahanadi Delta (India) B. C. Beheraa , R. R. Mishrab , J. K. Patrac , K. Sarangid , S. K. Duttae and H. N. Thatoic *


a Department

of Biotechnology, North Orissa University, Baripada, India; b Department of Biotechnology, MITS School of Biotechnology, Bhubaneswar, India; c Department of Biotechnology, College of Engineering and Technology, Biju Pattnaik University of Technology, Bhubaneswar, India; d Institute of Minerals and Materials Technology, Bhubaneswar, India; e Centre for Ecological Sciences, Indian Institute of Science, Bangalore 560012 (Received 15 November 2012; final version received 8 May 2013) This study aimed to assess soil nutrient status and heavy metal content and their impact on the predominant soil bacterial communities of mangroves of the Mahanadi Delta. Mangrove soil of the Mahanadi Delta is slightly acidic and the levels of soil nutrients such as carbon, nitrogen, phosphorous and potash vary with season and site. The seasonal average concentrations (μg/g) of various heavy metals were in the range: 14 810–63 370 (Fe), 2.8–32.6 (Cu), 13.4–55.7 (Ni), 1.8–7.9 (Cd), 16.6–54.7 (Pb), 24.4–132.5 (Zn) and 13.3–48.2 (Co). Among the different heavy metals analysed, Co, Cu and Cd were above their permissible limits, as prescribed by Indian Standards (Co = 17 μg/g, Cu = 30 μg/g, Cd = 3–6 μg/g), indicating pollution in the mangrove soil. A viable plate count revealed the presence of different groups of bacteria in the mangrove soil, i.e. heterotrophs, free-living N2 fixers, nitrifyers, denitrifyers, phosphate solubilisers, cellulose degraders and sulfur oxidisers. Principal component analysis performed using multivariate statistical methods showed a positive relationship between soil nutrients and microbial load. Whereas metal content such as Cu, Co and Ni showed a negative impact on some of the studied soil bacteria. Keywords: bacterial community, heavy metals, mangrove ecosystem, soil nutrients

1.

Introduction

Mangrove forests occurring at the interface between land and sea are a highly productive and biologically diverse ecosystem. This ecosystem is rich in nutrients and harbours diverse groups of microorganisms.[1] Various groups of bacteria such as nitrogen fixers, phosphate solubilisers, cellulose decomposers, nitrifiers, denitrifiers, sulfur oxidisers, iron oxidisers and iron reducers are usually present in the mangrove environment.[1] Complex interactions between these microbes maintain the nutritional status and ecological balance of the mangroves.[2] In the last few decades, mangrove forests have been disappearing all over the world due to climate change and pollution caused by the development of cities, ports and industries in the vicinity of the mangrove ecosystem. Determining the factors responsible for degradation of the mangrove environment is essential for their effective management. The Mahanadi Delta, Odisha is the second largest river delta in India after the Ganges, and the second largest mangrove ecosystem in Odisha next to Bhitarkanika. Near its estuary, the Mahanadi *Corresponding author. Email: [email protected] © 2013 Taylor & Francis

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B.C. Behera et al.

Delta is becoming polluted due to anthropogenic activities, the development of industries such as the Indian Farmer Fertiliser Corporation (IFFC), Paradeep Phosphate Limited, Skol Breweries and several prawn farms in and around the mangrove which continuously discharge effluents into the river course. These pollutants are continuously intermixed with the soil and may have an adverse effect on the soil microflora which plays a crucial role in increasing soil fertility and maintaining the ecological balance in the mangrove ecosystem. Hence, this study was designed to assess the soil nutrient and heavy metal contents, along with their influence on predominant soil bacterial communities of the Mahanadi Delta, a tropical mangrove ecosystem of Odisha, which will provide information on the pollution status for future management of the ecosystem.

2.

