Dynamic changes in microbial activity and community structure during biodegradation of petroleum compounds:A laboratory experiment

With 110-d incubation experiment in laboratory, the responses of microbial quantity, soil enzymatic activity, and bacterial community structure to different amounts of diesel fuel amendments were studied to reveal whether certain biological and biochemical characteristics could serve as reliable indicators of petroleum hydrocarbon contamination in meadow-brown soil, and use these indicators to evaluate the actual ecological impacts of 50-year petroleum-refining wastewater irrigation on soil function in Shenfu irrigation area. Results showed that amendments of ~ 1000 mg/kg diesel fuel stimulated the growth of aerobic heterotrophic bacteria, and increased the activity of soil dehydrogenase, hydrogenperoxidase, polyphenol oxidase and substrate-induced respiration. Soil bacterial diversity decreased slightly during the first 15 d of incubation and recovered to the control level on day 30. The significant decrease of the colony forming units of soil actinomyces and filamentous fungi can be taken as the sensitive biological indicators of petroleum contamination when soil was amended with 〉15000 mg/kg diesel fuel. The sharp decrease in urease activity was recommended as the most sensitive biochemical indicator of heavy diesel fuel contamination. The shifts in community structure to a community documented by Sphingomonadaceae within a-subgroup of Proteobacteria could be served as a sensitive and precise indicator of diesel fuel contamination. Based on the results described in this paper, the soil function in Shenfu irrigation area was disturbed to some extent.

Ecological effects of crude oil residues on the functional diversity of soil microorganisms in three weed rhizospheres

Ecological effects of crude oil residues on weed rhizospheres are still vague. The quantitative and diversity changes and metabolic responses of soil-bacterial communities in common dandelion （Taraxacum officinale）, jerusalem artichoke （Silphiurn perfoliatum L.） and evening primrose （A colypha australis L.） rhizospheric soils were thus examined using the method of carbon source utilization. The results indicated that there were various toxic effects of crude oil residues on the growth and reproduction of soil bacteria, but the weed rhizospheres could mitigate the toxic effects. Total heterotrophic counting colony-forming units （CFUs） in the rhizospheric soils were significantly higher than those in the non-rhizospheric soils. The culturable soil-bacterial CFUs in the jerusalem artichoke （S. perfoliatum） rhizosphere polluted with 0.50 kg/pot of crude oil residues were almost twice as much as those with 0.25 kg/pot and without the addition of crude oil residues. The addition of crude oil residues increased the difference in substrate evenness, substrate richness, and substrate diversity between non-rhizospheric and rhizospheric soils of T. officinale and A. australis, but there was no significant （p〉0.05） difference in the Shannon＇s diversity index between non-rhizospheric and rhizospheric soils of S. perfoliatum. The rhizospheric response of weed species to crude oil residues suggested that S. perfoliatum may be a potential weed species for the effective plant-microorganism bioremediation of contaminated soils by crude oil residues.

Distribution of Soil Enzyme Activities and Microbial Biomass Along a Latitudinal Gradient in Farmlands of Songliao Plain,Northeast China

Soil enzymes activities and microbial biomass have an important influence on nutrient cycling. The spatial distribution of soil enzymes activities and microbial biomass were examined along a latitudinal gradient in farmlands of Songliao Plain, Northeast China to assess the impact of climatic changes along the latitudinal transect on nutrient cycling in agroecosystems. Top soils （0-20 cm depth） were sampled in fields at 7 locations from north （Hallun） to south （Dashiqiao） in the end of October 2005 after maize harvest. The contents of total C, N, and P, C/N, available N, and available P increased with the latitude. The activities of invertase and acid phosphatase, microbial biomass （MB） C and N, and MBC/MBN were significantly correlated with latitude （P 〈 0.05, r^2 = 0.198, 0.635, 0.558, 0.211 and 0.317, respectively）, that is, increasing with the latitude. Significant positive correlations （P 〈 0.05） were observed between invertase activity and the total N and available P, and between acid phosphatase activity and the total C, C/N, available N, total P and available P. The urease, acid phosphatase, and dehydrogenase activities were significantly correlated with the soil pH and electrical conductivity （EC） （P 〈 0.05）. MBC and MBN were positively correlated with the total C, C/N, and available P （P 〈 0.05）. The MBC/MBN ratio was positively correlated with the total C, total N, C/N, and available N （P 〈 0.05）. The spatial distribution of soil enzyme activities and microbial biomass resulted from the changes in soil properties such as soil organic matter, soil pH, and EC, partially owing to variations in temperature and rainfall along the latitudinal gradient.

