- Email: [email protected]
Effects of lead and cadmium nitrate on biomass and substrate utilization pattern of soil microbial communities
Chemosphere 60 (2005) 508–514 www.elsevier.com/locate/chemosphere
Effects of lead and cadmium nitrate on biomass and substrate utilization pattern of soil microbial communities Muhammad Akmal, Xu Jianming *, Li Zhaojun, Wang Haizhen, Yao Huaiying Institute of Soil and Water Resources and Environmental Science, Zhejiang University, Hangzhou 310029, China Received 9 April 2004; received in revised form 18 August 2004; accepted 12 January 2005 Available online 17 February 2005
Abstract A study was conducted to evaluate the effects of different concentrations of lead (Pb) and cadmium (Cd) applied as their nitrates on soil microbial biomass carbon (Cmic) and nitrogen (Nmic), and substrate utilization pattern of soil microbial communities. The Cmic and Nmic contents were determined at 0, 14, 28, 42 and 56 days after heavy metal application (DAA). The results showed a significant decline in the Cmic for all Pb and Cd amended soils from the start to 28 DAA. From 28 to 56 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 declined significantly from 42 to 56 DAA. The Nmic contents also decreased significantly from start to 28 DAA for all other Pb and Cd treatments except for 200 mg kg 1 Pb which did not show significant difference from the control. Control and 200 mg kg 1 Pb had significantly lower soil microbial biomass C:N ratio as compared with other Pb treatments from 14 to 42 DAA, however at 56 DAA, only 1000 mg kg 1 Pb showed significantly higher C:N ratio compared with other treatments. No significant difference in C:N ratio for all Cd treated soils was seen from start to 28 DAA, however from 42 to 56 DAA, 100 mg kg 1 Pb showed significantly higher C:N ratio compared with other treatments. On 56 DAA, substrate utilization pattern of soil microbial communities was determined by inoculating Biolog ECO plates. The results indicated that Pb and Cd addition inhibited the functional activity of soil microbial communities as indicated by the intensity of average well color development (AWCD) during 168 h of incubation. Multivariate analysis of sole carbon source utilization pattern demonstrated that higher levels of heavy metal application had significantly affected soil microbial community structure. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Microbial biomass; Substrate utilization; Lead; Cadmium
1. Introduction 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 environ-
*
Corresponding author. Tel./fax: +86 571 86971955. E-mail address: [email protected] (J. Xu).
ment ecosystems (Knight et al., 1997; Giller et al., 1998). Soil is the primary supplier of heavy metals in atmosphere, hydrosphere and biota, and thus has a fundamental role in overall metal-cycling in nature. The natural concentrations of most heavy metals in soils vary widely and are mainly related to the soil parent materials. Anthropogenic sources such as smelters, mining, power stations, industry and the application of metal-containing pesticides, fertilizers, and metal-contaminated composts, and sewage sludges may contribute
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.01.001
M. Akmal et al. / Chemosphere 60 (2005) 508–514
to and at times exceed those from natural sources (McGrath et al., 1995). Several heavy metals are presently emitted in great quantities as a result of human activities. Their sudden releases, often in a biological available form, may damage or alter both natural and man-made ecosystems and subsequently menace human health. Heavy metals in soils above the ambient levels can adversely affect the surrounding ecosystem health. Microorganisms respond to excessive heavy metal levels in a variety of ways such as population loss (Knight et al., 1997), changes in population structure (Frostegard et al., 1993; Pennanen et al., 1996), and physiological activity (Valsecchi et al., 1995). Soil microorganisms play a vital role in maintaining soil productivity. Thus, any thing that disrupts these microorganisms and their functions in soil could be expected to affect the long term soil productivity and sustainability. Soil microbial processes, which play a significant role in nutrient cycling, have been found sensitive to increased heavy metal concentrations in the soils. The most serious aspect of the problem is that heavy metals persist in the soil and their negative effects are long lasting. Thus heavy metal contamination of soils could be a serious threat to long-term soil productivity—the main hindrance in the way of achieving the goal of sustainable ecosystem production. A number of soil microbiological parameters, notably microbial biomass carbon and basal respiration (Doran and Parkin, 1994; Sparling, 1997), have been suggested as possible indicators of soil environmental quality, and employed in the national and international monitoring programs (Yao et al., 2000). However, soil microbial community structure has also been recommended as a biological indicator of heavy metal stress (Pennanen et al., 1996; Yao et al., 2003). The Biolog ECO plates system uses 31 carbon substrates, 6 of which are not present in the GN plates; these substrates are predominantly amino acids, carbohydrates and carboxylic acids. The assay is based on measuring oxidative catabolism of the substrates to generate patterns of potential sole carbon source utilization. This technique is simple, uses an automated measuring apparatus and provides a more meaningful assay of community structure than isolate-based methods, because it measures utilization of carbon, the major factor that regulates microbial growth and community structure in soil (Knight et al., 1997; Baath et al., 1998). Toxicity of lead (Pb) and cadmium (Cd) has been widely reported in plants, animals, and human beings. In soil, Pb normally occurs in the range of 10–100 mg kg 1 and Cd below 1.0 mg kg 1 (Soon and Abboud, 1993), but in polluted soils especially near mining and smelting activities or sewage sludge treatment plants, Pb and Cd contents are even more than 1000 mg kg 1 and 100 mg kg 1, respectively (Peters and Shem, 1992; Fuge et al., 1993; Pichtel et al., 2000). The results of some long term field experiments have indicated nega-
509
tive effects of metals on microbial biomass and microbially-mediated processes in soils treated with metalcontaminated sludgeÕs in the past (McGrath et al., 1995; Baath et al., 1998). The effects were observed at surprisingly modest concentrations of heavy metals and the applied sludges contained mixtures of several metals. However, it is not well known which metal or groups of metals are producing these effects. Also some studies have shown the effects of these metals on microbial communities in soils near the smelters and mines (Kelly and Tate, 1998; Pichtel et al., 2000; Yao et al., 2003). These studies indicated that biomass and Biolog were useful methods for assessing long-term effects of heavy metals on soil community structure. At long-term field sites, soil microbial communities have had time to adapt to the stress presented by the elevated metal concentrations. Although comparison of metal-affected soil microbial communities and non-metal affected microbial communities at these sites can provide information on the changes that have occurred in the communities as a result of the metal contamination, such studies do not provide information on the time course of these changes. Hence, the information regarding the direct application of these heavy metals in inorganic form under better controlled, short-term experiments was required to assess the changes in soil microbial biomass and abilities of Biolog to detect the changes in the communitiesÕ structure following metal contamination. Accordingly, we selected different levels (from very low to significantly high) of Pb and Cd in nitrate forms to evaluate their individual effects on the changes in soil microbial biomass over time after their application; and to asses the pattern of substrate utilization of the soil microbial communities.