Materials and methods


2.1. Study site Soil samples were collected seasonally (summer, rainy, winter and autumn) during 2008–2009 from the mangrove forest of the Mahanadi river delta of Odisha. Six sampling sites inside the mangrove forest, viz., Jamboo, Kharnasi (which is greatly affected by daily anthropogenic activities due to fishing and transportation), Nuagada (receiving effluents from nearby prawn farms), Triveni (receiving effluents from nearby prawn farms), Atharbanki (adjacent to Paradip Phosphate Limited and Paradip township) and the IFFCO site (adjacent to IFFC) (Figure 1) were distributed along the Mahanadi estuaries near Paradip. Approximately 6 replicates of 100g of soil were collected from arbitrarily selected spots at each of the six locations, mixed thoroughly in sterile polythene bags and brought to the laboratory. 2.2. Soil physicochemical parameter To estimate soil physicochemical characteristics, six replicates of samples taken at random from each site were collected from 2–4 cm depth with the help of a spatula. The bulk samples were kept in airtight polyethylene containers at 4 ◦ C in an ice box and returned immediately to the laboratory for processing. Soil pH and electrical conductivity (EC) were determined using a digital pocket pH meter and a conductivity meter (Systronics, 341), respectively. Total nitrogen in the soil was determined using the Kjeidahl digestion method. Available phosphorus, organic carbon and potash of the soil were determined using the procedure described by Tabi and Ogunkunle.[3] 2.3. Soil heavy metal content For the determination of heavy metals, one gram of dried soil was digested with 20 mL of aqua regia (HCl=HNO3 3:1) in a beaker (open beaker digestion) on a thermostatically controlled hot plate. Then 5.0 mL of hydrogen peroxide was added to the sample to complete the digestion and the resulting mixture was heated to near dryness in a fume cupboard and filtered using a Whatman no. 42 filter paper, the volume was then made up to 50 mL with double-distilled water. Analyses of seven heavy metals, Pb, Cu, Zn, Cd, Co, Ni and Fe, were performed using Perkin-Elmer Model Analyst 200 flame atomic absorption spectrophotometer (AAS). 2.4. Bacterial population One gram of air-dried soil was diluted up to 10−5 in sterilised distilled water and 100-μL suspensions were separately spread plated on Petri plates (n = 3) containing different media

3


Chemistry and Ecology

Figure 1.

Map of Mahanadi river delta showing different study sites.

and incubated at 30 ± 0.1◦ C. Heterotrophic bacteria were enumerated on nutrient agar (NA) medium. To assess the Gram-negative bacterial population, soil suspensions were plated on NA medium containing crystal violet. Enumeration of nitrifying bacteria were carried out using the microplate most probable number (MPN) method [4] and denitrifying bacteria were enumerated using the method described by Binachi et al.[5] Asymbiotic nitrogen-fixing bacteria were cultured and enumerated using the method described by Guerinot and Colwell.[6] Starky solid medium and liquid medium described by Zhou et al. [7] were used for the isolation and enumeration of sulfur-oxidising bacteria. Bacterial colonies that formed halo zones on National Botanical Research Institute, Pune (NBRIP) agar medium were counted as phosphate-solubilising

4

B.C. Behera et al.

bacteria.[8] Anaerobic bacteria were cultivated and isolated following the method of Ramprasad et al.[9] Cellulose-degrading bacterial colonies were isolated from the carboxyl methyl cellulose agar medium.[10] 2.5. Statistical analysis Statistical analysis was performed with the multivariate statistical methods. Pearson’s correlation analysis and principal component analysis (PCA) were performed site-wise in different seasons by SPSS, v. 16 for Windows (SPSS Inc; Chicago, IL, USA). Test results were shown by the mean and standard errors of the replicates of each variable.


3.

Results

3.1. Soil physicochemical characteristics The spatial distribution of soil characteristics such as pH, EC, total N, P, K and total C were analysed from six different sites of the Mahanadi River Delta mangrove ecosystem and their average seasonal values are shown in Figure 2a–f. The pH at different sites was within a narrow range of 5.5–6.8 (Figure 2a), and was slightly acidic. The EC of the soil samples fluctuated at the different stations of the mangrove environment, with mean values between 0.5 and 4.9 (Figure 2b). Total nitrogen for the six sites ranged between 0.037 and 0. 094% (Figure 2c), with the maximum value in the winter season (0.094%). Soil phosphorus showed wide variation from 8.0 to 138.0 kg.ha−1 among the study sites with a maximum value in the rainy season. A significantly higher phosphorous content was observed in soils from the IFFCO and Atharbanki (near PPL) sites (Figure 2d). The potassium level varied from 289 to 498 kg.ha−1 among the study sites (Figure 2e). Total organic carbon among the study sites varied between 0.38 and 0.99%, showing a maximum in summer and minimum during winter (Figure 2f). 3.2.

