Variation in archaeal and bacterial community profiles and their functional metabolic predictions under the influence of pure and mixed fertilizers in paddy soil.

Impact of environmental perturbations i.e., nitrogen (N), phosphorus (P), potassium (K), and rice straw (Rs) on the dynamics of soil bacterial and archaeal communities are multifactor dependent and seeks a contemporary approach to study underlying mechanisms. The current study investigates the effect of pure and mixed fertilizers on soil physicochemical properties, the microbial community structure, and their functional metabolic predictions. It involved amendments with distinct combinations of N as C(H2N)2O, P and K as KH2PO4, K as KCl, and Rs in paddy soil microcosms with concentrations common in rice fields agriculture. Soil pH, electrical conductivity (EC), total carbon (TC), total nitrogen (TN), organic matter (OM), available K (AK), and total extractable P (TEP) were evaluated. To comprehend community variation and functional predictions, 16S rRNA-based high throughput sequencing (HTS) and phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) were employed, respectively. Our findings showed enhanced community richness and diversity in all amendments compared to control. Proteobacteria, Actinobacteria, and Firmicutes were dominant bacterial phyla. Regarding relative abundance, Chloroflexi, Bacteroidetes, and Verrucomicrobia showed positive while Actinobacteria, Acidobacteria, and Gemmatimonadetes showed negative trends compared to controls. Thaumarchaeota and Euryarchaeota were dominant archaeal phyla and exhibited increasing and decreasing trends, respectively. The PICRUSt analysis indicated functional prediction more towards amino acid, carbohydrate, energy, and lipid metabolism while less towards others. Concerning energy metabolism, most and least responsive treatments were KP and controls, respectively. These outcomes enhanced our understanding regarding soil quality, fertilizer composition and application, and functional metabolomics of archaea and bacteria.

Influence of variable biochar concentration on yield-scaled nitrous oxide emissions, Wheat yield and nitrogen use efficiency.

An important source of the destructive greenhouse gas, nitrous oxide (N2O) comes from the use of ammonium based nitrogen (N) fertilizers that release N2O in the incomplete conversion (nitrification) of NH4+ to NO3-1. Biochar has been shown to decrease nitrification rates and N2O emission. However, there is little information from semi-arid environments such as in Pakistan where conditions favor N2O emissions. Therefore, the object was to conduct field experiment to determine the impact of biochar rates in the presence or absence of urea amended soils on yield-scaled N2O emissions, and wheat yield and N use efficiency (NUE). The experiment on wheat (Triticum aestivum L.), had a randomized complete block design with four replications and the treatments: control, sole urea (150 kg N ha-1), 5 Mg biochar ha-1 (B5), 10 Mg biochar ha-1 (B10), urea + B5 or urea + B10. In urea amended soils with B5 or B10 treatments, biochar reduced total N2O emissions by 27 and 35%, respectively, over the sole urea treatment. Urea + B5 or + B10 treatments had 34 and 46% lower levels, respectively, of yield scaled N2O over the sole urea treatment. The B5 and B10 treatments had 24-38%, 9-13%, 12-27% and 35-43%, respectively greater wheat above-ground biomass, grain yield, total N uptake, and NUE, over sole urea. The biochar treatments increased the retention of NH4+ which likely was an important mechanism for reducing N2O by limiting nitrification. These results indicate that amending soils with biochar has potential to mitigate N2O emissions in a semi-arid and at the same time increase wheat productivity.