2. Materials and methods 2.1. Sampling and preparation of soil A bulk soil sample was collected at 0–15 cm depth from the research field of Hua Jia Chi campus of Zhejiang University, Hangzhou, China. The soil was brought to the laboratory, ground, passed through 2 mm sieve and mixed thoroughly. A subsample of the soil was taken, air-dried, mixed and analyzed for selected physico-chemical properties. The analyses showed that soil texture was silt loam with pH of 5.51and CEC of 11.3 cmol kg 1 soil. Soil total carbon was 19.3 g kg 1 and soil total nitrogen 1.6 g kg 1. Total background Pb and Cd contents in the soil were 28.5 and 0.045 mg kg 1, respectively. 2.2. Heavy metal addition The moist soil (equivalent to 150 g oven-dry weight) was transferred to 250 ml capacity glass beaker. Two
510
M. Akmal et al. / Chemosphere 60 (2005) 508–514
sets, each containing 90 beakers, were prepared to accommodate six levels of Pb and Cd with three replications for five times sampling. The soil samples were first adjusted to 40% of the soil water holding capacity (WHC) by adding distilled water and then pre-incubated at 25 °C for seven days (conditioning period). After conditioning, to one set of beakers Pb was applied as Pb(NO3)2 solution maintaining the concentrations of 0 (Ck), 200, 400, 600, 800 and 1000 mg kg 1 Pb. To the other set of beakers, Cd was added as solution of Cd(NO3)2 to obtain the concentration of 0 (Ck), 20, 40, 60, 80 and 100 mg kg 1 Cd. The moisture contents in the treated soil were adjusted to 50% of WHC and the soil samples were incubated at 25 °C for 56 days. The soil moisture was kept at the same level by adding distilled water at regular intervals throughout the incubation period. 2.3. Soil microbial biomass Eighteen beakers (either for Pb or Cd) were removed at 0, 14, 28, 42 and 56 days after heavy metal application (DAA) and analyzed for soil microbial biomass C (Cmic) and N (Nmic). The chloroform fumigation– extraction method was applied to measure Cmic and Nmic. Soil sample of 10 g (oven dry weight) was exposed to alcohol-free chloroform (CHCl3) vapor in a vacuum desiccator containing soda-lime at 25 °C for 24 h. The fumigated soil was transferred into empty desiccator and residual CHCl3 was removed from the fumigated soils by repeated evacuations. The fumigated soil was extracted immediately following CHCl3 removal by shaking for 30 min with 50 ml 0.5 mol l 1 K2SO4. The unfumigated 10 g soil (oven dry weight) was extracted at the time of fumigation commencement. Automatic analyzers were used to measure total organic carbon (Shimadzu, TOC-500) and nitrogen (Astoria, PacificInc.) in the samples. The data collected were adjusted to get the actual values of Cmic and Nmic (Anderson and Ingram, 1993). 2.4. Biolog ECO plate analyses Biolog ECO plates were used to study the substrate utilization pattern of soil microbial communities. Briefly, fresh soil (10 g) was added to 100 ml of distilled water in a 250-ml flask and was shaken at 200 rpm for 10 min. Ten-fold serial dilutions were made and 1000fold dilution was used to inoculate the Biolog ECO plates. Plates were incubated at 25 °C for seven days and color development was measured as absorbance (A) using an automated plate reader (VMAX, Molecular Devices, Crawley, UK) at 590 nm and the data were collected using Microlog 4.01 software (Biolog, Hayward, CA, USA).
2.5. Statistical analyses Soil microbial biomass carbon (Cmic), nitrogen (Nmic) and microbial biomass C:N ratio data were analyzed by ANOVA and means (n = 3) for different treatments were compared at 5% level of significance using DuncanÕs multiple test. The effects of different treatments were compared at specific as well as over different incubation periods (Gomez and Gomez, 1984). For Biolog data analysis, plates were read daily and average well color development (AWCD) over time for all C sources was calculated as a measure of total microbial activity. Means (n = 3) of AWCD for different treatments over time were compared at 5% level of significance to evaluate their effects (Garland, 1996; Campbell et al., 1997; Yao et al., 2000). For multivariate analysis of the Biolog data, the absorbance values were first transformed by dividing the AWCD to avoid bias among samples with different inoculumÕs density (Campbell et al., 1997; Garland, 1997) and then analyzed by principal component analysis technique.
3. Results 3.1. Microbial biomass C The dynamics of microbial biomass carbon (Cmic) affected by Pb(NO3)2 addition (Fig. 1a) showed a significant (P < 0.05) decline in the Cmic for all the levels of Pb from start to 28 days after heavy metal application (DAA). From 28 to 56 DAA, Cmic contents changed non-significantly for all other treatments except for 600 mg kg 1 Pb in which it declined significantly from 42 to 56 DAA. The soils amended with 800 and 1000 mg kg 1 Pb showed significantly (P < 0.05) lower Cmic contents compare with other treatments at all the stages of incubation. Up to 28 DAA, there was no statistical difference in Cmic contents among 200, 400 and 600 mg kg 1 Pb soils, but they were significantly lower in Cmic compared with the control. Until 56 DAA, the decreases in Cmic contents for 1000, 800, 600, 400 and 200 mg kg 1 Pb soils were 39.4%, 32.8%, 22.4%, 15.6% and 12.1%, respectively, compared to the initial contents. The soils treated with Cd(NO3)2 also showed a significant (P < 0.05) decline in Cmic for all the levels of Cd from start to 28 DAA (Fig. 1b). No significant change in Cmic was noted from 28 to 56 DAA for the lower levels of Cd but a significant decline was shown for 100 mg kg 1 Cd from 42 to 56 DAA. Up to 56 DAA, the decreases in Cmic contents were 46.7% and 35.7% for 80 and 100 mg kg 1 Cd treatments compared with their initial contents. 3.2. Microbial biomass nitrogen A significant decline in Nmic was reported from start to 28 DAA for all other Pb treatments except for
M. Akmal et al. / Chemosphere 60 (2005) 508–514 (a) Effect of Lead (Pb)
(b) Effect of Cadmium (Cd)
350 CK
300
200 mg/kg 400 mg/kg
250
600 mg/kg
200
800 mg/kg 1000 mg/kg
150
Cmic (mg kg-1)
Cmic (mg kg-1)
350
511
CK
300
20 mg/kg 40 mg/kg
250
60 mg/kg
200
80 mg/kg 100 mg/kg
150 100
100 0
14
28 42 Time (days)
56
0
14
28 Time (days)
42
56
Fig. 1. Effect of lead nitrate (a) and cadmium nitrate (b) on the dynamics of soil microbial biomass carbon (Cmic). The error bar is the standard error of the means (n = 3).
200 mg kg 1 Pb, which did not show significant difference from the control (Fig. 2a). From 28 to 56 DAA, 800 and 1000 mg kg 1 Pb soils again showed a significant (P < 0.05) decline in Nmic and up to 56 DAA more than 50% decrease in Nmic contents was noted for 800 and 1000 mg kg 1 Pb compared to initial contents. Although no significant difference in Nmic was shown between 400 and 600 mg kg 1 Pb from 28 to 56 DAA, but they were significantly lower in Nmic compared with the control. The data regarding the effect of Cd on the dynamics of Nmic (Fig. 2b) showed significantly (P < 0.05) lower in Nmic contents throughout the incubation period for all Cd treatments compared with the control. At 28 DAA, no significant difference in Nmic was noted between 40 and 60, and also between 80 and 100 mg kg 1 Cd treatments. The Nmic contents declined significantly from 42 to 56 DAA for 40, 60, 80 and 100 mg kg 1 Cd and until 56 DAA, these treatments had 55.3%, 61.1%, 66.4%, and 76.8% decreases in Nmic respectively, compared to the initial contents. 3.3. Microbial biomass C:N ratio The dynamics of soil microbial biomass C:N ratio affected by Pb addition is presented in Fig. 3a. The results showed no significant change in C:N ratio up to 14 DAA for all Pb treated soils, while from 14 to 42 DAA, control and 200 mg kg 1 Pb were significantly (P < 0.05) 60
lower in C:N ratio compared to other treatments. However, at 56 DAA only 1000 mg kg 1 Pb showed significantly higher in C:N ratio compared with other soils, which were non-significant with each other. Effect of Cd addition on the C:N ratio (Fig. 3b) indicated that there was no significant difference in C:N from start up to 28 DAA for all treatments, however from 42 to 56 DAA, control had significantly lower while 100 mg kg 1 Cd was significantly higher in C:N ratio compared with other treatments. At 56 DAA, the highest C:N ratio (13.3) was reported for 100 mg kg 1 Cd which was significantly (P < 0.05) higher than in all other treatments. 3.4. Soil microbial community structure The Biolog data were analyzed in two ways. First, the rate of color intensity on the Biolog plates over time was determined by calculating the average well color development (AWCD) on each plate at each reading time (Garland, 1996) and then Biolog profiles for different treatments were compared by principal component analysis (PCA). The effect of heavy metal addition on the activity of soil microbial communities (Fig. 4a and b) showed decrease in AWCD with the increasing levels of heavy metal, but the differences in AWCD for various Pb and Cd treatments were not significant up to 120 h of incubation. After 120 h of incubation, 1000 mg kg 1 Pb
(a) Effect of Lead (Pb)
(b) Effect of Cadmium (Cd)
60 CK
40
200 mg/kg
30
400 mg/kg
20
600 mg/kg 800 mg/kg
10
1000 mg/kg
Nmic (mg kg-1)
Nmic (mg kg-1)
50 50
CK
40
20 mg/kg
30
40 mg/kg 60 mg/kg
20
80 mg/kg
10
100 mg/kg
0
0 0
14
28 42 Time (days)
56
0
14
28 Time (days)
42
56
Fig. 2. Effect of lead nitrate (a) and cadmium nitrate (b) on the dynamics of soil microbial biomass nitrogen (Nmic). The error bar is the standard error of the means (n = 3).