Soil heavy metal

In this study, more attention has been paid to investigating the heavy metal content in intertidal sediment of the Mahanadi Delta mangrove environment. Seasonal average concentrations (μg/g) of various heavy metals in the soil sample at six different locations of the mangrove environment were: 14 810–63 370 (Fe), 2.8–32.6 (Cu), 13.4–55.7 (Ni), 1.8–7.9 (Cd), 16.6–54.7 (Pb), 24.4–132.5 (Zn) and 13.3–48.2 (Co) (Table 1). The normal value for Cu in soil is 30 μg/g and the observed values (2.8–32.6 μg/g) are above the prescribed limit. Average Ni concentrations among the six sites were found below the permissible limit (75–150 μg/g; Indian Standard) with a maximum (55.7 μg/g) at Kharnasi during the rainy season and a minimum (13.4 μg/g) at Atharbanki. Maximum concentrations of Cd (7.9 μg/g), Co (48 μg/g) and Zn (132.5 μg/g) were observed in mangrove forest adjacent to the IFFCO site. The Pb concentration varies from 16.6 to 54.7 μg/g in different sites with a maximum at Atharbanki (54.7 μg/g) and minimum at Kharnasi (16.6 μg/g). 3.3. Soil bacterial population To investigate the impact of soil nutrient content and heavy metals on the bacterial communities of the deltaic mangrove ecosystem of Mahanadi, some predominant bacterial communities were enumerated and their average seasonal variations are presented in Figure 3a–k. Among the different groups of bacteria studied, the heterotrophic bacterial population (46–167 × 105 cfu/g soil) was greater and reached a maximum during the rainy season (Figure 3a).

5



Figure 2.

(a) Soil pH. (b) Salinity. (c) Nitrogen. (d) Phosphorus. (e) Potash. (f) Carbon.

The free-living N2 -fixing bacterial population (28–113 × 105 cfu/g soil) was found to fluctuate during the study period, however, it reached a maximum during the winter and minimum during the rainy season (Figure 3b). The nitrifying bacterial population showed clear seasonal variation (8.33–104 × 105 cfu/g soil), being less in the summer season than in the rainy season (Figure 3c). The denitrifying bacteria population varied from 4.33 to 114 × 105 cfu/g soil, showing the highest peak during the winter season (Figure 3d). The Gram-negative bacterial population varied between 8 to 134 × 105 cfu/g soil in different sites, and was increased twofold at Kharnasi compared with the other sites during the rainy season (Figure 3e). The Gram-positive bacterial population varied from 9.33 to 110 × 105 cfu/g soil in different sites and seasons, with a peak during the winter season (Figure 3f). The sulfur-oxidising bacterial population was almost the same in autumn

6

Heavy metal content (μg/g) of soil samples collected from different locations of mangrove environment of Mahanadi delta estuary.

Station

Season

Cu

Zn

Co

Ni

Fe

Pb

Cd

Jambo

Summer Rainy Autumn Winter Summer Rainy Autumn Winter Summer Rainy Autumn Winter Summer Rainy Autumn Winter Summer Rainy Autumn Winter Summer Rainy Autumn Winter

9 ± 0.00 17.9 ± 1.15 11.7 ± 0.56 9 ± 0.57 9.7 ± 0.58 32.6 ± 2.53 4.4 ± 0.95 10 ± 0.57 16.5 ± 1.20 24.8 ± 1.19 18.2 ± 1.15 6.2 ± 0.63 15.8 ± 1.93 26.9 ± 1.41 13.8 ± 1.154 11.1 ± 1.04 2.8 ± 0.09 21.5 ± 1.62 5.7 ± 0.60 22.9 ± 1.53 17.7 ± 0.493 23.1 ± 1.12 16.5 ± 1.04 8.4 ± 0.99

47.5 ± 1.732 98.3 ± 1.65 49.2 ± 1.03 39 ± 0.60 47.7 ± 0.66 106 ± 1.15 49.2 ± 1.15 52.5 ± 1.03 51.1 ± 1.15 87.7 ± 0.91 44.9 ± 1.73 75.7 ± 2.3 53.2 ± 1.59 96.9 ± 1.76 40.9 ± 1.10 58.5 ± 0.66 118.7 ± 1.66 57.7 ± 1.73 24.4 ± 1.55 29.5 ± 2.69 55.1 ± 0.94 112.3 ± 0.81 55.1 ± 0.51 132.5 ± 2.45

15.9 ± 1.6 32 ± 1.15 25.4 ± 1.57 17 ± 1.99 16.3 ± 1.09 44.6 ± 1.10 18.3 ± 1.24 17.3 ± 0.63 19.1 ± 1.24 26.4 ± 1.16 22.3 ± 1.876 13.3 ± 1.13 18.5 ± 2.3 27.8 ± 1.7 19.7 ± 1.32 13.3 ± 1.27 26.1 ± 2.47 33.3 ± 1.7 17.2 ± 0.98 25.8 ± 0.57 18.8 ± 1.7 48.2 ± 2.39 38.4 ± 1.01 14.4 ± 2.28

26.2 ± 2.36 32.2 ± 2.36 19.3 ± 1.24 22.5 ± 1.81 48.9 ± 0.60 55.7 ± 0.002 31 ± 1.73 24.3 ± 0.90 37 ± 1.732 42.33 ± 1.14 23.7 ± 1.42 21.5 ± 1.26 38.2 ± 1.73 45.8 ± 2.3 16.4 ± 2.65 30.9 ± 1.18 36.6 ± 1.0 33.3 ± 1.76 17.2 ± 2.06 25.8 ± 1.78 35.9 ± 1.15 21.5 ± 1.26 27.4 ± 2.65 21.6 ± 2.0