Toxic effects of acetochlor and methamidophos on earthworm Eisenia fetida in phaiozem, northeast China

Acetochlor and methamidophos are two important agrochemicals which are widely applied to agricultural production in northeast China. The investigation on the earthworm Eisenia fetida as an important type of soil animals exposed to single and binary-combined contamination of acetochlor and methamidophos was thus carried out. The single toxic effect test showed that the two agrochemicals had their toxicity to the earthworms living in phaiozem. Acetochlor had a stronger acute toxic effect on the earthworms than methamidophos. The mortality of the earthworms exposed to individual acetochlor and methamidophos changed with an increase in the exposure time and the exposed concentrations. The LD50 value of acetochlor and methamidophos toxic to the earthworms was 115.6-275,3 and 29.5-228.6 mg/kg, respectively. The weight of the earthworms was a more sensitive index compared to the mortality in indicating toxic effects of acetochlor and methamidophos in phaiozem. When considering both the mortality and the body-weight change, the combined pollution of acetochlor and methamidophos in phaiozem resulted in their synergic toxic effects on the earthworms.

Single and joint stress of acetochlor and Pb on three agricultural crops in northeast China

In order to evaluate ecological risk of agrochemicals in agricultural environment, single and joint toxic effects of an important herbicide and a typical heavy metal on root elongation of crops were investigated. Seeds of the three crops including wheat （Triticum aestivum）, Chinese cabbage （Brassica pekimensis） and soybean （Glycine max） as the main crops in northeast China were exposed to acetochior as a herbicide and lead （Pb） as a heavy metal using the pot-culture method, and meadow brown soil as one of the main soils distributed in northeast China was applied in the investigation. The results indicated that the interactive effects of the two pollutants on root elongation of the three crops were very complicated although they had markedly significant （P〈0.01） linear interrelationships based on the regression analyses. When the concentration of added Pb^2＋ reached 200 mg/kg, acetochlor and Pb had an antagonistic effect on the inhibition of root elongation of the three crops. However, acetochlor and Pb had significantly （P〈0.05） synergic effects on the inhibition of root elongation when concentration of added Pb^2＋ was up to 1000 mg/kg. At the low concentration of added Pb, joint toxicity of acetochlor and Pb was more dependent on the concentration of Pb. Among the three crops, wheat was the most sensitive to the toxicity of Pb and Chinese cabbage was the most sensitive to the toxicity of acetochlor.

Effect of heavy metals on soil nematode communities in the vicinity of a metallurgical factory

The influence of Cu and Zn on soil nematode communities was examined along a pollution gradient with increasing distance from a metallurgical factory. Total and available heavy metal contents were used to study the effects of heavy metals on nematode abundance, trophic groups and ecological indices. The results demonstrated significant correlations between the number of total nematodes, bacterivores, plant-parasites and the total and available heavy metals. Bacterivores and plant-parasites were the dominant trophic groups. Significant differences in different sampling sites were found only in the number of bacterivores（P〈0.01）. The Shannon-Weaver diversity index（W）, trophic diversity index（TD）, evenness index（J＇） and dominance index（λ） were found to be sensitive to soil pH and C/N ratios. Significant correlations were found between the total nematodes （TNEM）, some genera （A crobeloides, Aphelenchoides, Cephalobus, Ditylenchus, Mesorhabditis, Tetylenchus and Tylenchus） and distance from the factory.