512
M. Akmal et al. / Chemosphere 60 (2005) 508–514 14
(a) Effect of Lead (Pb) CK
10
200 mg/kg
8
400 mg/kg
6
600 mg/kg
4
800 mg/kg 1000 mg/kg
2
Microbial biomas C:N
Microbial biomas C:N
12
(b) Effect of Cadmium (Cd)
12
CK
10
20 mg/kg
8
40 mg/kg 60 mg/kg
6
80 mg/kg
4
100 mg/kg
2 0
0 0
14
28 42 Time (days)
56
0
14
28 42 Time (days)
56
Fig. 3. Effect of lead nitrate (a) and cadmium nitrate (b) on the dynamics of soil microbial biomass C:N ratio. The error bar is the standard error of the means (n = 3).
0.7
(a) Effect of Lead (Pb)
0.7 CK
0.5
200 mg/kg
0.4
400 mg/kg
0.3
600 mg/kg
0.2
800 mg/kg 1000 mg/kg
0.1
AWCD 590 nm
AWCD 590 nm
0.6
(b) Effect of Cadmium (Cd)
0.6
CK
0.5
20 mg/kg
0.4
40 mg/kg
0.3
60 mg/kg
0.2
80 mg/kg 100 mg/kg
0.1 0
0 24
48
72 96 120 144 168 Time (hours)
24
48
72 96 120 144 168 Time (hours)
Fig. 4. Effect of lead nitrate (a) and cadmium nitrate (b) on the functional activity of soil microbial communities as indicated by average well color development (AWCD) at 590 nm. The error bar is the standard error of the means (n = 3).
and 100 mg kg 1 Cd showed significantly (P < 0.5) lower in AWCD compared with other treatments. Principal Component analysis using all 31 carbon sources, revealed a separation of soil samples, indicating the different patterns of potential C utilization and different microbial communities. The scores of the PC1 appeared to reflect the metal addition level, indicating that there was a change in carbon substrate utilization pattern associated with the different heavy metal contents. The PCA graphs (Fig. 5a and b) demonstrated the variability in the metabolic profiles affected by Pb and Cd addition. Correlation and analysis of the loadings of the most influential C sources on the PC1 indicated that microbial communities of Pb-amended soils had increased utilization of L-phenylalanine, D-mannitol, D-malic acid and aketobutyric acid and the lower utilization of tween 40, pyruvic acid methyl-ester, hyrdoxy butyric acid and Itaconic acid. However, the results of Cd treated soils showed an increased utilization of L-asparagine, L-phenylalanine, pyruvic acid methyl ester acid and L-threonine while the lower utilization of N-acetyl-D-glucosamine, D-galacturonic acid, a-cyclodextrin and D-xylose.
4. Discussion Pollution of agricultural soils by heavy metals from industrial and agricultural activities is a major environ-
mental concern. Heavy metal pollution cannot only result in adverse effects on various parameters relating to plant quality and yield, but also cause changes in the size, composition and activity of soil microbial communities (Giller et al., 1998). Abiotic stress caused by heavy metals, in inorganic and organic forms affects the growth, morphology and metabolism of soil microorganisms. In the present study, the dynamics of microbial biomass carbon (Cmic) and nitrogen (Nmic) affected by Pb and Cd addition showed a significant decline in Cmic and Nmic from start to 28 days after heavy metal application (DAA). The dynamics of Cmic 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 28 DAA. The differences in Cmic and Nmic among the treatments were caused by the different concentration of heavy metal added to the soil, which inhibited the growth of soil microorganisms. Higher levels of Cd (80 and 100 mg kg 1) and Pb (800 and 1000 mg kg 1) produced greater inhibitions of Cmic and Nmic than the lower levels of these metals. Also, a comparatively greater reduction of Cmic and Nmic with Cd than with Pb amended soils was seen in the present experiment which might be due to the higher solubility
M. Akmal et al. / Chemosphere 60 (2005) 508–514 (a) Effect of Lead (Pb)
(b) Effect of Cadmium (Cd) 10
10 CK
5
CK
5
20 mg/kg
200 mg/kg 400 mg/kg
0
600 mg/kg 800 mg/kg
-5
PC2
PC2
513
40 mg/kg
0
60 mg/kg 80 mg/kg
-5
100 mg/kg
1000 mg/kg -10 -10
-5
0 PC1
5
10
-10 -10
-5
0 PC1
5
10
Fig. 5. Plot of ordination of principal component (PC); PC1 against PC2 generated by Principal Component analysis showing the effect of lead nitrate (a) and cadmium nitrate (b) pollution on the pattern of substrate utilization by soil microbial communities at 96 h of incubation. The error bar is the standard error of the means (n = 3).
and more direct toxicity of Cd than Pb to soil microorganisms. Evidence for reduced soil microbial biomass under the heavy metal stress condition was also due to the additional energy cost to soil microorganisms (Cervantes, 1994). Such an additional energy cost can result in a decrease in the amount of substrate that is available for growth. For example, a decrease in the growth yield in response to metal stress was shown in a chemo-stat study using the marine bacterium Vibrio alginolyticus and the difference in the cellular energy budget was thought to be directed toward physiological processes required for detoxification mechanism (Gordon et al., 1993). The suggestion that soil microorganisms under stress divert energy from growth to cell maintenance functions may therefore be the possible explanation for the decreased in biomass for metal contaminated soils. The microbially-mediated nutrients cycling and their availability are significantly controlled by soil microbial biomass C:N ratio. Therefore, the microbial biomass C:N ratio is also an indicator of the effects of heavy metals on the functioning of a soil ecosystem (McGrath et al., 1995). Several investigators have reported that heavy metal stress can induce changes in the microbial biomass C:N ratio (McGrath et al., 1995; Huang and Khan, 1998; Khan et al., 1998). In our study, an increase in the microbial biomass C:N ratio was observed from the start to the end of incubation with Pb and Cd treated soils, which was due to the decline in the size of soil microbial community and a reduction of C mineralization in these soils. The direct toxic effects of higher levels of these heavy metals on soil microbial biomass are the main reason for significant increase in the C:N ratio in this study. Some studies have also shown an increase in the fungal population proportion as compared to bacteria in heavy metal amended soil due to the fact that fungi tend to be more resistant to heavy metals than bacteria (Hiroki, 1992; Kelly et al., 1999). Hence, this change in fungal to bacterial populations may also be a possible reason of changes in the C:N ratio for heavy metal treatment.