Distribution of ammonia-oxidizing archaea and bacteria along an engineered coastal ecosystem in subtropical China.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) are the crucial players in nitrogen cycle. Both AOA and AOB were examined along a gradient of human activity in a coastal ecosystem from intertidal zone, grassland, and Casuarina equisetifolia forest to farmland. Results showed that the farmland soils had noticeably higher nitrate-N, available P than soils in the other three sites. Generally, AOA and AOB community structures varied across sites. The farmland mainly had Nitrosotalea-like AOA, intertidal zone was dominated by Nitrosopumilus AOA, while grassland and C. equisetifolia forest primarily harbored Nitrososphaera-like AOA. The farmland and C. equisetifolia forest owned Nitrosospira-like AOB, intertidal zone possessed Nitrosomonas-like AOB, and no AOB was detected in the grassland. AOA abundance was significantly greater than AOB in this coastal ecosystem (p < 0.05, n = 8). AOB diversity and abundance in the farmland were significantly higher than those in the other three sites (p < 0.05, n = 2). The biodiversity and abundance of AOA were not significantly correlated with any soil property (p < 0.05, n = 8). However, the diversity of AOB was significantly correlated with pH, available P and total P (p < 0.05, n = 6). The abundance of AOB was significantly correlated with pH, nitrite, available N, available P and total P (p < 0.05, n = 6). This study suggested that the community structures of AOA and AOB vary in the different parts in the bio-engineered coastal ecosystem and agricultural activity appears to influence these nitrifiers.

Risk assessment and source analysis of soil heavy metal pollution from lower reaches of Yellow River irrigation in China.

The level of concentration of heavy metal in soil is detrimental to soil quality. The Heigangkou-Liuyuankou irrigation area in the lower-reach of Yellow River irrigation, as home to a large population and a major site to agricultural production, is vulnerable to heavy metal pollution. This study examined soil quality in Heigangkou-Liuyuankou irrigation areas of Kaifeng, China. Pollution in soil and potential risks introduced by heavy metal accumulation were assessed using Nemerow, Geoaccumulation, and Hakanson's ecological risk indices. Statistics and Geographic Information Systems (GIS) were used to model and present the spatiotemporal changes of the pollution sources and factors affecting the levels of pollution. The heavy metals found in the sampled soil are Cr, Ni, Cu, Zn, Cd, Pb, As, and Hg. Among them, Cd is more concentrated than the others. The southwestern region of the studied area confronts the most serious heavy metal pollution. There exist spatial disparities of low concentrations of different heavy metals in the study area. Hg and Cd are found to pose the highest potential ecological risks. However, their risk levels are not the same across the study area. Levels concentration of Ni, Cu, Zn, Cd, Pb, As, and Hg in soil are highly correlated. In combination, they post an additional threat to the ecological environment. Transportation, rural settlements, and water bodies are found to be the major sources of Cr, Ni, Cu, Zn, Cd, Pb, and Hg pollution in the soil; among the major sources, transportation is the most significant factor.

Rhamnolipid influences biosorption and biodegradation of phenanthrene by phenanthrene-degrading strain Pseudomonas sp. Ph6.

Given the sub-lethal risks of synthetic surfactants, rhamnolipid is a promising class of biosurfactants with the potential to promote the bioavailability of polycyclic aromatic hydrocarbons (PAHs), to provide a favorable substitute for synthetic surfactants. However, few previous studies have integrated the behavior and mechanism behind rhamnolipid-influenced PAH biosorption and biodegradation. This is, to our knowledge, the first report of a bacterial envelope regulated link between phenanthrene (PHE) biosorption and biodegradation by rhamnolipid-induced PHE-degrading strain Pseudomonas sp. Ph6. Rhamnolipid (0─400 mg L-1) can change the cell-surface zeta potential, cell surface hydrophobicity (CSH), cell ultra-microstructure and functional groups, and then alter PHE biosorption and biodegradation of Ph6. Greater amounts of PHE sorbed on cell envelopes results in more PHE diffusing into cytochylema, thus favoring PHE intracellular biodegradation of Ph6. Rhamnolipid (≤100 mg L-1) could change the microstructures and functional groups of cell envelopes of Ph6, enhance the cell-surface zeta potential and CSH, thus consequently favor PHE biosorption and biodegradation by strain Ph6. By contrast, rhamnolipid at higher concentrations (≥200 mg L-1) hindered PHE biosorption and biodegradation. Rhamnolipid, as a biosurfactant, can be successfully utilized as an additive to improve the microbial biodegradation of PAHs in the environments.

Changes in rhizosphere bacterial gene expression following glyphosate treatment.

