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Published online 11 May 2005
Published in J Environ Qual 34:1073-1080 (2005)
DOI: 10.2134/jeq2004.0438
© 2005 American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America
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TECHNICAL REPORTS

Organic Compounds in the Environment

Effect of Petroleum-Containing Wastewater Irrigation on Bacterial Diversities and Enzymatic Activities in a Paddy Soil Irrigation Area

H. Lia,b, Y. Zhanga, C. G. Zhanga and G. X. Chena,c,*

a Key Laboratory of Terrestrial Ecological Process, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China
b Graduate School of the Chinese Academy of Sciences, Beijing 10039, China
c Shenyang Key Laboratory of Environmental Engineering, Shenyang University, Shenyang 110044, China

* Corresponding author (gxchen{at}iae.ac.cn)

Received for publication November 17, 2004.

   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Effects of petroleum contamination on bacterial diversities and enzymatic activities in paddy soils were investigated in the Shenfu irrigation area, the largest area irrigated by oil-containing wastewater for more than 50 yr in northeastern China. Bacterial diversities were determined by conventional colony morphology typing techniques and 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Dehydrogenase, hydrogen peroxidase, polyphenol oxidase, urease, and substrate-induced respiration (SIR) were measured to evaluate the effects of petroleum-containing wastewater irrigation on soil biochemical characteristics. Results showed that paddy soil total petroleum hydrocarbon (TPH) concentration in the irrigation area varied from 277.11 to 5213.37 mg kg–1 dry soil. Soil TPH concentration declined along the gradient of the irrigation channel from up- to downstream. At the current pollution level, the paddy soil TPH concentration was positively correlated with the colony forming units (CFU) of aerobic heterotrophic bacteria (AHB) (r = 0.928, p < 0.001) and the genetic diversity based on DGGE profiles (r = 0.655, p < 0.05). The bacterial diversities in the soils based on colony morphotypes of AHB also increased with TPH concentration (r = 0.598), but not significant statistically (p = 0.052). Analysis of soil enzyme activities indicated a significant positive correlation between soil TPH concentration and activities of dehydrogenases (r = 0.974, p < 0.001), hydrogen peroxidases (r = 0.957, p < 0.001), polyphenol oxidases (r = 0.886, p < 0.001), and SIR (r = 0.916, p < 0.001). On the contrary, the urease activity showed a negative correlation with paddy soil TPH concentration (r = –0.814, p = 0.002), and could be used as a sensitive indicator of petroleum contamination.

Abbreviations: AHB, aerobic heterotrophic bacteria • CFU, colony forming units • DGGE, denaturing gradient gel electrophoresis • PAH, polycyclic aromatic hydrocarbon • PCR, polymerase chain reaction • SIR, substrate-induced respiration • TPH, total petroleum hydrocarbon


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
WASTEWATER PLAYS an important role in suburban agriculture irrigation in different parts of the world, especially in countries that are short of water, since it contains nutrients that can be used by crops (Mark, 2004). Major wastewater irrigation areas in China are distributed in suburban areas of the northern part of the country (Liu and Xu, 2002). The Shenfu irrigation area, located between the cities of Shenyang and Fushun in Liaoning province, is the largest petroleum-containing wastewater irrigation area in China. The wastewater mainly comes from an oil refinery in Fushun. The up- and mid-stream areas of the Shenfu irrigation area have been irrigated by wastewater since 1940s. A 70-km irrigation channel was constructed in 1960, and then the irrigation area was extended to 10000 ha (Liu et al., 1981). Soils in the irrigation area have been seriously contaminated by the petroleum-containing wastewater (Wu et al., 1985).