Single and joint effects of pesticides and mercury on soil urease

The influence of two pesticides including chlorimuron-ethyl and furadan and mercury （Hg） on urease activity in 4 soils （meadow burozem and phaeozem） was investigated. The soils were exposed to various concentrations of the two pesticides and Hg individually and simultaneously. Results showed that there was a close relationship between urease activity and organic matter content in soil. Chlorimuron-ethyl and furadan could both activate urease in the 4 soils. The maximum increment of urease activity by chlorimuronethyl was up to 14%-18%. There was almost an equal increase （up to 13%-21%） in the urease activity by furadan. On the contrary, Hg markedly inhibited soil urease activity. A logarithmic equation was used to describe the relationship （P〈0.05） between the concentration of Hg and the activity of soil urease in the 4 tested soils. Semi-effect dose （ED50） values by the stress of Hg based on the inhibition of soil urease in the 4 soils were 88, 5.5, 24 and 20 mg/kg, respectively, according to the calculation of the corresponding equations. The interactive effect of chlorimuron-ethyl or furadan with metal Hg on soil urease was mainly synergic at the highest tested concentrations.

Influences of Cu or Cd on the neurotoxicity induced by petroleum hydrocarbons in ragworm Perinereis aibuhitensis

The ecotoxicological effects of heavy metals and petroleum hydrocarbons （PHCs） on ragworms are still vague. The relationships between toxicological indices （mortality and acetylcholinesterase （ACHE） activity） and concentrations of toxicants （Cu, Cd, and PHCs） were examined in the estuary keystone species Perinereis aibuhitensis in laboratory conditions. The results of single toxicant indicated that three toxicants had potentially physiological toxicity to P. aibuhitensis. The estimated 4-d and 10-d LC50 for Cu, Cd, and PHCs was derived from the relationships between mortality and toxicants concentrations. Notable changes in the morphological signs and symptoms of P. aibuhitensis exposed to PHCs were observed. The ACHE activity of P. aibuhitensis was more sensitive to the toxicity of PHCs than the others. The results of combined toxicants implied that the combined toxicity of Cu or Cd and PHCs to P. aibuhitensis was related to the concentration combination of toxicants. Compared to single PHCs treatment, the addition of Cu or Cd significantly mitigated the neurotoxicity of PHCs to ACHE activity in P. aibuhitensis, which showed an antagonistic effect.

Influences of phosphate nutritional level on the phytoavailability and speciation distribution of cadmium and lead in soil

A pot experiment was conducted to examine the influence of phosphate levels on the phytoavailability and speciation distribution of cadmium （Cd）, lead （Pb） in soil. Spring wheat （Triticum aestivum L.） was selected as the tested plant. There were 5 phosphate fertilizer（Ca（H2PO4）2） levels including 0, 50, 100, 200, and 400 mg P2O5/kg soil, marked by P0, P1, P2, P3, and P4, respectively. CdCl2·2.5H2o and Pb（NO3）2 were added to soil as the following levels： Cd ＋ Pb = 25＋0, 0＋1000, and 25＋1000 mg/kg, marked by T1, T2, and T3, respectively. The results showed that the P fertilizer promoted the dry weight of wheat in all treatments and alleviated the contamination induced by Cd and Pb. With increasing levels of the additional P fertilizer, Cd concentration in different parts （root, haulm, chaffand grain） of wheat decreased at the P1 level at first and then increased. The soluble plus exchangeable （SE） fraction of Cd in soil decreased at the P1 level and then increased from P2 to P4 levels. The moderate P fertilizer reduced the phytoavailability of Cd. The application of P could obviously restrain the uptake of Pb by wheat and there were significantly negative correlations between the levels of P and the uptake of Pb. Phosphorus supply resulted in a decrease in the SE fraction of Pb and there was a significantly negative correlation between the levels of P and the SE fraction of Pb in soil. All the levels of the P fertilizer in this experiment could reduce the phytoavailability of Pb. Thus, it is feasible to apply the P fertilizer （Ca（H2PO4）2） to Pb contaminated soils. However, the levels of P application should be restricted in case that redundant P may increase the phytoavailability of Cd.