In commercial agriculture, populations and interactions of rhizosphere microflora are potentially affected by the use of specific agrichemicals, possibly by affecting gene expression in these organisms. To investigate this, we examined changes in bacterial gene expression within the rhizosphere of glyphosate-tolerant corn (Zea mays) and soybean (Glycine max) in response to long-term glyphosate (PowerMAX™, Monsanto Company, MO, USA) treatment. A long-term glyphosate application study was carried out using rhizoboxes under greenhouse conditions with soil previously having no history of glyphosate exposure. Rhizosphere soil was collected from the rhizoboxes after four growing periods. Soil microbial community composition was analyzed using microbial phospholipid fatty acid (PLFA) analysis. Total RNA was extracted from rhizosphere soil, and samples were analyzed using RNA-Seq analysis. A total of 20-28 million bacterial sequences were obtained for each sample. Transcript abundance was compared between control and glyphosate-treated samples using edgeR. Overall rhizosphere bacterial metatranscriptomes were dominated by transcripts related to RNA and carbohydrate metabolism. We identified 67 differentially expressed bacterial transcripts from the rhizosphere. Transcripts downregulated following glyphosate treatment involved carbohydrate and amino acid metabolism, and upregulated transcripts involved protein metabolism and respiration. Additionally, bacterial transcripts involving nutrients, including iron, nitrogen, phosphorus, and potassium, were also affected by long-term glyphosate application. Overall, most bacterial and all fungal PLFA biomarkers decreased after glyphosate treatment compared to the control. These results demonstrate that long-term glyphosate use can affect rhizosphere bacterial activities and potentially shift bacterial community composition favoring more glyphosate-tolerant bacteria.

Biodegradation of 3,3',4,4'-tetrachlorobiphenyl by Sinorhizobium meliloti NM.

A rhizobial strain, Sinorhizobium meliloti NM, could use 3,3',4,4'-tetrachloro-biphenyl (PCB 77) as the sole carbon and energy source for growth in mineral salt medium. The degradation efficiency of PCB 77 by strain NM and the bacterial growth increased with a decrease in PCB 77 concentration (5-0.25mgL(-1)). The addition of secondary carbon sources, phenolic acids and one surfactant influenced PCB 77 degradation, rhizobial growth and biofilm formation. The highest degradation efficiency was observed in the presence of caffeic acid. Benzoate and chloride ions were detected as the PCB 77 metabolites. The up-regulation of benzoate metabolism-related gene expression was also observed using quantitative reverse transcription-polymerase chain reaction. This report is the first to demonstrate Sinorhizobium using coplanar tetrachlorobiphenyl as a sole carbon and energy source, indicating the potential wide benefit to the field of rhizobia-assisted bioremediation.

Glyphosate effects on soil rhizosphere-associated bacterial communities.

Glyphosate is one of the most widely used herbicides in agriculture with predictions that 1.35 million metric tons will be used annually by 2017. With the advent of glyphosate tolerant (GT) cropping more than 10 years ago, there is now concern for non-target effects on soil microbial communities that has potential to negatively affect soil functions, plant health, and crop productivity. Although extensive research has been done on short-term response to glyphosate, relatively little information is available on long-term effects. Therefore, the overall objective was to investigate shifts in the rhizosphere bacterial community following long-term glyphosate application on GT corn and soybean in the greenhouse. In this study, rhizosphere soil was sampled from rhizoboxes following 4 growth periods, and bacterial community composition was compared between glyphosate treated and untreated rhizospheres using next-generation barcoded sequencing. In the presence or absence of glyphosate, corn and soybean rhizospheres were dominated by members of the phyla Proteobacteria, Acidobacteria, and Actinobacteria. Proteobacteria (particularly gammaproteobacteria) increased in relative abundance for both crops following glyphosate exposure, and the relative abundance of Acidobacteria decreased in response to glyphosate exposure. Given that some members of the Acidobacteria are involved in biogeochemical processes, a decrease in their abundance could lead to significant changes in nutrient status of the rhizosphere. Our results also highlight the need for applying culture-independent approaches in studying the effects of pesticides on the soil and rhizosphere microbial community.

Application of organic amendments to restore degraded soil: effects on soil microbial properties.