There is a growing interest in the changes in microbial community structure and diversity as a response to environmental stress (Macnaughton et al., 1999; Raeid et al., 2002). The changes in microbial diversity may result in the changes in soil function (Lawton, 1994). Petroleum hydrocarbons are the main pollutants in the Shenfu irrigation area. Some components of petroleum hydrocarbon have been identified as genotoxicants in a short-term mutagenicity test such as an Ames test, and as animal carcinogens in a long-term carcinogenicity test (Dibble et al., 1990; Verschueren, 1983). The effects of petroleum hydrocarbons, especially polycyclic aromatic hydrocarbons (PAHs), on soil bacterial diversities have been reported recently (Macnaughton et al., 1999; Raeid et al., 2002; Steven et al., 2003). Little information is available on the effects of petroleum wastewater irrigation on cropped paddy soils. Before the development of molecular techniques for estimating genetic diversity, studies on microbial community structure and diversity were restricted to cultivation-based methods, covering only the aerobic heterotrophic fraction of the total bacterial population capable of forming colonies on a solid media. Such an obvious limitation commonly causes underestimation of the population diversity present in the natural environment (Torsvik et al., 1990; Amann et al., 1995). In this study, culture-independent molecular techniques were used, including PCR amplification of 16S rDNA genes and DGGE. The DGGE technique has the potential needed for community monitoring since it analyzes the major constituents of microbial communities by generating fingerprints (Muyzer et al., 1993). Similar to other molecular approaches, PCR-DGGE allows the detection of both culturable and unculturable microorganisms and eliminates the problem of selectivity during culturing. The conventional colony morphology typing technique and the PCR-DGGE profiling technique will be used simultaneously in this study to access the community diversities of the soil bacteria in the Shenfu irrigation area.

Soil enzymatic activities mainly originate from soil microorganisms. Soil enzymes participate in many biological processes in soils and offer a useful assessment of soil "function." It can be taken as one of the indicators of soil health (Dick, 1997; Killham and Staddon, 2002). The impacts of heavy metals (Kandeler et al., 1996) and pesticides (Gianfreda et al., 1994) on soil enzymes have been previously reported, but the effects of petroleum hydrocarbons on soil enzymes have been scarcely studied. Oxidoreductases, such as the widely studied dehydrogenase, hydrogen peroxidase, and polyphenol oxidase, catalyze a wide range of oxidation–reduction reactions in soils. Urease was chosen because it plays a key role in the N cycle, transforming urea to ammonium (Dick, 1992; Gianfreda et al., 1994). Soil respiration is one of the most common measurements of microbial mineralization and is taken as another important soil function (Prosser, 1997). The immediate respiration of a microbial community following a glucose addition is quantified in a manner avoiding a significant contribution of cell multiplication (Anderson and Domsch, 1978). In this research, dehydrogenase, hydrogen peroxidase, polyphenol oxidase, urease, and SIR were chosen as the parameters to evaluate the effects of petroleum-containing wastewater irrigation on soil biochemical characteristics.

Both microbial diversities and soil enzymatic activities were related to the soil function. The effects of long-term petroleum-containing wastewater irrigation on soil microbial diversities and soil enzymatic activities in the Shenfu irrigation area have been scarcely studied. To understand if soil functions are affected by long-term petroleum wastewater irrigation, soil bacterial diversities and enzymatic activities in the Shenfu irrigation area were evaluated in this study.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Site Description and Soil Sampling
Soil samples were collected from the paddy fields in the Shenfu wastewater irrigation area that is located in the suburban area between the cities of Fushun and Shenyang, Liaoning province, China. Soil samples were collected in July 2003 when the paddy fields were flooded by petroleum-containing wastewater for nearly 3 mo. Thirteen sampling sites differing in their relative position and irrigation history were selected. In the following text the 13 sites are referred as Sites A to M (Fig. 1) . Topsoil (0–20 cm) from each site was collected using a stainless steel auger. The auger was washed by water and 70% ethanol between two sampling sites. Five individual soil cores from each site were combined for analysis. The soil samples were placed in airtight bags and stored on ice before being transported to the laboratory. Subsequently, all samples were kept at 4°C until analysis.