20 670 ± 1 215.03 37 810 ± 1 185.99 17 500 ± 655.76 21 312 ± 1 187.05 21 110 ± 655.76 43 370 ± 1 172.73 14 810 ± 5 009.01 21 560 ± 1 115.03 29 950 ± 25.16 33 620 ± 1 218.24 27 370 ± 585.87 21 310 ± 1 561.78 28 880 ± 1 732.13 35 310 ± 1 183.11 20 810 ± 1 069.80 21 370 ± 1 581.29 35 900 ± 577.36 54 620 ± 43.71 41 750 ± 1 100.16 63 370 ± 1 810.08 48 710 ± 1 159.71 39 370 ± 155.66 31 250 ± 1 161.19 22 250 ± 32.14

15.9 ± 0.60 34.7 ± 1.03 28.9 ± 0.58 22.6 ± 1.41 16.6 ± 1.15 43.3 ± 0.57 ND 29.4 ± 2.43 18.8 ± 1.21 32.6 ± 1.78 16.9 ± 1.16 ND 19.1 ± 0.49 22.9 ± 1.15 39.9 ± 2.07 34.1 ± 1.47 24.1 ± 2.19 14.3 ± 1.47 14.3 ± 2.88 ND 22.3 ± 1.47 16.8 ± 1.64 52.1 ± 2.19 54.7 ± 0.57

3 ± 0.57 4.4 ± 0.61 2 ± 0.57 2.2 ± 0.57 3.5 ± 0.2 3.4 ± 0.75 4.2 ± 0.41 2.4 ± 0.66 2.9 ± 0.05 2.6 ± 0.099 1.8 ± 0.05 2.4 ± 0.099 3 ± 0.5 3.3 ± 0.24 5.1 ± 0.60 2.3 ± 0.17 3.4 ± 0.11 3.2 ± 0.05 2.1 ± 0.099 2.2 ± 0.05 2.8 ± 0.099 6.2 ± 0.36 5.6 ± 0.11 7.9 ± 0.60

Kharnasi

Triveni

Nuagada

Atharabanki

IFFCO

Note: ND, not determined.

B.C. Behera et al.


Table 1.

7



Figure 3. (a) Heterotrophic bacteria. (b) Nitrogen-fixing bacteria. (c) Nitrifying bacteria. (d) Denitrifying bacteria. (e) Gram-negative bacteria. (f) Gram-poitive bacteria. (g) Sulfur-oxidising bacteria. (h) Spore-forming bacteria. (i) Anaerobic bacteria. (j) Phosphate-solubilising bacteria. (k) Cellulose-degrading bacteria.

and winter, but gradually increased towards summer (6.33–78 × 105 cfu/g soil) (Figure 3g). The spore-forming bacterial population followed a common trend which was greater in the summer season (5.33–60 × 105 cfu/g soil) and declined steadily towards the rainy season (Figure 3h). The population of anaerobic bacteria varied from 2 to 12 × 105 cfu/g soil, being less in the rainy season and increasing towards winter and summer (Figure 3i). The phosphate-solubilising

B.C. Behera et al.


8

Figure 3.

Continued

bacteria (1.00–7.00 × 105 cfu/g soils) reached a maximum during the rainy season (Figure 3j). The cellulose-degrading bacteria varied from 1 to 34 × 105 cfu/g soil) and showed a maximum value in the winter season (Figure 3k). The results of principal component (PCA) and correlation analysis (CA) of the heavy metal content with the microbial load (Table 2) and the physicochemical characteristics with the microbial load (Table 3) in soil samples from six different sites in the Mahanadi Delta during the four different seasons are presented. In this study, the PC between the heavy metal content and microbial load (Figure 4) showed seven factors or the PCs explain 76.409% of the total variance with a KMO Adequacy of 0.451 during four seasons. The results of PCA between the heavy metal

Copper Zinc Cobalt Nickel Iron Lead Cadmium HB GIB CDB SPB NFB NB DNB AB GPB SOB PSB

Correlations between heavy metal content and microbial load. Copper

Zinc

Cobalt

Nickel

Iron

Lead

Cadmium

HB

1.0 0.173 0.462a 0.566a 0.030 −0.006 −0.006 −0.017 −0.207 −0.330a 0.261b −0.109 0.150 −0.135 0.146 0.076 −0.372a −0.191