Topsoil removal, compaction, and other practices in urban and industrial landscapes can degrade soil and soil ecosystem services. There is growing interest to remediate these for recreational and residential purposes, and urban waste materials offers potential to improve degraded soils. Therefore, the objective of this study was to compare the effects of urban waste products on microbial properties of a degraded industrial soil. The soil amendments were vegetative yard waste compost (VC), biosolids (BioS), and a designer mix (DM) containing BioS, biochar (BC), and drinking water treatment residual (WTR). The experiment had a completely randomized design with following treatments initiated in 2009: control soil, VC, BioS-1 (202 Mg ha(-1)), BioS-2 (403 Mg ha(-1)), and DM (202 Mg BioS ha(-1) plus BC and WTR). Soils (0-15-cm depth) were sampled in 2009, 2010, and 2011 and analyzed for enzyme activities (arylsulfatase, ß-glucosaminidase, ß-glucosidase, acid phosphatase, fluorescein diacetate, and urease) and soil microbial community structure using phospholipid fatty acid analysis (PLFA). In general, all organic amendments increased enzyme activities in 2009 with BioS treatments having the highest activity. However, this was followed by a decline in enzyme activities by 2011 that were still significantly higher than control. The fungal PLFA biomarkers were highest in the BioS treatments, whereas the control soil had the highest levels of the PLFA stress markers (P < 0.10). In conclusion, one-time addition of VC or BioS was most effective on enzyme activities; the BioS treatment significantly increased fungal biomass over the other treatments; addition of BioS to soils decreased microbial stress levels; and microbial measures showed no statistical differences between BioS and VC treatments after 3 years of treatment.

Association of shifting populations in the root zone microbiome of millet with enhanced crop productivity in the Sahel region (Africa).

This study characterized specific changes in the millet root zone microbiome stimulated by long-term woody-shrub intercropping at different sites in Senegal. At the two study sites, intercropping with woody shrubs and shrub residue resulted in a significant increase in millet [Pennisetum glaucum (L.) R. Br.] yield (P < 0.05) and associated patterns of increased diversity in both bacterial and fungal communities in the root zone of the crop. Across four experiments, operational taxonomic units (OTUs) belonging to Chitinophaga were consistently significantly (P < 0.001) enriched in the intercropped samples, and "Candidatus Koribacter" was consistently significantly enriched in samples where millet was grown alone. Those OTUs belonging to Chitinophaga were enriched more than 30-fold in residue-amended samples and formed a distinct subgroup from all OTUs detected in the genus. Additionally, OTUs belonging to 8 fungal genera (Aspergillus, Coniella, Epicoccum, Fusarium, Gibberella, Lasiodiplodia, Penicillium, and Phoma) were significantly (P < 0.005) enriched in all experiments at all sites in intercropped samples. The OTUs of four genera (Epicoccum, Fusarium, Gibberella, and Haematonectria) were consistently enriched at sites where millet was grown alone. Those enriched OTUs in intercropped samples showed consistently large-magnitude differences, ranging from 30- to 1,000-fold increases in abundance. Consistently enriched OTUs in intercropped samples in the genera Aspergillus, Fusarium, and Penicillium also formed phylogenetically distinct subgroups. These results suggest that the intercropping system used here can influence the recruitment of potentially beneficial microorganisms to the root zone of millet and aid subsistence farmers in producing higher-yielding crops.

Comparison of whole-cell fatty acid (MIDI) or phospholipid fatty acid (PLFA) extractants as biomarkers to profile soil microbial communities.

The whole-cell lipid extraction to profile microbial communities on soils using fatty acid (FA) biomarkers is commonly done with the two extractants associated with the phospholipid fatty acid (PLFA) or Microbial IDentification Inc. (MIDI) methods. These extractants have very different chemistry and lipid separation procedures, but often shown a similar ability to discriminate soils from various management and vegetation systems. However, the mechanism and the chemistry of the exact suite of FAs extracted by these two methods are poorly understood. Therefore, the objective was to qualitatively and quantitatively compare the MIDI and PLFA microbial profiling methods for detecting microbial community shifts due to soil type or management. Twenty-nine soil samples were collected from a wide range of soil types across Oregon and extracted FAs by each method were analyzed by gas chromatography (GC) and GC-mass spectrometry. Unlike PLFA profiles, which were highly related to microbial FAs, the overall MIDI-FA profiles were highly related to the plant-derived FAs. Plant-associated compounds were quantitatively related to particulate organic matter (POM) and qualitatively related to the standing vegetation at sampling. These FAs were negatively correlated to respiration rate normalized to POM (RespPOM), which increased in systems under more intensive management. A strong negative correlation was found between MIDI-FA to PLFA ratios and total organic carbon (TOC). When the reagents used in MIDI procedure were tested for the limited recovery of MIDI-FAs from soil with high organic matter, the recovery of MIDI-FA microbial signatures sharply decreased with increasing ratios of soil to extractant. Hence, the MIDI method should be used with great caution for interpreting changes in FA profiles due to shifts in microbial communities.