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  		Fig. 1. Distribution and irrigated history of soil sampling sites in the Shenfu irrigation area. Site A is located at the upstream area and was changed to ground water irrigation more than 30 yr ago. Site B is located at the upstream area and was changed to upland 30 yr ago. Site C is located at the third plot of the branch channel in the upstream area and has been irrigated by wastewater. Site D is located at the sixth plot of the branch channel in the upstream area and has been irrigated by wastewater. Site E is located at the ninth plot of the branch channel in the upstream area and has been irrigated by wastewater. Site F is located at the upper reach of the mid-stream area and has been irrigated by wastewater. Site G is located at the middle reach of the mid-stream area and has been irrigated by wastewater. Site H is located at the lower reach of the mid-stream area and has been irrigated by wastewater. Site I is located at the upper reach of the downstream area and was changed to ground water irrigation 10 yr ago. Site J is located at the middle reach of the downstream area and has been irrigated by wastewater. Site K is located at the lower reach of the downstream area and was changed to upland 10 yr ago. Site L is located at the lower reach of the downstream area and was changed to ground water irrigation 10 yr ago. Site M is located at the end of the channel and was changed to ground water irrigation 20 yr ago.

 
Physical and chemical properties of the studied soils are presented in Table 1. Soil moisture contents were determined by drying the soils at 110°C for 48 h. Particle size analyses were performed by a modified hydrometer method, in which the clay content was determined after 8 h (Day, 1965). Soil pH was determined in a 1:5 ratio of soil to water. Organic carbon content was determined by the Walkley–Black method using FeSO4 for titration (Nelson and Sommers, 1982).


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  		Table 1. Physical and chemical properties of soil samples.

 
Extraction of Total Petroleum Hydrocarbon from Petroleum-Contaminated Soil
Soil samples were air-dried and sieved (2 mm) for the analysis of TPH content. Total petroleum hydrocarbon in soil was extracted as described before (Sanjeet et al., 2001). In brief, TPH in 10 g of soil was consecutively extracted with hexane, methylene chloride, and chloroform (20 mL each). All the three extracts were pooled and dried at room temperature by evaporation of the solvents under a gentle nitrogen stream in a fume hood. After evaporation, the amount of residual TPH recovered was determined gravimetrically.

Enumeration of Bacterial Colony Forming Units and Colony Morphology
One gram of soil was added to a test tube containing 9 mL of sterile 0.85% NaCl in distilled water. This suspension was vortexed at maximum velocity for 60 s. From this suspension, 0.1-mL soil dilutions appropriate to ensure 30 to 100 colonies per plate were spread on meat-peptone agar plates supplemented with fungicide (25 µg natamycin mL–1) (Pedersen, 1992). The plates were inspected after 4 d of incubation at 25°C.

Diversity analysis based on colony morphology was performed by grouping colonies appearing on the plates according to visual differences, for example, color, diameter, edge, surface, shape, and size (Palumbo et al., 1996). The plate containing approximately 100 colonies was selected for calculating Shannon–Weaver indices (n = 3) on the basis of these groupings.

DNA Extraction and Purification
DNA extractions were performed as described by Zhou et al. (1996) with a minor modification. Soil samples of 0.5 g were mixed with 1000 µL of DNA extraction buffer (100 mM Tris-HCl [pH 8.0], 100 mM sodium EDTA [pH 8.0], 100 mM sodium phosphate [pH 8.0], 1.5 M NaCl, 1% CTAB) and 10 µL of proteinase K (10 mg mL–1) in microcentrifuge tubes, vortexed for 10 s, and incubated at 37°C for 1 h. One hundred microliters of 20% SDS was added and the samples were incubated in a 65°C water bath for 2 h with gentle end-over-end inversions every 15 to 20 min. The supernatants were collected after centrifugation at 6000 x g for 10 min at room temperature and transferred into other microcentrifuge tubes. Supernatants were mixed with an equal volume of chloroform isoamyl-alcohol (24:1, v/v). The aqueous phase was recovered by centrifugation and precipitated with 0.6 volume of isopropanol at room temperature for 1 h. The pellet of crude nucleic acids was obtained by centrifugation at 16000 x g for 20 min at room temperature, washed with cold 70% ethanol, and resuspended in TE buffer to give a final volume of 50 µL. The crude DNA extract was subjected to agarose gel electrophoresis and the DNA bands were excised, melted, and purified by TaKaRa agarose gel DNA purification kit (Takara Bio, Shiga, Japan) as described by the manufacturer. The eluted DNA was then further purified by a Wizard minicolumn containing 1 mL of Wizard DNA clean-up resin (Promega, Madison, WI). The DNA was eluted from the resin twice with 50 µL of hot (70°C) Tris-EDTA buffer to facilitate release of high-molecular-weight DNA.