The analysis of Biolog data in the present study depicted marked differences in the activity of soil microbial communities among the heavy metal loadings. Average well color development (AWCD) seems to mainly reflect the species metabolic activity and the ability of the bacterial community to respond to the substrates. The reduction of the microbial activity as shown by AWCD for heavy metal treated soils was mainly due to the reduction in the numbers and species diversity of the biota. In this study, AWCD values in the Biolog plates were closely related to the soil microbial biomass or heavy metal concentrations, as the lower AWCD values occurred for the higher metal levels. It is contrary to the findings of Yao et al. (2003), which did not show relation of AWCD values with the microbial biomass or heavy metal concentrations. It appears that AWCD also provides some information about the differences in soil microbial biomass along with the community structure. Due to high concentrations of C sources in the Biolog plates, some bacterial species which can use these C sources will grow and reproduce quickly and will change the original community structure. Kelly and Tate (1998) found that elevated metal loadings resulted in changes in the structure of soil microbial communities, as indicated by changes in their metabolic profiles. Further, Knight et al. (1997) has reported that both the metal concentration and reduced pH values had significant effects on the Biolog pattern of soil microbial communities. Our results also indicated a change in the C substrate utilization pattern associated with the heavy metal contents. However, control and lower levels of heavy metals applied did not differ much from each other, possibly suggesting that there was a threshold level for a pollution effect on the substrate utilization pattern. So it was shown that the threshold level for Pb and Cd toxicity to soil microbiota in the present study was between 600– 800 mg kg 1 and 40–60 mg kg 1 respectively, which could be useful information in evaluating soil quality and ecosystems sustainability.
514
M. Akmal et al. / Chemosphere 60 (2005) 508–514
Acknowledgement The research was supported by the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of China; and National Key Project for Science and Technology of China (No. 2001BA804A25).
References Anderson, J.M., Ingram, J.S.I., 1993. Chemical analysis. In: Anderson, J.M., Ingram, J.S.I. (Eds.), Tropical Soil Biology and Fertility: a Hand Book of Methods. CAB International, UK, pp. 62–87. Baath, E., Diaz-Ravina, M., Frostegard, A., Campbell, C.D., 1998. Effect of metal-rich sludge amendments on the soil microbial community. Appl. Environ. Microbiol. 64, 238– 245. Campbell, C.D., Grayston, S.J., Hirst, D.J., 1997. Use of rhizosphere carbon sources in sole carbon source tests to discriminate soil microbial communities. J. Microbiol. Meth. 30, 33–41. Cervantes, C., 1994. Copper resistance mechanisms in bacteria and fungi. FEMS Microbiol. Rev. 14, 121–138. Doran, J.W., Parkin, T.B., 1994. Defining and assessing soil quality. In: Doran, J.W., Coleman, D.C., Bezdicek, D.F., Stewart, B.A. (Eds.), Defining Soil Quality for a Sustainable Environment. Soil Science Society of America, Madison, pp. 3–21. Frostegard, A., Tunlid, A., Baath, E., 1993. Phospholipid fatty acid composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals. Appl. Environ. Microbiol. 59, 3605– 3617. Fuge, R., Pearce, F.M., Pearce, N.J.G., Perkins, W.T., 1993. Geochemistry of Cd in the secondary environment near abandoned metalliferous mines, Wales. Appl. Geochem. (Suppl.) 2, 29–35. Garland, J.L., 1996. Analytical approaches to the characterization of samples of microbial communities using patterns of potential C source utilization. Soil Biol. Biochem. 28, 213–221. Garland, J.L., 1997. Analysis and interpretation of communitylevel physiological profiles in microbial ecology. FEMS Microbiol. Ecol. 24, 289–300. Giller, K.E., Witter, E., McGrath, S.P., 1998. Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: a review. Soil Biol. Biochem. 30, 1389– 1414. Gomez, A.K., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research. John Wiley and Sons, New York, USA. Gordon, A.S., Harwood, V.J., Sayyar, S., 1993. Growth, copper-tolerant cells, and extracellular protein production
in copper stressed cultures of Vibrio alginolyticus. Appl. Environ. Microbiol. 59, 60–66. Hiroki, M., 1992. Effects of heavy metal contamination on soil microbial population. Soil Sci. Plant Nutri. 38, 141–147. Huang, C.Y., Khan, K.S., 1998. Effects of cadmium, lead and their interaction on the size of microbial biomass in a red soil. Soil Environ. 1, 227–236. Kelly, J.J., Tate, R.L., 1998. Effects of heavy metal contamination and remediation on soil microbial communities in the vicinity of a zinc smelter. J. Environ. Qual. 27, 609– 617. Kelly, J.J., Haggblom, R.L., Tate, R.L., 1999. Changes in soils microbial communities over time resulting from one time application of zinc: a laboratory microcosm study. Soil Biol. Biochem. 31, 1455–1465. Khan, K.S., Xie, Z.M., Huang, C.Y., 1998. Relative toxicity of lead chloride and lead acetate to microbial biomass in red soil of China. J. Appl. Environ. Biol. 4, 179–184. Knight, B., McGrath, S.P., Chaudri, A.M., 1997. Biomass carbon measurements and substrate utilization patterns of microbial populations from soils amended with cadmium, copper, or zinc. Appl. Environ. Microbiol. 63, 39–43. McGrath, S.P., Chaudri, A.M., Giller, K.E., 1995. Long-term effects of metals in sewage sludges on soils, microorganisms and plants. J. Ind. Microbiol. 14, 94–104. Pennanen, T., Frostegard, A., Fritze, H., Baath, E., 1996. Phospholipid fatty acid composition and heavy metal tolerance of soil microbial communities along two heavy metal-polluted gradients in coniferous forests. Appl. Environ. Microbiol. 62, 420–428. Peters, R.W., Shem, L., 1992. Adsorption/desorption characteristics of lead on various types of soil. Environ. Prog. 11, 234–240. Pichtel, J., Kuroiwa, K., Sawyerr, H.T., 2000. Distribution of Pb, Cd and Ba in soils and plants of two contaminated sites. Environ. Pollut. 110, 171–178. Soon, Y.K., Abboud, S., 1993. Lead, chromium, and nickel. In: Carter, M.R. (Ed.), Soil Sampling and Methods of Analysis. Lewis, Boca Raton, pp. 101–108. Sparling, G.P., 1997. Soil microbial biomass, activity and nutrient cycling as indicators of soil health. In: Pankhurst, C.E., Doube, B.M., Gupta, V.V.S.R. (Eds.), Biological Indicators of Soil Health. CAB International, Wallingford, UK, pp. 97–119. Valsecchi, G., Gugliotti, C., Farini, A., 1995. Microbial biomass, activity, and organic matter accumulation in soils contaminated with heavy metals. Biol. Fertil. Soils 20, 253–259. Yao, H., He, Z., Huang, C., Campbell, C.D., 2000. Microbial biomass and community structure in a sequence of soils with increasing fertility and changing land use. Microb. Ecol. 40, 223–237. Yao, H., Xu, J., Huang, C., 2003. Substrate utilization pattern, biomass and activity of microbial communities in a sequence of heavy metal-polluted paddy soils. Geoderma 115, 139–148.