1.0 0.268b 0.042 0.349a 0.566a 0.566a 0.058 −0.419a −0.231b 0.015 −0.112 0.280b −0.249b −0.069 0.072 −0.320a −0.034

1.0 0.219 −0.066 −0.110 −0.110 0.001 0.301a −0.388a 0.071 −0.220 0.462a −0.006 0.029 −0.275b −0.446a 0.055

1.0 −0.103 −0.107 −0.107 −0.072 −0.207 −0.375a 0.536a −0.311a 0.056 0.046 0.053 −0.039 −0.265b 0.083

1.0 0.428a 0.428a −0.238b −0.271b 0.111 −0.196 −0.059 0.147 −0.052 0.357a 0.045 0.178 −0.087

1.0 1.000a −0.024 −0.263b −0.187 −0.146 −0.125 0.190 −0.086 0.103 0.053 0.110 −0.067

1.0 −0.024 −0.263b −0.187 −0.146 −0.125 0.190 −0.086 0.103 0.053 0.110 −0.067

1.0 0.071 −0.125 0.098 −0.030 0.220 0.028 −0.038 0.046 −0.252b 0.022

GIB

CDB

SPB

NFB

NB

DNB

AB

GPB

SOB

1.0 −0.114 1.0 −0.170 −0.338a 1.0 −0.283b 0.553a −0.135 1.0 0.235b −0.091 0.125 −0.128 1.0 0.135 0.162 −0.340a −0.033 0.121 1.0 0.025 0.077 −0.020 0.039 0.029 −0.278b 1.0 −0.015 0.158 −0.051 −0.132 −0.144 −0.207 0.031 1.0 0.083 0.413a −0.177 0.272b −0.229 −0.103 0.462a 0.115 1.0 0.041 0.250b −0.053 0.148 0.184 0.329a −0.310a −0.182 −0.262b

PSB



Table 2.

1.0

a

Notes: Correlation is significant at the 0.01 level (two-tailed). HB, heterotrophic bacteria; GIB, Gram-negative bacteria; CDB, cellulose-degrading bacteria; SPB, spore-forming bacteria; NFB, nitrogen-fixing bacteria; DNB, denitrifying bacteria; AB, anaerobic bacteria; GPB, Gram-positive bacteria; SOB, sulfur-oxidising bacteria; PSB, phosphate-solubilising bacteria. b Correlation is significant at the 0.05 level (two-tailed).

9

Correlations between microbial load and the physicochemical characteristics.

Parameter

Carbon

Carbon Potash Salinity pH Phosphorus Nitrogen HB GNB GPB CD SPB NB NFB DNB AB SOB PSB

1.0 0.300a 0.549b 0.184 −0.010 0.490b −0.104 0.393b −0.089 −0.058 0.097 −0.034 0.488b 0.018 −0.173 0.008 0.273a

a

Potash

Salinity

pH

1.0 0.017 1.0 0.090 0.260a 1.0 −0.152 0.150 0.230 0.401b 0.438b 0.462b −0.105 −0.019 −0.126 0.095 0.273a 0.060 0.095 0.066 −0.026 0.166 0.060 −0.221 −0.009 0.108 0.225 b 0.411 −0.257a −0.124 −0.018 0.242a −0.123 −0.360b 0.101 −0.148 0.189 −0.144 −0.040 0.231 0.465b 0.093 −0.136 0.374b −0.192

Phosphorus Nitrogen

1.0 0.223 −0.137 −0.351b −0.154 −0.190 0.371b −0.150 −0.079 0.133 0.092 0.103 0.085 b

1.0 −0.120 0.032 0.135 0.087 0.210 0.092 0.220 −0.106 0.187 0.396b −0.039

HB

GNB

GPB

CD

SPB

NB

NFB

DNB

AB

SOB

1.0 0.060 1.0 0.066 0.016 1.0 −0.125 −0.115 0.161 1.0 0.091 −0.188 −0.031 −0.340b 1.0 a 0.553b −0.139 1.0 −0.033 −0.291 −0.128 0.223 0.240a −0.152 −0.091 0.128 −0.128 1.0 0.018 0.119 −0.186 0.163 −0.357b −0.038 0.125 1.0 −0.030 0.039 0.012 0.077 −0.011 0.043 0.027 −0.270a 1.0 −0.242a 0.108 0.087 0.418b −0.165 0.281a −0.235a −0.086 0.455b 1.0 0.017 0.034 −0.176 0.251a −0.059 0.147 0.185 0.326b −0.307b −0.258a

PSB

B.C. Behera et al.


10

Table 3.