Single and joint effects of petroleum hydrocarbons and cadmium on the polychaete Perinereis aibuhitensis Grube

Using the concentration gradient and combined pollutant exposure method, the single and joint effects of petroleum hydrocarbons （PHCs） and cadmium （Cd） on polychaete Perinereis aibuhitensis Grube, an ecologically keystone species in estuarine and coastal environment, have been investigated. The results indicate that the toxicity of PHCs to P. aibuhitensis is stronger than that of Cd to the organism. There are positive correlations between the mortality of worms and the exposed concentration of single Cd or PHCs in solution. Similarly, the accumulation of Cd or PHCs in worms increased with increasing Cd- or PHC-exposed concentrations. All the correlation relationships can be described using unitary quadratic equations （Y or Z = aX^2 ＋ bX ＋ c）. It is calculated, on the basis of these expressions, that the median lethal dose （LC50） ofP aibuhitensis exposed to a single Cd or PHCs is 793.4-13567.3 and 28.0-119.9 μg/L, respectively. The exposed time has some stimulative effect on the two pollutants and on the mortality of the worms. Thus, even a low concentration of a single Cd or PHCs may have strong toxic effects on the worms when the exposed time becomes longer. The accumulation of Cd or PHCs in worms differs with an increase in exposure time at the given exposed concentration of a single Cd or PHCs. Noticeably, the accumulation of PHCs in worms decreases with an increase in exposure time at the given high concentration of PHCs in solution. The joint effect of PHCs and Cd on P. aibuhitensis is very complicated and changes with the exposed concentrations of the two pollutants. At the given concentration of PHCs, the joint toxicity of the two pollutants on the worms changes from synergism to antagonism with an increase in Cd concentration. The accumulation of Cd in the worms significantly decreases with the addition of PHCs to exposure solution.

Toxic effects of acetochlor, methamidophos and their combination on nifH gene in soil

Toxic effects of two agrochemicals on nifH gene in agricultural black soil were investigated using denaturing gradient gel electrophoresis （DGGE） and sequencing approaches in a microcosm experiment. Changes of soil nifH gene diversity and composition were examined following the application of acetochlor, methamidophos and their combination. Acetochlor reduced the nifH gene diversity （both in gene richness and diversity index values） and caused changes in the nifH gene composition. The effects of acetochlor on nifH gene were strengthened as the concentration of acetochlor increased. Cluster analysis of DGGE banding patterns showed that nifH gene composition which had been affected by low concentration of acetochlor （50 mg/kg） recovered firstly. Methamidophos reduced nifH gene richness that except at 4 weeks. The medium concentration of methamidophos （150 mg/kg） caused the most apparent changes in nifH gene diversity at the first week while the high concentration of methamidophos （250 mg/kg） produced prominent effects on nifH gene diversity in the following weeks. Cluster analysis showed that minimal changes of nifH gene composition were found at 1 week and maximal changes at 4 weeks. Toxic effects of acetochlor and methamidophos combination on nifH gene were also apparent. Different nifH genes （bands） responded differently to the impact of agrochemicals： four individual bands were eliminated by the application of the agrochemicals, five bands became predominant by the stimulation of the agrochemicals, and four bands showed strong resistance to the influence of the agrochemicals. Fifteen prominent bands were partially sequenced, yielding 15 different nifH sequences, which were used for phylogenetic reconstructions. All sequences were affiliated with the alpha- and beta-proteobacteria, showing higher similarity to eight different diazotrophic genera.

Isolation and characterization of Sporobolomyces sp. LF1 capable of degrading chlorimuron-ethyl

A yeast strain which was capable of degrading sulfonylurea herbicide chlorimuron-ethyl named as LF1 was isolated from a chlorimuron-ethyl contaminated soil near the warehouse of the factory producing chlorimuron-ethyl in Shenyang City, Northeast China. The strain was identified as Sporobolomyces sp., based on its morphological and physiological characteristics and the phylogenetic analysis of 18S rRNA gene sequence. So far, this is the only yeast strain of Sporobolomyces sp. which is able to degrade chlorimuronethyl. Incubation tests showed that when the initial concentration of chlorimuron-ethyl in culture was 5 mg/L, LF1 could degrade more than 77% of the herbicide after incubation for 4 d at 30℃. The possible mechanism of chlorimuron-ethyl degradation by LF1 could be the acidic hydrolysis caused by the acids from the metabolism of the yeast strain. Further study should be conducted to examine the pathways of chlorimuron-ethyl degradation by LF1 and to approach the feasibility of using LF1 to degrade the chlorimuron-ethyl in soil system.