Effects of lead and cadmium nitrate on biomass and substrate utilization pattern of soil microbial communities Muhammad Akmal, Xu Jianming *, Li Zhaojun, Wang Haizhen, Yao Huaiying Institute of Soil and Water Resources and Environmental Science, Zhejiang University, Hangzhou 310029, China Received 9 April 2004; received in revised form 18 August 2004; accepted 12 January 2005 Available online 17 February 2005
Abstract A study was conducted to evaluate the effects of different concentrations of lead (Pb) and cadmium (Cd) applied as their nitrates on soil microbial biomass carbon (Cmic) and nitrogen (Nmic), and substrate utilization pattern of soil microbial communities. The Cmic and Nmic contents were determined at 0, 14, 28, 42 and 56 days after heavy metal application (DAA). The results showed a significant decline in the Cmic for all Pb and Cd amended soils from the start to 28 DAA. From 28 to 56 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 declined significantly from 42 to 56 DAA. The Nmic contents also decreased significantly from start to 28 DAA for all other Pb and Cd treatments except for 200 mg kg 1 Pb which did not show significant difference from the control. Control and 200 mg kg 1 Pb had significantly lower soil microbial biomass C:N ratio as compared with other Pb treatments from 14 to 42 DAA, however at 56 DAA, only 1000 mg kg 1 Pb showed significantly higher C:N ratio compared with other treatments. No significant difference in C:N ratio for all Cd treated soils was seen from start to 28 DAA, however from 42 to 56 DAA, 100 mg kg 1 Pb showed significantly higher C:N ratio compared with other treatments. On 56 DAA, substrate utilization pattern of soil microbial communities was determined by inoculating Biolog ECO plates. The results indicated that Pb and Cd addition inhibited the functional activity of soil microbial communities as indicated by the intensity of average well color development (AWCD) during 168 h of incubation. Multivariate analysis of sole carbon source utilization pattern demonstrated that higher levels of heavy metal application had significantly affected soil microbial community structure. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Microbial biomass; Substrate utilization; Lead; Cadmium
1. Introduction 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 environ-
*
Corresponding author. Tel./fax: +86 571 86971955. E-mail address: [email protected] (J. Xu).
ment ecosystems (Knight et al., 1997; Giller et al., 1998). Soil is the primary supplier of heavy metals in atmosphere, hydrosphere and biota, and thus has a fundamental role in overall metal-cycling in nature. The natural concentrations of most heavy metals in soils vary widely and are mainly related to the soil parent materials. Anthropogenic sources such as smelters, mining, power stations, industry and the application of metal-containing pesticides, fertilizers, and metal-contaminated composts, and sewage sludges may contribute
0045-6535/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2005.01.001
M. Akmal et al. / Chemosphere 60 (2005) 508–514
to and at times exceed those from natural sources (McGrath et al., 1995). Several heavy metals are presently emitted in great quantities as a result of human activities. Their sudden releases, often in a biological available form, may damage or alter both natural and man-made ecosystems and subsequently menace human health. Heavy metals in soils above the ambient levels can adversely affect the surrounding ecosystem health. Microorganisms respond to excessive heavy metal levels in a variety of ways such as population loss (Knight et al., 1997), changes in population structure (Frostegard et al., 1993; Pennanen et al., 1996), and physiological activity (Valsecchi et al., 1995). Soil microorganisms play a vital role in maintaining soil productivity. Thus, any thing that disrupts these microorganisms and their functions in soil could be expected to affect the long term soil productivity and sustainability. Soil microbial processes, which play a significant role in nutrient cycling, have been found sensitive to increased heavy metal concentrations in the soils. The most serious aspect of the problem is that heavy metals persist in the soil and their negative effects are long lasting. Thus heavy metal contamination of soils could be a serious threat to long-term soil productivity—the main hindrance in the way of achieving the goal of sustainable ecosystem production. A number of soil microbiological parameters, notably microbial biomass carbon and basal respiration (Doran and Parkin, 1994; Sparling, 1997), have been suggested as possible indicators of soil environmental quality, and employed in the national and international monitoring programs (Yao et al., 2000). However, soil microbial community structure has also been recommended as a biological indicator of heavy metal stress (Pennanen et al., 1996; Yao et al., 2003). The Biolog ECO plates system uses 31 carbon substrates, 6 of which are not present in the GN plates; these substrates are predominantly amino acids, carbohydrates and carboxylic acids. The assay is based on measuring oxidative catabolism of the substrates to generate patterns of potential sole carbon source utilization. This technique is simple, uses an automated measuring apparatus and provides a more meaningful assay of community structure than isolate-based methods, because it measures utilization of carbon, the major factor that regulates microbial growth and community structure in soil (Knight et al., 1997; Baath et al., 1998). Toxicity of lead (Pb) and cadmium (Cd) has been widely reported in plants, animals, and human beings. In soil, Pb normally occurs in the range of 10–100 mg kg 1 and Cd below 1.0 mg kg 1 (Soon and Abboud, 1993), but in polluted soils especially near mining and smelting activities or sewage sludge treatment plants, Pb and Cd contents are even more than 1000 mg kg 1 and 100 mg kg 1, respectively (Peters and Shem, 1992; Fuge et al., 1993; Pichtel et al., 2000). The results of some long term field experiments have indicated nega-
509
tive effects of metals on microbial biomass and microbially-mediated processes in soils treated with metalcontaminated sludgeÕs in the past (McGrath et al., 1995; Baath et al., 1998). The effects were observed at surprisingly modest concentrations of heavy metals and the applied sludges contained mixtures of several metals. However, it is not well known which metal or groups of metals are producing these effects. Also some studies have shown the effects of these metals on microbial communities in soils near the smelters and mines (Kelly and Tate, 1998; Pichtel et al., 2000; Yao et al., 2003). These studies indicated that biomass and Biolog were useful methods for assessing long-term effects of heavy metals on soil community structure. At long-term field sites, soil microbial communities have had time to adapt to the stress presented by the elevated metal concentrations. Although comparison of metal-affected soil microbial communities and non-metal affected microbial communities at these sites can provide information on the changes that have occurred in the communities as a result of the metal contamination, such studies do not provide information on the time course of these changes. Hence, the information regarding the direct application of these heavy metals in inorganic form under better controlled, short-term experiments was required to assess the changes in soil microbial biomass and abilities of Biolog to detect the changes in the communitiesÕ structure following metal contamination. Accordingly, we selected different levels (from very low to significantly high) of Pb and Cd in nitrate forms to evaluate their individual effects on the changes in soil microbial biomass over time after their application; and to asses the pattern of substrate utilization of the soil microbial communities.
