Scholarly article on topic 'Influence of 1,2-Dichlorobenzene on Microbial and Enzyme Activities in Wetland Soil'

Influence of 1,2-Dichlorobenzene on Microbial and Enzyme Activities in Wetland Soil Academic research paper on "Biological sciences"

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{"1 / 2-Dichlorobenzene" / "Microbial Community" / Urease / Catalase}

Abstract of research paper on Biological sciences, author of scientific article — Huixing Liang, Aihui Chen, Cheng Ding, Zhaoxia Li

Abstract The potential effect of 1,2-dichlorobenzen(1,2-DCB) on microorganisms and enzyme activities was investigated in a flooded wetland soil. The results showed that the number of actinomycetes declined significantly after the treatment of 1,2-DCB at different concentrations, and the amout of bacteria and fungi were significantly decreased in soil samples treated with higher than 200mg/Kg. 1,2-DCB at over the range of selected concentrations could completely inhibit urease activityin the first 28 days.1,2-DCB at the concentration lower than 100mg/Kg dried soil stimulated the activity of catalase, while the others showed an inhibition during period of incubation. Results indicated that these two enzymes are sensitive to 1,2-DCB contamination.

Academic research paper on topic "Influence of 1,2-Dichlorobenzene on Microbial and Enzyme Activities in Wetland Soil"

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Procedía

Environmental Sciences

ELSEVIER

Procedía Environmental Sciences 10 (2011) 122 - 127

2011 3rd International Conference on Environmental Science and Information Application Technology (ESIAT 2011)

Influence of 1,2-Dichlorobenzene of Microbial and Enzyme

Activities in Wetland Soil

Huixing Lianga'b, Aihui Chena, Cheng Dinga*, Zhaoxia Lia

aSchool of Environmental Science and Engineering, Yancheng Institute of Technology, Yancheng, China b College of Forest Resources and Environment Nanjing Forestry University Nanjing, China

Abstract

The potential effect of 1,2-dichlorobenzen(1,2-DCB) on microorganisms and enzyme activities was investigated in a flooded wetland soil. The results showed that the number of actinomycetes declined significantly after the treatment of 1,2-DCB at different concentrations, and the amout of bacteria and fungi were significantly decreased in soil samples treated with higher than 200 mg/Kg. 1,2-DCB at over the range of selected concentrations could completely inhibit urease activityin the first 28 days.1,2-DCB at the concentration lower than 100 mg/Kg dried soil stimulated the activity of catalase , while the others showed an inhibition during period of incubation. Results indicated that these two enzymes are sensitive to 1,2-DCB contamination.

© 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of Conference ES IAT2011 Organizat ion Committee.

Keyword: 1,2-Dichlorobenzene; Microbial Community; Urease; Catalase

1. Introduction

The functioning of modern society is highly dependent on artificial chlorinated organic chemicals, such as chlorobenzenes, which are produced in large quantities as chemical intermediates and in the manufacture of degreasers, solvents, and deodorants[1]. Their prolific use inevitably results in increasing quantities being released into the environment. Chlorobenzenes are chemically stable and resistant to processes such as photolysis, so their removal from the environment is dependent on the degradative activities of microbial communities. Because of the potential of microorganisms for the prevention and reversal of environmental contamination, their ability to catabolize substituted benezenes, such as 1,2-

* Corresponding author. Tel.: +86 0515 88298191 E-mail address:hx_liang1980@yahoo.com.cn ;dingc@163.com.

1878-0296 © 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of Conference ESIAT2011 Organization Committee. doi: 10.1016/j .proen v .2011.09.022

dichlorobenzene (1,2-DCB), has been extensively studied and documented[2,3]. However, most of these studies have investigated the interactions and degradative pathway of single organisms in flask-based liquid systems. If the potential of microbial communities to remove contaminants from soil, either naturally or as part of a wastedecontamination system, is to be realized and effectively exploited, more needs to be known about the ability of soil microbial communities to respond to a range of concentrations of common organic pollutants, such as 1,2-DCB. Furthermore, the response of soil microbial communities to pollution stress also deserves greater study, as their potential as rapid indicators of environmental contamination and ecosystem response is enormous.