Joint effects of cadmium and lead on seedlings of four Chinese cabbage cultivars in northeastern China

In northeastern China, large area of vegetable land has been simultaneously polluted by cadmium （Cd） and lead （Pb）. Joint effects of Cd and Pb on Chinese cabbage （Brassica pekinensis L.） were investigated using the seed germination and sand culture method. Four Chinese cabbage cultivars including Kangbingjinchun （KB）, Dongyangchunxia （DY）, Qinglvwang （QL） and Qiangshi （QS） from Shenyang in northeastern China were adopted in this study. The results showed that there were positive linear relationships between the inhibitory rate of biomass, root and shoot elongation and the concentrations of Cd and Pb. In particular, root elongation was more sensitive to joint stress of Cd and Pb. The activity of superoxide dismutase and the content of malondialdehyde （MDA）, soluble protein （SP） and proline （PRO） changed significantly with increasing exposure concentration of Cd and Pb. The decrement in the activity of antioxidative enzymes, the content of SP and accumulation of MDA were relatively low in KB and QS. PRO played an important role in resisting Cd and Pb stress.

Vertical Distribution and Seasonal Fluctuation of Nematode Trophic Groups as Affected by Land Use

A field investigation was conducted at the Shenyang Experimental Station of Ecology, Chinese Academy of Sciences, in an aquic brown soil of Northeast China under three land use types (cropland, abandoned cropland, and woodland) in order to evaluate whether the vertical distribution and seasonal fluctuation for the number of total nematodes and trophic groups could reflect soil ecosystem differences and to determine the relationships between soil chemical properties and soil nematodes. The majority of soil nematodes were present in the 0-20 cm soil layers, and for these land use types plant parasites were the most abundant trophic group. In the abandoned cropland the numbers of plant parasites reached a peak on the August sampling date, whereas the cropland and woodland peaked on the October sampling date. Meanwhile, in all land use types the number of total nematodes, bacterivores, plant parasites, and omnivores-predators was negatively (P < 0.05, except for bacterivores in cropland, which was not significant) correlated with bulk density, and positively (P < 0.05, except for fungivores in abandoned cropland, which was not significant) correlated with total organic carbon and total nitrogen.

Effects of heavy metal pollution of highway origin on soil nematode guilds in North Shenyang, China

Soil samples were collected with distance at 5, 20, 40, 80, 160, and 320 m from the Shen-Ha （Shenyang-Harbin） Highway, Northeast China, to investigate the effect of heavy metals of highway origin on soil nematode guilds. The contents of soil Pb, Cu, Zn, and the nematode community structure were analyzed. The results showed that the contents of total and available Pb, Ca, Zn varied significantly with the different distances from the highway. Pb was the main pollutant in the soils in the vicinity of Shen-Ha Highway. The zone from 20 to 40 m away from the highway was the most polluted area. The highest abundance of soil nematodes was found at 5 m while the lowest at 20 m away from the highway. Thirty six genera of nematodes belonging to 23 families were identified. Nematode guilds having different responses to soil heavy metals were classified into four types. Soil nematode guilds may act as a prominent indicator to heavy metal pollution of highway origin.

Technical Innovation of Land Treatment Systems for Municipal Wastewater in Northeast China

On the basis of ecological principles including holistic optimization, cycling and regeneration, and regional differentiation, land treatment systems (LTSs) for municipal wastewater were continuously explored and updated in the western Shenyang area and the Huolinhe area, China. Intensified pretreatment, addition of a man-made soil filtration layer, and use of an ecologically diversified secondary plant cover were proved to be technically feasible. Hydraulic loading was determined according to the assimilation capacity of soil ecosystems, thus ensuring safe operation of wastewater treatment. This modernized and alternative approach to wastewater treatment had been widely applied in middle-sized and small cities and towns of Northeast China, and these innovative systems in some areas had indicated favorable ecological, social, and economic benefits.