1.0

Notes: Correlation is significant at the 0.05 level (two-tailed). Correlation is significant at the 0.01 level (two-tailed CDB, cellulose-degrading bacteria; SPB, spore-forming bacteria; NFB, nitrogen-fixing bacteria; DNB, denitrifying bacteria; AB, anaerobic bacteria; GPB, Gram-positive bacteria; SOB, sulfur-oxidising bacteria; PSB, phosphate-solubilising bacteria.


11

Cadmium

0.8000

Lead Zinc Iron

0.4000 PC1

NB

CDB

AB

Copper

PSB HB

NFB

DNB

Cobalt

Nickel SPB

GIB

–0.4000

0.0000

0.4000

0.8000

PC2 Figure 4.

PCA of bacterial population with heavy metals.

CARBON

0.800

SALINITY NITROGEN

0.600 NITRIFIN

Pc1


–0.4000

GPB

SOB

0.0000

0.400

GNEGATIV

pH

PSOLUBIZ

POTASH

0.200

SPORE

SOXIDISI PHOSPHOR GPOSITIV

0.000 NITROFIX

–0.400

DENITRIF

CD

H.BACTER ANAEROBI

0.000

0.400

0.800

PC3 Figure 5.

PCA of bacterial population with soil nutrient content.

content and bacterial load revealed that with the increase in the concentrations of Cu, Ni and Co, there is decrease in the bacterial load of Gram-negative, heterotrophic, denitrifying, cellulosedegrading, nitrifying and sulfur-oxidising bacteria, indicating a negative impact or possibly toxic effect of the heavy metal on the bacterial communities inhabiting in the mangrove environment (Figure 4). Similarly, the PC between the physicochemical characteristics and microbial load (Figure 5) showed seven factors or the PCs explain 76.805% of the total variance with a KMO Adequacy of 0.539 during four different seasons. The overall results showed that in all seven PCs, there is positive correlation between the physicochemical characteristics (C, N, salinity and pH) and microbial load (Gram negative, sulfur oxidising, phosphate solubilising, nitrifying).

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4.

B.C. Behera et al.

Discussion


4.1. Soil physicochemical characteristics Since the soil quality concept encompasses not only productivity, but also environmental quality and land use, changes in soil with perturbations need to be fully described to assist in the rebuilding or maintenance of an ecosystem. Soil biological investigations give information on the the impact of environmental conditions on the metabolic activity of soil.[11] For the microbiological characterisation of soils contaminated with heavy metals, the evaluation of soil physicochemical properties is a useful parameter to assess the nutritional status of the soil. In the present study, a slightly acidic pH (Figure 2a) is observed in the mangrove soil. Similar sediment pH values have been reported in other mangrove swamps.[12–15] The acidic pH was partly due to active microbial decomposition of mangrove litter and hydrolysis of tannin in mangrove plants, which released various types of organic acids, and partly resulted from oxidation of FeS2 and FeS to H2 SO4 .[13] Unlike fresh water, where pH is the controlling factor, in estuarine water, salinity is the controlling factor for the bioavailability and the toxicity of the pollutant bound to sediment.[16] In the present investigation, maximum EC (Figure 2b) in summer also supports the previous findings of Essien et al. [17] who also observed similar EC values during summer in the intertidal mangrove soil of Qua Iboe Estuary of Nigeria. The high nitrogen content in winter (Figure 2c) supports the observation of Ashok Kumar et al. [18] who observed almost the same amount of nitrogen in the soil of the Muthupetai mangrove in Tamilnadu, India. In the present study, the high nitrogen content in the mangrove soil in winter may be due to the significant increase in oxidation of dead plant organic matter which has settled on the top layer.[19] Maximum phosphate content (Figure 2d) was observed at site-5 and site-6 because these two sampling sites are located on the bank of the Mahanadi River and are greatly affected by the continuous release of effluent from the nearby IFFCO and PPL fertiliser plants. In addition to the above, the high flush of water from upstream and agriculture run-off water during the rainy season are also responsible for the high P levels in the samples, which supports the previous findings of Sravanakumar et al.[19] There is significant variation in the potash content (Figure 2e) observed among the study sites, which supports Rammurthy et al.[20] Sodium and potassium content in the soil varied considerably among samples, particularly in nutrient level.[20] The organic carbon content (Figure 2f) is lower during the rainy season. This contradicts the findings of Martinez et al. [21] who observed a relatively higher carbon content during the rainy season, which is attributed due to replenishment by run-off water, but supports the finding of Routray et al. [22] and Senthilkumar et al. [23] who also observed the highest carbon peak in the summer and low organic carbon content in the rainy season in Bhitarkanika mangrove of Odisha and Pichavaram mangrove estuary near southeast coast of India, respectively. The higher organic carbon content during the dry season is due to the greater degradation of litter at higher temperature and low fresh water inflow into the mangrove forest.[23] In summer, the land becomes dry and there is an increase in soil temperature and salinity, resulting in a decrease in moisture content which hampers the flourishing of microbial populations, resulting in the accumulation of organic carbon in the soil.[22]

4.2.