Few studies of the impact of increasing concentrations of organic xenobiotics on soil microbial communities have been conducted. This is partly due to the complex nature of solidassociated toxicity measures, certainly when compared to liquid studies[4]. Furthermore, soil microbial communities are heterogenous, being composed of taxa with a range of sensitivities; because of this, their response to chemical stress cannot be expressed by a single dose-response relationship[5]. As microbial communities play a vital role in soil health and sustainability, it is essential to understand how they respond to contamination exposure. This has been more intensively investigated in the case of heavy metals, contamination by which has been shown to alter soil microbial community composition and metabolic potential.

The present paper aims to investigate the effect of 1,2-DCB on microbial populations and soil enzymes in wetland soil. It is helpful to understand the environmental toxicological effect of 1,2-DCB and enrich isolates and activated sludge capable of degrading 1,2-DCB to treat wastewater with it.

2. Materials And Methods

The soil was collected from the red-crowned national nature reserve of Sheyang county in Jiangsu province, China. We taked the soil near the reed root for the material. Fresh soil samples were collected from plant residues, and the soil was air-driedat room temperature and sieved (2 mm) sieve to remove plant debris,soil micro fauna and stones.

The experiments were conducted using 1, 2-DCB concentrations of 0, 100, 200, 400 and 800 mg/kg dried soil in soil. For each treatment, 1000 g dried soil were put into the same plastic pot. Then 1500 ml distilled water was added to submerge the soil. The flooded soil was incubated in the dark at 28 ± 1 °C. About 200 g of Soil samples were taken from each bucket ta time intervals (days 7, 14, 21, 28, 35 and 50) after treatment and incubation for microbal numeration and enzyme activity assays. Distilled water was added periodically to keep the soil flooded[6]. Soil samples that was not applied with 1, 2-DCB was used as a control(CK).

Total soil bacteria, actinomycetes and fungi were counted by a 10-fold serial dilution plate techbique. Compositions of the medium were as follows: (1) bacteria(g/L): beef extract 5.0, peptone 10.0, NaCl 5.0, distilled water 1000 mL, agar 18.0, pH 7.2-7.4. (2) actinomycetes:(g/L): soluble starch 10.0, (NH4)2SO4 2.0, K2HPO41.0, MgSO4.7H2O 1.0, NaCl 7.0, CaCO3 3.0, distilled water 1000 mL, agar 18.0, pH 7.2-7.4. (3) fungi (g/L): potato without peel 200.0, sucrose 20.0, distilled water 1000 mL, agar 18.0, pH natural. The amount of microbial was accounted by the logarithm of per gram in dry soil .

Soil urease and catalase activity were analyzed following the methods by Yan[7]and Min[6]. Briefly, 5 g of air-dried soil was mixed with 1.5 ml methylbenzene, 10 ml buffer and 5 ml substratesolution in a reaction flask and incubation at 37 °C for 24 hours. The following bufferand substrate solution were used: 0.1 mol/L citratebuffer with pH 6.7 and 10% (w/v) urea solution. After incubation, the culture solutions were brought to 100 ml with 37 °C distilled water, mixed carefully and filtered using Xinhua filter paper. One milliliter filtrate was added to the following reaction solutions composed of: 4 ml 0.1 mol/L phenolate and 3 ml 0.9% hypochlorite solution. The soil urease activities were expressed as mg NH3-

N.Catalase activity was measured by back-titrating residual H2O2 with KMnO4. Five grams of soil sampleswere added to 40 ml distilled water and 5 ml 0.3% hydrogen peroxide solution. The mixture was incubated at 37 °C for 20 min. After incubation, the reaction was stopped by adding 5 ml 1.5 mol/L sulfuric acid. The mixture was filtered and titrated using 0.1 mol/L KMnO4. Consumed amount of 0.1 mol/L KMnO4 per g dried soil was used to express the activity of catalase. Soil enzymes were expressed in realative value.