2. Materials and methods 2.1. Sampling and preparation of soil A bulk soil sample was collected at 0–15 cm depth from the research field of Hua Jia Chi campus of Zhejiang University, Hangzhou, China. The soil was brought to the laboratory, ground, passed through 2 mm sieve and mixed thoroughly. A subsample of the soil was taken, air-dried, mixed and analyzed for selected physico-chemical properties. The analyses showed that soil texture was silt loam with pH of 5.51and CEC of 11.3 cmol kg 1 soil. Soil total carbon was 19.3 g kg 1 and soil total nitrogen 1.6 g kg 1. Total background Pb and Cd contents in the soil were 28.5 and 0.045 mg kg 1, respectively. 2.2. Heavy metal addition The moist soil (equivalent to 150 g oven-dry weight) was transferred to 250 ml capacity glass beaker. Two
510
M. Akmal et al. / Chemosphere 60 (2005) 508–514
sets, each containing 90 beakers, were prepared to accommodate six levels of Pb and Cd with three replications for five times sampling. The soil samples were first adjusted to 40% of the soil water holding capacity (WHC) by adding distilled water and then pre-incubated at 25 °C for seven days (conditioning period). After conditioning, to one set of beakers Pb was applied as Pb(NO3)2 solution maintaining the concentrations of 0 (Ck), 200, 400, 600, 800 and 1000 mg kg 1 Pb. To the other set of beakers, Cd was added as solution of Cd(NO3)2 to obtain the concentration of 0 (Ck), 20, 40, 60, 80 and 100 mg kg 1 Cd. The moisture contents in the treated soil were adjusted to 50% of WHC and the soil samples were incubated at 25 °C for 56 days. The soil moisture was kept at the same level by adding distilled water at regular intervals throughout the incubation period. 2.3. Soil microbial biomass Eighteen beakers (either for Pb or Cd) were removed at 0, 14, 28, 42 and 56 days after heavy metal application (DAA) and analyzed for soil microbial biomass C (Cmic) and N (Nmic). The chloroform fumigation– extraction method was applied to measure Cmic and Nmic. Soil sample of 10 g (oven dry weight) was exposed to alcohol-free chloroform (CHCl3) vapor in a vacuum desiccator containing soda-lime at 25 °C for 24 h. The fumigated soil was transferred into empty desiccator and residual CHCl3 was removed from the fumigated soils by repeated evacuations. The fumigated soil was extracted immediately following CHCl3 removal by shaking for 30 min with 50 ml 0.5 mol l 1 K2SO4. The unfumigated 10 g soil (oven dry weight) was extracted at the time of fumigation commencement. Automatic analyzers were used to measure total organic carbon (Shimadzu, TOC-500) and nitrogen (Astoria, PacificInc.) in the samples. The data collected were adjusted to get the actual values of Cmic and Nmic (Anderson and Ingram, 1993). 2.4. Biolog ECO plate analyses Biolog ECO plates were used to study the substrate utilization pattern of soil microbial communities. Briefly, fresh soil (10 g) was added to 100 ml of distilled water in a 250-ml flask and was shaken at 200 rpm for 10 min. Ten-fold serial dilutions were made and 1000fold dilution was used to inoculate the Biolog ECO plates. Plates were incubated at 25 °C for seven days and color development was measured as absorbance (A) using an automated plate reader (VMAX, Molecular Devices, Crawley, UK) at 590 nm and the data were collected using Microlog 4.01 software (Biolog, Hayward, CA, USA).
2.5. Statistical analyses Soil microbial biomass carbon (Cmic), nitrogen (Nmic) and microbial biomass C:N ratio data were analyzed by ANOVA and means (n = 3) for different treatments were compared at 5% level of significance using DuncanÕs multiple test. The effects of different treatments were compared at specific as well as over different incubation periods (Gomez and Gomez, 1984). For Biolog data analysis, plates were read daily and average well color development (AWCD) over time for all C sources was calculated as a measure of total microbial activity. Means (n = 3) of AWCD for different treatments over time were compared at 5% level of significance to evaluate their effects (Garland, 1996; Campbell et al., 1997; Yao et al., 2000). For multivariate analysis of the Biolog data, the absorbance values were first transformed by dividing the AWCD to avoid bias among samples with different inoculumÕs density (Campbell et al., 1997; Garland, 1997) and then analyzed by principal component analysis technique.
3. Results 3.1. Microbial biomass C The dynamics of microbial biomass carbon (Cmic) affected by Pb(NO3)2 addition (Fig. 1a) showed a significant (P < 0.05) decline in the Cmic for all the levels of Pb from start to 28 days after heavy metal application (DAA). From 28 to 56 DAA, Cmic contents changed non-significantly for all other treatments except for 600 mg kg 1 Pb in which it declined significantly from 42 to 56 DAA. The soils amended with 800 and 1000 mg kg 1 Pb showed significantly (P < 0.05) lower Cmic contents compare with other treatments at all the stages of incubation. Up to 28 DAA, there was no statistical difference in Cmic contents among 200, 400 and 600 mg kg 1 Pb soils, but they were significantly lower in Cmic compared with the control. Until 56 DAA, the decreases in Cmic contents for 1000, 800, 600, 400 and 200 mg kg 1 Pb soils were 39.4%, 32.8%, 22.4%, 15.6% and 12.1%, respectively, compared to the initial contents. The soils treated with Cd(NO3)2 also showed a significant (P < 0.05) decline in Cmic for all the levels of Cd from start to 28 DAA (Fig. 1b). No significant change in Cmic was noted from 28 to 56 DAA for the lower levels of Cd but a significant decline was shown for 100 mg kg 1 Cd from 42 to 56 DAA. Up to 56 DAA, the decreases in Cmic contents were 46.7% and 35.7% for 80 and 100 mg kg 1 Cd treatments compared with their initial contents. 3.2. Microbial biomass nitrogen A significant decline in Nmic was reported from start to 28 DAA for all other Pb treatments except for
M. Akmal et al. / Chemosphere 60 (2005) 508–514 (a) Effect of Lead (Pb)
(b) Effect of Cadmium (Cd)
350 CK
300
200 mg/kg 400 mg/kg
250
600 mg/kg
200
800 mg/kg 1000 mg/kg
150
Cmic (mg kg-1)
Cmic (mg kg-1)
350
511
CK
300
20 mg/kg 40 mg/kg
250
60 mg/kg
200
80 mg/kg 100 mg/kg
150 100
100 0
14
28 42 Time (days)
56
0
14
28 Time (days)
42
56
Fig. 1. Effect of lead nitrate (a) and cadmium nitrate (b) on the dynamics of soil microbial biomass carbon (Cmic). The error bar is the standard error of the means (n = 3).
200 mg kg 1 Pb, which did not show significant difference from the control (Fig. 2a). From 28 to 56 DAA, 800 and 1000 mg kg 1 Pb soils again showed a significant (P < 0.05) decline in Nmic and up to 56 DAA more than 50% decrease in Nmic contents was noted for 800 and 1000 mg kg 1 Pb compared to initial contents. Although no significant difference in Nmic was shown between 400 and 600 mg kg 1 Pb from 28 to 56 DAA, but they were significantly lower in Nmic compared with the control. The data regarding the effect of Cd on the dynamics of Nmic (Fig. 2b) showed significantly (P < 0.05) lower in Nmic contents throughout the incubation period for all Cd treatments compared with the control. At 28 DAA, no significant difference in Nmic was noted between 40 and 60, and also between 80 and 100 mg kg 1 Cd treatments. The Nmic contents declined significantly from 42 to 56 DAA for 40, 60, 80 and 100 mg kg 1 Cd and until 56 DAA, these treatments had 55.3%, 61.1%, 66.4%, and 76.8% decreases in Nmic respectively, compared to the initial contents. 3.3. Microbial biomass C:N ratio The dynamics of soil microbial biomass C:N ratio affected by Pb addition is presented in Fig. 3a. The results showed no significant change in C:N ratio up to 14 DAA for all Pb treated soils, while from 14 to 42 DAA, control and 200 mg kg 1 Pb were significantly (P < 0.05) 60
lower in C:N ratio compared to other treatments. However, at 56 DAA only 1000 mg kg 1 Pb showed significantly higher in C:N ratio compared with other soils, which were non-significant with each other. Effect of Cd addition on the C:N ratio (Fig. 3b) indicated that there was no significant difference in C:N from start up to 28 DAA for all treatments, however from 42 to 56 DAA, control had significantly lower while 100 mg kg 1 Cd was significantly higher in C:N ratio compared with other treatments. At 56 DAA, the highest C:N ratio (13.3) was reported for 100 mg kg 1 Cd which was significantly (P < 0.05) higher than in all other treatments. 3.4. Soil microbial community structure The Biolog data were analyzed in two ways. First, the rate of color intensity on the Biolog plates over time was determined by calculating the average well color development (AWCD) on each plate at each reading time (Garland, 1996) and then Biolog profiles for different treatments were compared by principal component analysis (PCA). The effect of heavy metal addition on the activity of soil microbial communities (Fig. 4a and b) showed decrease in AWCD with the increasing levels of heavy metal, but the differences in AWCD for various Pb and Cd treatments were not significant up to 120 h of incubation. After 120 h of incubation, 1000 mg kg 1 Pb
(a) Effect of Lead (Pb)
(b) Effect of Cadmium (Cd)
60 CK
40
200 mg/kg
30
400 mg/kg
20
600 mg/kg 800 mg/kg
10
1000 mg/kg
Nmic (mg kg-1)
Nmic (mg kg-1)
50 50
CK
40
20 mg/kg
30
40 mg/kg 60 mg/kg
20
80 mg/kg
10
100 mg/kg
0
0 0
14
28 42 Time (days)
56
0
14
28 Time (days)
42
56
Fig. 2. Effect of lead nitrate (a) and cadmium nitrate (b) on the dynamics of soil microbial biomass nitrogen (Nmic). The error bar is the standard error of the means (n = 3).