Polymerase Chain Reaction Amplification of 16S rDNA from Soil
The community DNA extracted from the soil was amplified with eubacterial primers 338f and 518-GC as previously described (Øvreås et al., 1997). Polymerase chain reaction was performed in a total volume of 50 µL, containing 20 pM of each primer, 200 µM deoxynucleoside triphosphates, 2.5U of Taq DNA polymerase, and the buffer supplied with the enzyme (Takara Bio) and 1 µL of template DNA. Thermocycling was performed with a PTC-100 programmable thermal controller (MJ Research, Waltham, MA) using the following conditions: 95°C for 1 min, 65°C for 1 min, 72°C for 3 min with a touchdown of 0.5°C per cycle for the first 20 cycles, thereafter followed by 10 cycles at the annealing temperature of 55°C. The last cycle was followed by a final elongation of 72°C for 8 min (Müller et al., 2002).

Denaturing Gradient Gel Electrophoresis Separation of 16S rDNA and Analysis of Profiles
The Bio-Rad D-Code Universal Detection Mutation System (Bio-Rad, Hercules, CA) was used for DGGE as described by Muyzer et al. (1993) with a minor modification. The PCR products were loaded onto 1-mm-thick 8% (w/v) polyacrylamide (37.5:1 acrylamide to bisacrylamide) gels containing a 40 to 70% linear denaturing gradient. One hundred percent denaturant is 7 M urea and 40% (v/v) deionized formamide. The denaturing gradient gel was prepared and allowed to polymerize for 1.5 h. A top gel (6% acrylamide, 0% denaturant, approximately 7.5 mm in height) into which the 20-well comb was inserted was applied on top of the denaturant gel to minimize denaturant gradient disturbance during comb insertion and was allowed to polymerize for 0.5 h. Wells were washed with 1x TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0), and approximately 350 ng of amplified PCR product was loaded per lane. Gels were run in 1x TAE buffer at 60°C and 35 V for 16 h. The resulting gel was stained in 1x TAE buffer containing SYBR Green I (diluted 1:10000; Sigma, St. Louis, MO) and a digital image of the gel was obtained using Gel Doc 2000 gel documentation systems (Bio-Rad). Band patterns were analyzed using image analysis software (Quantity One 4.2.3; Bio-Rad). Lanes in the gel image were defined after background subtraction. One representative experimental lane that contained most of the bands we were interested in was selected and all the bands in the gel image were automatically matched and the remaining unmatched bands were manually matched. A densitometric curve was calculated for each gel tracks. Band positions were converted to Rf values between 0 and 1, and profile similarity was calculated by determining Dice's coefficient for the total number of lane patterns. Dendrograms were constructed by using the unweighted pair group method with mathematical averages (UPGMA) (Ibekwe et al., 2001). Intensities of the bands as judged by peak areas in the densitometric curves were exported to Excel files to calculate the Shannon–Weaver index (H).

Enzyme Assays
Activity of soil dehydrogenase was estimated as described by Casida et al. (1964) with a minor modification. Five grams of soil was mixed with 10 mL of 0.25% aqueous triphenyltetrazolium chloride (TTC) and incubated in a sealed tube at 30°C for 6 h. The absorbance at 485 nm of methanol extracts of the triphenylformazan (TPF) produced was then measured using methanol as a blank. The activity of dehydrogenase was expressed as mg TPF g–1 dry soil 6 h–1.

Soil hydrogen peroxidase activity was determined by the potassium permanganate titration method and expressed as mL 0.1 mol L–1 KMnO4 g–1 dry soil (Rodríguez-Kábana and Truelove, 1982; Alef and Nannipieri, 1995).