Values of microbal numeration and enzyme activity were averages of three replications and compared statistically by t-test 5% level with SPSS 12.0 software. Differences between values at P > 0.05 were considered not statistically significant.

3. Results

The effect of various concentration of 1, 2-DCB on soil bacteria , actinomycetes and fungi with the increasing incubation time are showed in Table1-3.

The table 1 showed that the various concentrations of 1, 2-DCB all decreased the amount of bacteria in soil. The first 14 days, The bacteria number in soil treated with 1, 2-DCB were lower than that of control soil samples. Through correlation analysis, the inhibition of the bacteria number and 1, 2-DCB concentrations were positively correlated in the 7th day and 14th day. The bacterial amount in soil treated with higher than 100mg/kg soil of 1, 2-DCB was significant different from that in the control(P<0.05) in the first 21 days. During the experiment, the amount of bacteria in soils treated with 800mg/kg soil was inhibited and significantly different (P<0.05) to that in control. The number of bacteria in soil samples treated with 200mg/kg rised to nearly the same level as that in the control after 28 days incubation.

Table 1. Effect of 1,2-dichlorobenzene on bacterial amounts in wetland soil

Incubation 1,2-dichlorobenzene (mg/kg dried soil)

(day) 0 100 200 400 800

7 7.335 a 7.325 a 7.204 b 7.127 c 7.108c

14 7.360 a 7.314 a 7.233 b 7. 153 b 7.036 c

21 7.498 a 7.502 a 7.267 b 7.181 b 7.193 b

28 7. 308 a 7.387 a 7.217 a 7.116 b 7.102 b

35 7.412 a 7.452 a 7.326 a 7.302 a 7.131b

50 7.358 a 7.383 a 7.289 a 7.261 a 7.063 b

Note:Values in the table is the means of logarithm of the microbial number; the letters in the same line mean the difference in different 1,2-DCB concentration are significant ( n = 3, P < 0. 05) , the same below

The table 2 showed that during the experiment, fungi number in soil samples treated with 400mg/kg and 800mg/kg soil 1,2-DCB was inhibited and significantly different(P<0.05) from that in control. The population of fungi in the soil samples treated with 100mg/kg and 200mg/kg was less than that in the contral, but there were no differences(P>0.05) between them. The number of fungi in the soil sample with a high-level of 1,2-DCB, however, was suppressed from the beginning to the end. When the concentration of 1, 2-DCB was lower than 400mg/kg soil, the fungi in soil had tolerance to some extent.

Table 2. Effect of 1,2-dichlorobenzene on fungi amounts in wetland soil

Incubation 1,2-dichlorobenzene (mg/kg dried soil)

(day) 0 100 200 400 800

7 3.403 a 3.332 a 3.361 a 3.069 b 3.126 b

14 3.392 a 3.214 a 3.338 a 3.018 b 3.118 b

21 3.487 a 3.384a 3.436 a 3.197 b 3.138 b

28 3.558 a 3.447 a 3.453 a 3.139 b 2.942 c

35 3.430 a 3.292 a 3.319 a 2.929 b 2.940 b

50 3.528 a 3.535 a 3.471 a 3.010 b 3.157 b

From table 3, we can see that the soil actinomycetes were very sensitive to 1, 2-DCB. The population of actinomycetes in soil samples treated with 1, 2-DCB was all inhibited, which was significantly different to wetland soils amended with non-1,2-DCB at the whole incubation stage. Wetland soil actinomycetes can be considered as an indicator of 1, 2-DCB pollution.