512
M. Akmal et al. / Chemosphere 60 (2005) 508–514 14
(a) Effect of Lead (Pb) CK
10
200 mg/kg
8
400 mg/kg
6
600 mg/kg
4
800 mg/kg 1000 mg/kg
2
Microbial biomas C:N
Microbial biomas C:N
12
(b) Effect of Cadmium (Cd)
12
CK
10
20 mg/kg
8
40 mg/kg 60 mg/kg
6
80 mg/kg
4
100 mg/kg
2 0
0 0
14
28 42 Time (days)
56
0
14
28 42 Time (days)
56
Fig. 3. Effect of lead nitrate (a) and cadmium nitrate (b) on the dynamics of soil microbial biomass C:N ratio. The error bar is the standard error of the means (n = 3).
0.7
(a) Effect of Lead (Pb)
0.7 CK
0.5
200 mg/kg
0.4
400 mg/kg
0.3
600 mg/kg
0.2
800 mg/kg 1000 mg/kg
0.1
AWCD 590 nm
AWCD 590 nm
0.6
(b) Effect of Cadmium (Cd)
0.6
CK
0.5
20 mg/kg
0.4
40 mg/kg
0.3
60 mg/kg
0.2
80 mg/kg 100 mg/kg
0.1 0
0 24
48
72 96 120 144 168 Time (hours)
24
48
72 96 120 144 168 Time (hours)
Fig. 4. Effect of lead nitrate (a) and cadmium nitrate (b) on the functional activity of soil microbial communities as indicated by average well color development (AWCD) at 590 nm. The error bar is the standard error of the means (n = 3).
and 100 mg kg 1 Cd showed significantly (P < 0.5) lower in AWCD compared with other treatments. Principal Component analysis using all 31 carbon sources, revealed a separation of soil samples, indicating the different patterns of potential C utilization and different microbial communities. The scores of the PC1 appeared to reflect the metal addition level, indicating that there was a change in carbon substrate utilization pattern associated with the different heavy metal contents. The PCA graphs (Fig. 5a and b) demonstrated the variability in the metabolic profiles affected by Pb and Cd addition. Correlation and analysis of the loadings of the most influential C sources on the PC1 indicated that microbial communities of Pb-amended soils had increased utilization of L-phenylalanine, D-mannitol, D-malic acid and aketobutyric acid and the lower utilization of tween 40, pyruvic acid methyl-ester, hyrdoxy butyric acid and Itaconic acid. However, the results of Cd treated soils showed an increased utilization of L-asparagine, L-phenylalanine, pyruvic acid methyl ester acid and L-threonine while the lower utilization of N-acetyl-D-glucosamine, D-galacturonic acid, a-cyclodextrin and D-xylose.
4. Discussion Pollution of agricultural soils by heavy metals from industrial and agricultural activities is a major environ-
mental concern. Heavy metal pollution cannot only result in adverse effects on various parameters relating to plant quality and yield, but also cause changes in the size, composition and activity of soil microbial communities (Giller et al., 1998). Abiotic stress caused by heavy metals, in inorganic and organic forms affects the growth, morphology and metabolism of soil microorganisms. In the present study, the dynamics of microbial biomass carbon (Cmic) and nitrogen (Nmic) affected by Pb and Cd addition showed a significant decline in Cmic and Nmic from start to 28 days after heavy metal application (DAA). The dynamics of Cmic 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 28 DAA. The differences in Cmic and Nmic among the treatments were caused by the different concentration of heavy metal added to the soil, which inhibited the growth of soil microorganisms. Higher levels of Cd (80 and 100 mg kg 1) and Pb (800 and 1000 mg kg 1) produced greater inhibitions of Cmic and Nmic than the lower levels of these metals. Also, a comparatively greater reduction of Cmic and Nmic with Cd than with Pb amended soils was seen in the present experiment which might be due to the higher solubility
M. Akmal et al. / Chemosphere 60 (2005) 508–514 (a) Effect of Lead (Pb)
(b) Effect of Cadmium (Cd) 10
10 CK
5
CK
5
20 mg/kg
200 mg/kg 400 mg/kg
0
600 mg/kg 800 mg/kg
-5
PC2
PC2
513
40 mg/kg
0
60 mg/kg 80 mg/kg
-5
100 mg/kg
1000 mg/kg -10 -10
-5
0 PC1
5
10
-10 -10
-5
0 PC1
5
10
Fig. 5. Plot of ordination of principal component (PC); PC1 against PC2 generated by Principal Component analysis showing the effect of lead nitrate (a) and cadmium nitrate (b) pollution on the pattern of substrate utilization by soil microbial communities at 96 h of incubation. The error bar is the standard error of the means (n = 3).
and more direct toxicity of Cd than Pb to soil microorganisms. Evidence for reduced soil microbial biomass under the heavy metal stress condition was also due to the additional energy cost to soil microorganisms (Cervantes, 1994). Such an additional energy cost can result in a decrease in the amount of substrate that is available for growth. For example, a decrease in the growth yield in response to metal stress was shown in a chemo-stat study using the marine bacterium Vibrio alginolyticus and the difference in the cellular energy budget was thought to be directed toward physiological processes required for detoxification mechanism (Gordon et al., 1993). The suggestion that soil microorganisms under stress divert energy from growth to cell maintenance functions may therefore be the possible explanation for the decreased in biomass for metal contaminated soils. The microbially-mediated nutrients cycling and their availability are significantly controlled by soil microbial biomass C:N ratio. Therefore, the microbial biomass C:N ratio is also an indicator of the effects of heavy metals on the functioning of a soil ecosystem (McGrath et al., 1995). Several investigators have reported that heavy metal stress can induce changes in the microbial biomass C:N ratio (McGrath et al., 1995; Huang and Khan, 1998; Khan et al., 1998). In our study, an increase in the microbial biomass C:N ratio was observed from the start to the end of incubation with Pb and Cd treated soils, which was due to the decline in the size of soil microbial community and a reduction of C mineralization in these soils. The direct toxic effects of higher levels of these heavy metals on soil microbial biomass are the main reason for significant increase in the C:N ratio in this study. Some studies have also shown an increase in the fungal population proportion as compared to bacteria in heavy metal amended soil due to the fact that fungi tend to be more resistant to heavy metals than bacteria (Hiroki, 1992; Kelly et al., 1999). Hence, this change in fungal to bacterial populations may also be a possible reason of changes in the C:N ratio for heavy metal treatment.