Adaptability of microbial inoculators and their contribution to degradation of mineral oil and PAHs

Five dominant bacteria strains（A cetobacter sp., Alcdigenes sp., Micrococcus sp., Arthrobacter sp. and Bacillus sp.） and five fungi strains （Cephalosporium sp. Ⅰ, Cephalosporlum sp. Ⅱ, Aspergillus sp. Ⅰ, Aspergillus sp. Ⅱ and Fusarium sp.） isolated from petroleum-contaminated soil were used to assess the potential capability of mineral oil and PAH enhanced degradation separately and jointly using the batch liquid medium cultivation with diesel oil spiked at 1000 mg/L. The experiment was performed on a reciprocal shaker in the darkness at 25℃ to 30℃ for 100 d. The dynamic variation in the activity of microbial inoculators in each treatment and the degradation of the target pollutants during the period of experiment were monitored. Results showed a more rapid biodegradation of mineral oil and PAHs at the beginning of the experiment （about 20 d） by dominant bacteria, fungi and their mixture than that of the indigenous microorganisms, however, thereafter an opposite trcnd was exhibited that the removal ratio by indigenous microorganisms was superior to any other dominant treatments and the tendency lasted till the end of the experiment, indicating the limited competitive capability of dominant microorganisms to degrade the contaminants, and the natural selection of indigenous microorganisms for use in the removal of the contaminants. At the end of the experiment, the removal ratio of mineral oil ranged from 56.8 % to 79.2 % and PAHs ranged from 96.8 % to 99.1% in each treatment by microbial inoculators,

Nematode Diversity in Phaeozem Agroecosystems of Northeast China

The diversity and distribution patterns of soil nematode communities in phaeozem agroecosystems of Northeast China were assessed to evaluate nematode taxonomic diversity and functional diversity in relation to climatic condition and soil characteristics in human modified landscape. Along the latitudinal gradient, soil samples were collected from north （Hailun） to south （Gongzhuling） down to a depth of 100 cm with intervals of 030, 20-40, 40-60, 60-80, and 80-100 cm. The nematode abundance and taxonomic diversity （generic richness） were lower at Hailun than at other sites, and higher values of evenness were observed at Hailun and Harbin than at Dehui and Gongzhuling. Nematode faunal analysis revealed that soil food web at Hailun was sueeessionally more mature or structured, and the environment little disturbed, while at Harbin and Gongzhuling, the soil food web was degraded with stressed environment. The environmental variables relevant in explaining the patterns of nematode distribution and diversity in phaeozem agroecosystems, using canonical correspondence analysis （CCA）, were the mean annual temperature, total nitrogen, electrical conductivity, mean annual precipitation, and other soil properties. Among these variables, the mean annual temperature was a relatively important factor, which could explain 29.05% of the variations in nematode composition.

PCR-DGGE Analysis of Nematode Diversity in Cu-Contaminated Soil

A wheat pot experiment was conducted under greenhouse conditions to assess the effect of copper contamination on soil nematode diversity by polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） method and morphological analysis. The soil was treated with CuSO4.5H2O at the following concentrations： 0, 50, 100, 200, 400, and 800 mg kg^-1 dry soil, and the soil samples were collected at wheat jointing and ripening stages. Nematode diversity index （H′） from morphological analysis showed no difference between the control and the treated samples in either of the sampling dates. At the wheat ripening stage, nematode diversity obtained by the PCR-DGGE method decreased noticeably in the Cu800 treatment in comparison with the control. With optimization of the method of nematode DNA extraction, PCR-DGGE could give more information on nematode genera, and the intensity of the bands could reflect the abundance of nematode genera in the assemblage. The PCR-DGGE method proved promising in distinguishing nematode diversity in heavy metal coritaminated soil.

Horizontal distribution and levels of heavy metals in the biggest snowstorm in a century in Shenyang,China

The horizontal distribution and levels of heavy metals in the biggest snowstorm in Shenyang since 1904 were investigated by analyzing 4 metals (As,Cd,Pb,and Cu) in a series of ultraclean samples collected from 17 sites distributed in different regions of the Shenyang area,China.The results showed that the concentrations of all the 4 heavy metals in snow from the industrial regions were high,up to 7.3 (As),2.2 (Cd),850.0 (Pb),and 0.197-20.2 (Cu)μg/kg,respectively.In the suburb,in contrast,their concentration...