Table 3. Effect of 1,2-dichlorobenzene on actinomycetic amounts in wetland soil

Incubation 1,2-dichlorobenzene (mg/kg dried soil)

(day) 0 100 200 400 800

7 6.337 a 5.996 b 5.898 b 5.797 b 5.707 b

14 6.170 a 6.076 b 6.051 b 5.637 c 5.877 c

21 6.311 a 6.099 b 6.016 b 5.951c 5.854 c

28 6.394 a 6.111 b 6.057 b 5.841 c 5.700 c

35 6.467 a 6.196 b 6.117 b 5.986 c 5.951 c

50 6.581 a 6.373 b 6.192 b 5.623 c 5.757 c

Fig 1 showed that 1, 2-DCB mainly inhibited urease activity in the soil within the incubation stage. The extent of inhibition on urease activity ranged from 29% to 60% in this study after 7 days. 1, 2-DCB application and the inhibition due to 1,2-DCB was positive proportionate to the applied concentration, which the urease activities in soil s treated with 1,2-DCB were significantly different (P<0.05) from that of control. Then urease activities were followed by a recovery during the later period. No significant difference in urease activity was observed in soils treated with 100mg/kg soil and 200mg/kg after 28 days, and urease activity for treatment with 100mg/kg was even stimulated after 35 days. But urease activities treated with 400mg/kg soil and 800mg/kg was seriously inhibited until to 35 days, and were nearly the same level as that in the control after 50 days incubation.

Incubation time (day)

Fig. 1. Effeet of 1,2-dichlorobenzene on the activity of urease in wetland soil

The catalase activities(Fig. 2) of soils samples with 1,2-DCB concentration higher than 100mg/kg soil were inhibited from the beginning to the end of the experiment, however the 100mg/kg soil treatments had stimulated them. In the first 14 days, it was observed the higher the concentration of 1,2-DCB, the greater the inhibition detected on the catalase activities, and the enxyme activities declined in positive to the applied concentration of 1,2-DCB. The extent of inhibiton on catalase activity ranged from 14% to 49% within the initial 28 days, which there were significant differences(P<0.05) between treated soils and the control . Then the catalase activities were recovered nearly to the level of control.

Incubation time (day)

Fig. 2. Effeet of 1,2-dichlorobenzene on the activity of catalase in wetland soil

4. Discussion

The concentration of 1,2-DCB was an important factor affecting population of bacteria and fungi. The number of bacteria in soil samples treated by lower than 100mg/Kg had no difference to that in control, while the 1,2-DCB in soils higher than 200mg/kg had significant inhibited the growth of fungi during the experimen. The results demonstrated that significant difference in the actinomycetes between soils

supplemented with 1,2-DCB and non-1,2-DCB within the whole incubation stage. The present study pointed out that the majority of bacteria in environmental samples cannot be cultured by traditional cultivation techniques, which the culturability of bacteria from natural habiats ranged from 0.1% in seawater to 1%[8]. Some other menthods, such as phospholipid fatty acid analysis(PLFA), community level physiological profiling (CLPP) and PCR-based method(DGGE, RAPD, T-RFLP et al), would be valuable to further assess the influence of 1,2-DCB on the microbial community in the wetland soils.

In this study, 1,2-DCB caused obviously influence on urease activity, because there was significant difference(P<0.05) in enzymatic activity between the treated soil and the soil without 1,2-DCB(CK). The higher the 1, 2-DCB concentration applied ,the longer the time needed to recover to the level of control. Catalase can split hydrogen peroxide into molecular oxygen and water and keep cells from damage caused by reactive oxygen species[9]. There was no obvious influence on catalase activity after application of 1,2-DCB with 50mg/kg dried soil, while the other treatments could significant inhibite the catalase activity for 28 days and the higher the concentrations used, the stronger the effect on soil catalase.

Soil health and quality is a product of the efficiency of biogeochemical cycling processes, many of which are driven by microorganisms, soil health is in part a function of microbial community structure. Therefore, as illustrated here, the effect of pollution on soils needs to be monitored using ecologically relevant parameters.

Acknowledgements

This study was supported by Natural Science Fund of Jiangsu Province (No.BK2009171) and Yancheng Institute of Technology(XKY2010002 2009008).

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