Soil polyphenol oxidase activity was measured by the colorimetric method based on the purpurogallin formation in the pyrogallic acid–amended soil sample (after 3 h of incubation at 30°C) and expressed as mg purpurogallin g–1 dry soil 3 h–1 (Ma et al., 2003).

Soil urease activity was determined by the colorimetric method according to the NH3–N formation in the urea-amended soil sample (after 48 h incubation at 37°C) and expressed as mg NH3–N g–1 dry soil 24 h–1 (Nannipieri et al., 1980; Kandeler and Gerber, 1988).

Substrate-Induced Respiration Measurements
The CO2 released from 15 g of glucose-amended soils after 24 h of incubation at 30°C was trapped in 20 mL of 0.1 mol L–1 NaOH and determined by titration with 0.1 mol L–1 HCl (Alef and Nannipieri, 1995). Substrate-induced respiration was expressed as mL CO2 kg–1 dry soil.

Diversity Index
The results of the two different community analyses were evaluated by determining the Shannon diversity index calculated as:

[1]
where Pi is the percentage of (i) the total (s) intensity accounted for by the ith band and (ii) the total number of colonies accounted for by the ith morphotype, respectively (Shannon and Weaver, 1949).

Statistical Analysis
Analysis of all data was performed by ANOVA using SPSS 10.0 for Windows (SPSS, 1999). Linear correlation coefficients were determined between different biological and biochemical parameters. Significance of all statistical analysis was accepted at {alpha} = 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Total Petroleum Hydrocarbon Accumulation and Distribution
The analysis of TPH concentration showed that TPH concentration varied between 277.11 and 5213.37 mg kg–1 dry soil in the Shenfu irrigation area (Fig. 2) . The TPH concentration in paddy soils (except Sites B and K) showed a significant (r = 0.691, p = 0.018) correlation with organic matter contents (Table 1). Soils at up- and mid-stream areas of the irrigation channel have a higher accumulation of TPH than soils at the downstream area. The most seriously polluted site was Site F, located at the upper reaches of the mid-stream area, where TPH concentration was up to 5213.37 mg kg–1 dry soil. The TPH concentration of Site M located at the end of the channel was only 277.11 mg kg–1. The TPH concentrations of Sites C, D, and E, which were located on the branch channel, were 1266.52, 1207.72, and 715.29 mg kg–1, respectively. It also significantly decreased with the increase of distance from the inflow.



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  		Fig. 2. Total petroleum hydrocarbon (TPH) concentrations of soil samples in the Shenfu irrigation area. Error bars (n = 3) indicate standard deviations.

 
Sites A and C were two adjacent paddy fields located at the upstream area of the irrigation area (Fig. 1). These two fields were different only in their irrigation history. Site A was changed to ground water–irrigated fields about 30 yr ago, whereas Site C has been irrigated by wastewater for more than 50 yr. The situation of Sites J and L is similar to that of Sites A and C (Fig. 1). Site L was changed to ground water–irrigated fields about 10 yr ago, whereas Site J has been irrigated by wastewater until the present time. The TPH concentration of Site A was lower than Site C, but not significant (p = 0.164). However, TPH concentration of Site L was significantly (p = 0.005) lower than Site J. These results indicated that transformation from wastewater irrigation to ground water irrigation could reduce the accumulation of TPH in paddy soils.

Two sites (B and K) that have been changed to upland use decades ago were selected to compare the TPH accumulation with the geographically adjacent paddy fields (A and L), which are irrigated by ground water. Sites A and B were two adjacent fields located at the upstream area but different in their irrigation history (Fig. 1). Site B was changed to upland field about 30 yr ago, while Site A was changed to ground water–irrigated paddy fields at the same time. The situation of Sites L and K is similar to that of Sites A and B (Fig. 1). Site K was changed to upland field about 10 yr ago, while Site L was changed to ground water irrigated paddy fields at the same period. The TPH concentration of Sites B and K was significantly lower than Sites A (p = 0.002) and L (p = 0.013), respectively. These results indicated that uplands had a higher TPH degradation rate than paddy fields even if the paddy fields were irrigated by ground water.