Soil heavy metal

Heavy metal pollution is a serious environmental problem. The general distribution pattern, as well as the cycling of heavy metals in coastal areas, are largely controlled by both abiotic and biotic parameters. Hence, an investigation of heavy metals in the sediment is essential to assess the extent of any pollution. The concentration of Fe (14 810–63 370 μg/g) in the present study



13

is higher than previously found by Chakrapani and Subramanian [24] from sediment of the Mahanadi Basin of Odisha and also Ramanathan et al. [25] who observed 16 200–46 900 μg/g Fe from the sediments of the Sundarban mangroves of India. In the present study, the observed higher Fe concentration in the mangrove soil might be a result of the textural and mineralogical characteristics of the mangrove sediment.[25] The observed Cu content in this study is higher than the normal Cu content in soil. A lower Cu content (2–20 μg/g) was also reported by Chakrapani and Subramanian [24] from sediment of the Mahanadi Basin. We found that Ni is slightly lower than the permissible limit and also lower than that found by Ramanathan et al. [25] who reported 19–86 μg/g Ni from the Pichavaram mangrove ecosystem of Tamilnadu, India. The maximum permissible levels of Cd and Zn in soil as per the Indian Standard are 3–6 and 300–600 μg/g, respectively.[26] In this study, the level of Cd (7.9) is above the permissible limit, indicating the pollution status of the ecosystem, which is free of Zn contamination (132.5 μg/g). Among heavy metals, Cd, commonly associated with soil pollution, is also considered to be particularly toxic and is responsible for significant decreases in biological activity in soils.[27] The threshold value of Co in soil is 17 μg/g [28,29] and the observed value (48.2 μg/g) is much higher than the permissible limit, indicating that the soil of the Mahanadi Delta is polluted with Co and Cd. Estuaries receive inputs from multiple sources of organic and inorganic matter, such as pesticides and fertilisers derived from livestock and agricultural activities around the urban development through drainage basin in to the river and the intrusion of marine water from ocean during high tidal periods which contain multiple ionic as well as metallic sources, which may increase the Cd and Co content in the study sites. In addition to this, industrial effluents from the nearby Paradeep ports, IFFCO and PPL fertiliser industries transport a continuous supply of chemical contaminants into estuaries. Over time, these contaminants accumulate at significant concentrations in fine-bedded sediments. The permissible limit of Pb in soil as per Indian Standards is 250–500 μg/g, which is higher than the present finding and indicates that mangrove soil of Mahanadi River Delta is below the pollution limits for Pb. The present finding revealed that there is a marginal heavy metals pollution in the Mahanadi delta which supports the previous report of Chakrapani and Subramanian.[24] The observed heavy metals pollution in soil of the Mahanadi river basin could be attributed to weathering, leaching and uptake by vegetation, as reported by Ramanathan et al. [25] in Pichavaram mangrove. 4.3.

Soil bacterial population

Plate counts have been seen as a more appropriate method for determining the effect of heavy metals on soil bacteria than culture-independent approaches.[30] The diversity and activity of microbial communities are important indices of soil quality. Soil microbes play significant roles in the recycling of plant nutrients, the maintenance of soil structure, the detoxification of noxious chemicals and the control of plant pests and plant growth.[31] Alterations in the composition of microbial communities have often been proposed as an easy and sensitive indicator of anthropogenic effects on soil ecology.[32] Among the different groups of bacteria, heterotrophic bacteria play key role in the degradation and transformation of organic matter in the marine environment because they are the dominant group of bacteria. In the present investigation, the largest heterotrophic bacterial population (Figure 3a) was found during the rainy season, which may be due to the enrichment of nutrients in the soil. It has been also reported that an increase in water temperature negatively affected the abundance of culturable heterotrophic bacteria [33], which supports the results of the present investigation. Next to heterotrophic bacteria, N2 -fixing bacteria (Figure 3b) are efficient at using a variety of mangrove substrates despite differences in carbon content and phenol concentrations. However, their abundance may be dependent on physical conditions and mangrove community composition. Both symbiotic and asymbiotic N2 -fixing