The analysis of Biolog data in the present study depicted marked differences in the activity of soil microbial communities among the heavy metal loadings. Average well color development (AWCD) seems to mainly reflect the species metabolic activity and the ability of the bacterial community to respond to the substrates. The reduction of the microbial activity as shown by AWCD for heavy metal treated soils was mainly due to the reduction in the numbers and species diversity of the biota. In this study, AWCD values in the Biolog plates were closely related to the soil microbial biomass or heavy metal concentrations, as the lower AWCD values occurred for the higher metal levels. It is contrary to the findings of Yao et al. (2003), which did not show relation of AWCD values with the microbial biomass or heavy metal concentrations. It appears that AWCD also provides some information about the differences in soil microbial biomass along with the community structure. Due to high concentrations of C sources in the Biolog plates, some bacterial species which can use these C sources will grow and reproduce quickly and will change the original community structure. Kelly and Tate (1998) found that elevated metal loadings resulted in changes in the structure of soil microbial communities, as indicated by changes in their metabolic profiles. Further, Knight et al. (1997) has reported that both the metal concentration and reduced pH values had significant effects on the Biolog pattern of soil microbial communities. Our results also indicated a change in the C substrate utilization pattern associated with the heavy metal contents. However, control and lower levels of heavy metals applied did not differ much from each other, possibly suggesting that there was a threshold level for a pollution effect on the substrate utilization pattern. So it was shown that the threshold level for Pb and Cd toxicity to soil microbiota in the present study was between 600– 800 mg kg 1 and 40–60 mg kg 1 respectively, which could be useful information in evaluating soil quality and ecosystems sustainability.
514
M. Akmal et al. / Chemosphere 60 (2005) 508–514
Acknowledgement The research was supported by the Teaching and Research Award Program for Outstanding Young Teachers in Higher Education Institutions of China; and National Key Project for Science and Technology of China (No. 2001BA804A25).
References Anderson, J.M., Ingram, J.S.I., 1993. Chemical analysis. In: Anderson, J.M., Ingram, J.S.I. (Eds.), Tropical Soil Biology and Fertility: a Hand Book of Methods. CAB International, UK, pp. 62–87. Baath, E., Diaz-Ravina, M., Frostegard, A., Campbell, C.D., 1998. Effect of metal-rich sludge amendments on the soil microbial community. Appl. Environ. Microbiol. 64, 238– 245. Campbell, C.D., Grayston, S.J., Hirst, D.J., 1997. Use of rhizosphere carbon sources in sole carbon source tests to discriminate soil microbial communities. J. Microbiol. Meth. 30, 33–41. Cervantes, C., 1994. Copper resistance mechanisms in bacteria and fungi. FEMS Microbiol. Rev. 14, 121–138. Doran, J.W., Parkin, T.B., 1994. Defining and assessing soil quality. In: Doran, J.W., Coleman, D.C., Bezdicek, D.F., Stewart, B.A. (Eds.), Defining Soil Quality for a Sustainable Environment. Soil Science Society of America, Madison, pp. 3–21. Frostegard, A., Tunlid, A., Baath, E., 1993. Phospholipid fatty acid composition, biomass, and activity of microbial communities from two soil types experimentally exposed to different heavy metals. Appl. Environ. Microbiol. 59, 3605– 3617. Fuge, R., Pearce, F.M., Pearce, N.J.G., Perkins, W.T., 1993. Geochemistry of Cd in the secondary environment near abandoned metalliferous mines, Wales. Appl. Geochem. (Suppl.) 2, 29–35. Garland, J.L., 1996. Analytical approaches to the characterization of samples of microbial communities using patterns of potential C source utilization. Soil Biol. Biochem. 28, 213–221. Garland, J.L., 1997. Analysis and interpretation of communitylevel physiological profiles in microbial ecology. FEMS Microbiol. Ecol. 24, 289–300. Giller, K.E., Witter, E., McGrath, S.P., 1998. Toxicity of heavy metals to microorganisms and microbial processes in agricultural soils: a review. Soil Biol. Biochem. 30, 1389– 1414. Gomez, A.K., Gomez, A.A., 1984. Statistical Procedures for Agricultural Research. John Wiley and Sons, New York, USA. Gordon, A.S., Harwood, V.J., Sayyar, S., 1993. Growth, copper-tolerant cells, and extracellular protein production
in copper stressed cultures of Vibrio alginolyticus. Appl. Environ. Microbiol. 59, 60–66. Hiroki, M., 1992. Effects of heavy metal contamination on soil microbial population. Soil Sci. Plant Nutri. 38, 141–147. Huang, C.Y., Khan, K.S., 1998. Effects of cadmium, lead and their interaction on the size of microbial biomass in a red soil. Soil Environ. 1, 227–236. Kelly, J.J., Tate, R.L., 1998. Effects of heavy metal contamination and remediation on soil microbial communities in the vicinity of a zinc smelter. J. Environ. Qual. 27, 609– 617. Kelly, J.J., Haggblom, R.L., Tate, R.L., 1999. Changes in soils microbial communities over time resulting from one time application of zinc: a laboratory microcosm study. Soil Biol. Biochem. 31, 1455–1465. Khan, K.S., Xie, Z.M., Huang, C.Y., 1998. Relative toxicity of lead chloride and lead acetate to microbial biomass in red soil of China. J. Appl. Environ. Biol. 4, 179–184. Knight, B., McGrath, S.P., Chaudri, A.M., 1997. Biomass carbon measurements and substrate utilization patterns of microbial populations from soils amended with cadmium, copper, or zinc. Appl. Environ. Microbiol. 63, 39–43. McGrath, S.P., Chaudri, A.M., Giller, K.E., 1995. Long-term effects of metals in sewage sludges on soils, microorganisms and plants. J. Ind. Microbiol. 14, 94–104. Pennanen, T., Frostegard, A., Fritze, H., Baath, E., 1996. Phospholipid fatty acid composition and heavy metal tolerance of soil microbial communities along two heavy metal-polluted gradients in coniferous forests. Appl. Environ. Microbiol. 62, 420–428. Peters, R.W., Shem, L., 1992. Adsorption/desorption characteristics of lead on various types of soil. Environ. Prog. 11, 234–240. Pichtel, J., Kuroiwa, K., Sawyerr, H.T., 2000. Distribution of Pb, Cd and Ba in soils and plants of two contaminated sites. Environ. Pollut. 110, 171–178. Soon, Y.K., Abboud, S., 1993. Lead, chromium, and nickel. In: Carter, M.R. (Ed.), Soil Sampling and Methods of Analysis. Lewis, Boca Raton, pp. 101–108. Sparling, G.P., 1997. Soil microbial biomass, activity and nutrient cycling as indicators of soil health. In: Pankhurst, C.E., Doube, B.M., Gupta, V.V.S.R. (Eds.), Biological Indicators of Soil Health. CAB International, Wallingford, UK, pp. 97–119. Valsecchi, G., Gugliotti, C., Farini, A., 1995. Microbial biomass, activity, and organic matter accumulation in soils contaminated with heavy metals. Biol. Fertil. Soils 20, 253–259. Yao, H., He, Z., Huang, C., Campbell, C.D., 2000. Microbial biomass and community structure in a sequence of soils with increasing fertility and changing land use. Microb. Ecol. 40, 223–237. Yao, H., Xu, J., Huang, C., 2003. Substrate utilization pattern, biomass and activity of microbial communities in a sequence of heavy metal-polluted paddy soils. Geoderma 115, 139–148.