Colony Forming Units and Morphology Diversity of Aerobic Heterotrophic Bacteria in Polluted Soil
The CFU of AHB in different sampling sites is shown in Fig. 3 . Results showed that the CFU of AHB in paddy soil samples varied between 4 and 37 x 107 CFU g–1 dry soil and showed a significant correlation (r = 0.928, p < 0.001) with the TPH concentration at the current pollution level. The quantities of AHB in two upland fields (Sites B and K) were much higher than that of paddy soils.



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  		Fig. 3. Total numbers of aerobic heterotrophic bacteria (AHB) in the Shenfu irrigation area. Error bars (n = 4) indicate standard deviations.

 
The Shannon–Weaver indices of AHB based on colony morphology presented in Table 2 are the average values (n = 3) for the replicates of each soil. The number of colony types varied between 6 and 14. Results showed that the bacterial diversity of upland was a little higher than that of paddy soils and the diversity of AHB in paddy soils showed a positive correlation with TPH concentration (r = 0.598), but it was not significant (p = 0.052). The bacterial diversities based on colony morphology in paddy soils increased with TPH concentration at the current pollution level, but not significantly.


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  		Table 2. Bacterial diversity (mean ± SE) of different soil samples in the Shenfu irrigated area.

 
Genetic Diversity of Eubacteria in Polluted Soil
Genetic diversity was investigated by total DNA extraction and PCR amplification of 16S rDNA fragments followed by separation by DGGE (Fig. 4) . We detected bands at 41 different Rf locations, with each sample containing 16 to 23 bands. Shannon–Weaver index calculated on the basis of intensities of DGGE bands (Table 2) showed a significant positive correlation with TPH concentration (r = 0.655, p = 0.029). Site F, with the highest accumulation of TPH, has the highest bacterial diversity. It was indicated that at the current pollution level, petroleum-containing wastewater irrigation significantly increased the bacterial diversity based on DGGE analysis.



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  		Fig. 4. Denaturing gradient gel electrophoresis (DGGE) analysis of soil bacterial communities in the Shenfu irrigation area.

 
A cluster analysis of DGGE profiles based on band Dice's coefficient is shown in Fig. 5 . It was clear that two upland field samples (Sites B and K) were grouped together. The paddy soil samples can be divided into two parts. Site A was separated from all the other paddy soil samples, while the other paddy soil samples can also be grouped into two large branches. Sites F, G, and H, located at the mid-stream area of the irrigation channel, were grouped together. The most seriously polluted field, Site F, was separated from the other two due to its high TPH concentration. The other paddy soil samples were assigned to a different branch according to the sampling sites and pollution degree.



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  		Fig. 5. A dendrogram representation of a hierarchical cluster analysis of the denaturing gradient gel electrophoresis (DGGE) profiles.

 
Soil Enzymatic Activities
Table 3 shows the results of the effect of long-term petroleum-containing wastewater irrigation on the enzymatic activities. In this study, the activities of dehydrogenases, hydrogen peroxidases, and polyphenol oxidases showed a close correlation with TPH concentration and their correlation coefficients were 0.974 (p < 0.001), 0.957 (p < 0.001), and 0.886 (p < 0.001), respectively. The activities of dehydrogenases also significantly correlated with the CFU of AHB (r = 0.852, p = 0.001). On the contrary, urease activity was significantly (p = 0.002) negatively correlated with TPH concentration (r = –0.814).


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  		Table 3. Effect of wastewater irrigation on soil enzymatic activities (mean ± SE) in the Shenfu irrigated area.