14

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bacteria play a vital role in the nitrogen enrichment of mangrove ecosystems.[1] In the present study, a higher population of N2 -fixing bacteria was observed during the winter season. Routray et al. [22] also described higher populations of N2 -fixing bacteria in the winter season in Bhitarakanika mangrove, indicating that this was the most favourable period of biodegradation, as the litter fall during the spring is ready for accelerated transformation in winter and more influenced by suitable moisture and temperature. After nitrogen fixation, nitrification and denitrification are key process that complete the nitrogen cycle in mangrove. During the study, more nitrifying bacteria (Figure 3c) were observed in the rainy season than in summer, supporting the observation of Hansen et al. [34] who concluded that during the summer, the number and specific activity of nitrifying bacteria in coastal marine sediment diminished. This is probably due to heterotrophic bacteria and autotrophic algae, which are more successful in competing for ammonia during summer as they utilise this substrate more efficiently than the nitrifiers. In the rainy and winter seasons, this competition is reduced and the nitrifying bacteria are able to take up ammonia even at low concentrations.[35] Denitrification is a dissimilatory process in which oxidised nitrogen is used as an alternative electron acceptor for energy production when oxygen is limiting; it consists of four reaction steps in which nitrate is reduced to dinitrogen gas.[36] The occurrence of denitrifying bacteria in the mangrove rhizosphere is of interest because it may imply that loss of fixed nitrogen via denitrification and nitrogen is frequently a limiting nutrient in such systems.[37] In the present investigation, the maximum population of denitrifying bacteria (Figure 3d) in the winter season indicates that this is the most favourable period for the denitrification process in mangrove soil. The population of Gram-negative bacteria (Figure 3e) was greater than that of Gram-poitive bacteria (Figure 3f) at all the study sites throughout the study period because the cell walls of Gram-negative bacteria are better adapted to survival in a marine environment.[38] The summer is more favourable for sulfur oxidation and the winter for sulfur reduction,[39] which reflected in this study in which the population of sulfur-oxidising bacteria (Figure 3g) increased towards summer (Figure 3e). The larger population in summer probably resulted from mineralisation of soil organic sulfur under favourable moisture and temperature conditions and a lack of plant uptake. The smaller populations n winter and spring were probably due to leaching, plant uptake and low rates of mineralisation at low soil temperatures.[39] More nutrition is available during the rainy season and the environmental conditions are favourable for their growth and reproduction, hence, fewer spore-forming bacteria (Figure 3h) are observed in the rainy season than in the summer. Because of the increase in soil temperature and low inflow of fresh water in mangrove sediment during the summer, the land becomes dry, resulting in decreasing moisture and rate of oxygen penetration.[23,34] This anoxic condition favours the growth and activity of anaerobic bacteria. But in the rainy season, the mangrove sediment is inundated with water and the continuous water turbulence results in increases in the oxygen level and may decrease the anaerobic bacterial population. The maximum P-solubilising bacterial population occuring during the rainy season (Figure 3j) supports the previous finding of Ramanathan et al. [40] in Sundarban mangrove forest of India. In the present study, fewer cellulose-degrading bacteria (Figure 3k) in the soil of mangroves of the Mahanadi Delta contradicts the findings of Ramanathan et al. [40] who observed comparatively large numbers of cellulose-degrading bacteria from Sundarban mangroves of West Bengal, India. Relatively more nutrition in the rainy season would increase the microbial population, which was reflected in the increase in heterotrophic, phosphate-solubilising, nitrifying and Gram-negative bacteria in the Mahanadi Delta mangrove soil. Because soil quality is strongly influenced by microbe-mediated processes and function can be related to diversity, it is likely that microbial community structure will have the potential to serve as an early indication of soil degradation or soil improvement. Thus, analysis of microbial communities might provide data to elucidate the links between soil biotic and abiotic factors.



5.

15

Conclusions

The mangrove soils of the Mahanadi Delta are enriched with nutrients like N, P and K which vary with season and site. More nutrients in the mangrove soil favours the growth of heterotrophs, nitrogen fixers, nitrifiers and denitrifiers, indicating the key biological processes taking place in the partially anaerobic mangrove soil. Analysis of the soil samples revealed that the mangrove environment is marginally polluted with heavy metals like Cu, Cd, Zn and Fe, which vary among the different study sites. The results of PCA between heavy metal content and bacterial load revealed that with the increase in the concentration of Cu, Ni and Co, there is a decrease in the load of Gram-negative, heterotrophic, denitrifying, cellulose-degrading, nitrifying and sulfuroxidising bacteria, indicating that heavy metals may have a negative impact on or may be toxic for bacterial communities inhabiting in the mangrove environment. Baseline information of the soil physicochemical and heavy metal content, along with microbial characteristics, would be a useful tool for ecological assessment and monitoring the most sensitive and fragile coastal mangrove ecosystems for the development of future management strategies. Acknowledgements The authors are grateful to the authorities of North Orissa University for providing laboratory facilities to carry out this study. The help and cooperation of the field staff of forest department of Mangrove Forest Division, Rajnagar is gratefully acknowledged.

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