 
The SIR of polluted soils was significantly (p < 0.001) correlated with TPH concentration with the correlation coefficient of 0.916. Compared with the data of dehydrogenase activity and CFU of aerobic heterotrophic bacteria, it could be concluded that SIR was also significantly correlated with dehydrogenase activity (r = 0.903, p < 0.001) and heterotrophic bacterial CFU (r = 0.770, p = 0.006) at the current pollution level.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
Soils at up- and mid-stream areas of the main channel and branch channel have a higher accumulation of TPH than soils at the downstream area. Total petroleum hydrocarbon accumulation in the soil is affected by the absorption of soil particles and the degradation of microorganisms. The highest accumulation of TPH occurred at the mid-stream area of the irrigation area. This may due to the fact that soil texture in the upstream area was sandy soil, which has a lower adsorption of petroleum than the loam soil in the mid-stream area (Table 1).

The paddy fields changed to ground water–irrigated fields more than a decade ago (Sites A and L) have a lower TPH accumulation than the paddy fields irrigated by wastewater until now. This phenomenon indicated that ground water irrigation could reduce the accumulation of TPH in paddy soils. But TPH concentration of Site A was only 96.63 mg kg–1 lower than Site C. In fact, the irrigation water of Site A comes from a 15-m-deep well. It can be deduced that TPH may pollute the ground water by infiltration and eluviation. Transformation from wastewater-irrigated paddy fields to uplands (Sites B and K) can efficiently accelerate the degradation of TPH, because the degradation of TPH by microorganisms is mainly an oxygen-participated process. Aerobic heterotrophic bacteria, the chief decomposer of organic matter, were activated under the aerobic condition. This indicated that changing of paddy fields to uplands is an effective method to alleviate the petroleum pollution in this area.

The CFU of AHB in paddy soils was significantly correlated with TPH concentration. This may be due to the fact that several petroleum aliphatic and polycyclic hydrocarbons can act as sources of carbon and energy for the growth of soil microorganisms (Galli, 1998). The great deal of nutrients (including C, N, P, and K) in wastewater stimulated the growth of heterotrophic bacteria.

Diversity calculated on the basis of colony morphology and DGGE profiles showed positive correlation with TPH concentration, but differed in their significance (p = 0.052 for colony morphology and p = 0.029 for DGGE bands). In addition, the Shannon–Weaver indices calculated based on DGGE profiles were little higher than that calculated from colony morphology. This may due to the limitations of these two methods. The culturable part of the microbial community represents only a minor fraction (about 20%) of the total community (Muyzer et al., 1993; Macnaughton et al., 1999) and only of the dominant bacteria. It is uncertain whether it is representative of the whole community or even of the active part of the community. Therefore, the diversity of colony morphotypes may underestimate the total diversity. Moreover, the Shannon–Weaver indices based on colony morphotypes are very similar among all samples. It may be due to the media used in this study. Meat-peptone agar is a high nutrient media and permits many kinds of bacteria grow on it. Thus, the differences among all the samples were reduced. Compared to the colony morphology approach, DGGE includes the unculturable bacteria. However, as the profiles contain a maximum of about 41 band types, they clearly do not include all species. It is important to bear in mind that some bacteria produce more than one band on the DGGE (Nübel et al., 1996), and that DNA sequences from different bacteria can have identical melting behavior and hence cannot be separated on DGGE gels. Furthermore, it is doubtful whether extracted and amplified DNA reflects the quantitative abundance of the species (Farrelly et al., 1995; von Wintzingerode et al., 1997). Whether the bands represent the most abundant species, the most easily extractable species, the most active species, or a combination of all these groups is uncertain. Nevertheless, DGGE might be a sensitive method for detecting differences in community diversity.

The hierarchical cluster analysis separated Site A from all the other paddy soil samples. Site A was changed to be irrigated by ground water 30 yr ago, but the TPH accumulation was still up to 1169.89 mg kg–1 dry soil, because the dominant component of TPH in Site A is PAHs such as phenanthrene, pyrene, fluoranthene, and benzofluoranthene, whereas the PAH concentrations in other sampling sites are much lower (Song et al., 1997). The changes of TPH components may result in the changes of microbial diversity and community structure, which needs to be studied further. Many other factors, such as soil pH, water capacity, climate, and human behavior would also contribute to the change of soil microbial community.

Oxidoreductase plays an important role in energy transformation in the respiration chain and participates in the synthesis of soil humics and the soil formation process (Gramss et al., 1999; Dick, 1997). In our study, the activities of dehydrogenase, hydrogen peroxidase, and polyphenol oxidase increased with TPH concentration at the current pollution level. It was also found that dehydrogenase activities have a significant correlation with CFU of AHB. Dehydrogenase is active as intracellular enzymes only. It was often considered as an index of overall microbial activity (Nannipieri et al., 1990; García et al., 1997).

Hydrogen peroxidase is another key oxidoreductase associated with aerobic microbial activities (Rodríguez-Kábana and Truelove, 1982). As a product of the biological respiration process and the biochemical oxidation process of organic materials, hydrogen peroxide exists widely in organisms and soils. Hydrogen peroxide is toxic to plants and soil organisms. Hydrogen peroxidase may decompose hydrogen peroxide into molecular oxygen and water, thus alleviating its toxicity to organisms (Daniel et al., 1992). Hydrogen peroxidase activity showed a significant correlation with TPH concentration at the current pollution level. It was suggested that, to some extent, the addition of organic matter is needed to activate soil hydrogen peroxidase activity. Similar phenomena have been observed in organic waste compost process (Pascual et al., 1998; García-Gil et al., 2000) and anthracene-contaminated soil (Ma et al., 2003).

Polyphenol oxidase is associated with hydroxylation of aromatic rings and leads ultimately to their mineralization or humification. Therefore, polyphenol oxidase plays an important role in the process of conversion of aromatic organic compounds and was negatively correlated to the level of humification (Zhou et al., 1981; Ma et al., 2003). A great deal of PAHs and polyphenols in petroleum-containing wastewater induced an increase in polyphenol oxidase activities as substrate at the current pollution level.

Urease was susceptible for many disturbances (Kandeler and Gerber, 1988; Gianfreda et al., 1994; García-Gil et al., 2000). It was also sensitive to petroleum contamination. In our research, urease activity was significantly decreased with the TPH concentration. It may be due, in part, to the high concentration of available nitrogen in irrigation wastewater (Table 1). Therefore, activity of urease can be taken as a sensitive biological indicator of petroleum contamination.

To some extent, SIR has a close relationship with the concentration of organic matter in soil (Alef and Nannipieri, 1995). Much organic matter in the Shenfu irrigation area enhanced soil SIR at the current pollution level. It was also found that SIR was significantly correlated with dehydrogenase activities and CFU of aerobic heterotrophic bacteria. This phenomenon confirms the fact that SIR can be taken as the measurement of the biomass of active microbes (Anderson and Domsch, 1978; Tate, 2000). As previously mentioned, dehydrogenase activities represented the overall microbial activities. Increasing bacterial quantity resulted in increasing intracellular dehydrogenase activities and soil SIR. Furthermore, increasing dehydrogenase activities promoted the consumption of oxygen, which was the acceptor of hydrogen atoms. The increasing oxygen consumption directly resulted in increasing soil SIR. These three factors interacted closely with each other.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
In this study, we evaluated the effects of long-term petroleum-containing wastewater irrigation on bacterial diversities and soil enzymatic activities. The community structure and diversity of microbial communities and enzymatic activities in the soil ecosystem could be changed under the pollution stress. On the other hand, soil microorganisms and soil enzymatic activities are sensitive biological indicators of soil pollution. The petroleum pollution in the Shenfu irrigation area resulted in the increase of the number of AHB and the diversities of eubacteria at the present pollution level. The activities of oxidoreductases were increased due to the presence of TPH. The decrease in activity of urease can be taken as a sensitive indicator of degradation of soil function.


   ACKNOWLEDGMENTS
 
We thank the Ministry of Science and Technology of China for financial support through Projects 2004 AA649060 and 2004 CB418500. The authors also gratefully thank Shenyang Key Laboratory of Environmental Engineering for funding this research.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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C. H. Nakatsu
Soil Microbial Community Analysis Using Denaturing Gradient Gel Electrophoresis
Soil Sci. Soc. Am. J., March 12, 2007; 71(2): 562 - 571.
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