Lipid profiling of the soybean pathogen Phytophthora sojae using Fatty Acid Methyl Esters (FAMEs).

Phytophthora sojae is a destructive soilborne pathogen of soybean, but currently there is no rapid or commercially available testing for its infestation level in soil. For growers, such information would greatly improve their ability to make management decisions to minimize disease damage to soybean crops. Fatty acid profiling of P. sojae holds potential for determining the prevalence of this pathogen in soil. In this study, the Fatty Acid Methyl Ester (FAME) profile of P. sojae was determined in pure culture, and the profile was subsequently evaluated for its potential use in detecting the pathogen in soil. The predominant fatty acids in the FAME profile of P. sojae are the unsaturated 18C fatty acids (18:1ω9 and 18:2ω6) followed by the saturated and unsaturated 16C fatty acids (16:0 and 16:1ω7). FAME analysis of P. sojae zoospores showed two additional long-chain saturated fatty acids (20:0 and 22:0) that were not detected in the mycelium of this organism. Addition of a known number of zoospores of P. sojae to soil demonstrated that fatty acids such as 18:1ω9, 18:2ω6, 20:1ω9, 20:4ω6, and 22:1ω9 could be detected and quantified against the background levels of fatty acids present in soil. These results show the potential for using selected FAMEs of P. sojae as a marker for detecting this pathogen in soybean fields.

Standardizing methylation method during phospholipid fatty acid analysis to profile soil microbial communities.

Phospholipid fatty acid (PLFA) as biomarkers, is widely used to profile microbial communities in environmental samples. However, PLFA extraction and derivatization protocols are not standardized and have widely varied among published studies. Specifically investigators have used either HCl/MeOH or KOH/MeOH or both for the methylation step of PLFA analysis, without justification or research to support either one. It seems likely that each method could have very different outcomes and conclusions for PLFA based studies. Therefore, the objective of this study was to determine the effect of catalyst type for methylation on detecting PLFAs and implications for interpreting microbial profiling in soil. Fatty acid samples extracted from soils obtained from a wetland, an intermittently flooded site, and an adjacent upland site were subjected to HCl/MeOH or KOH/MeOH catalyzed methylation procedures during PLFA analyses. The methylation method using HCl/MeOH resulted in significantly higher concentrations of most PLFAs than the KOH/MeOH method. Another important outcome was that fatty acids with a methyl group (18:1ω,7c 11Me, TBSA 10Me 18:0, 10Me 18:0, 17:0 10Me and 16:0 10Me being an actinomycetes biomarker) could not be detected by HCl/MeOH catalyzed methylation but were found in appreciable concentrations with KOH/MeOH method. From our results, because the HCl/MeOH method did not detect the fatty acids containing methyl groups that could strongly influence the microbial community profile, we recommend that the KOH/MeOH catalyzed transesterification method should become the standard procedure for PLFA profiling of soil microbial communities.

Stigmasterol and cholesterol regulate the expression of elicitin genes in Phytophthora sojae.

Sterol acquisition by soilborne plant pathogens of the genus Phytophthora is presumed to involve extracellular proteins belonging to class-I elicitins. However, little is known about the relationship between sterol availability and elicitin secretion. The objective of this study was to determine the expression of class-I elicitin genes in Phytophthora sojae when grown in a medium containing stigmasterol or cholesterol. P. sojae growth was stimulated by nanomolar concentrations of stigmasterol and cholesterol, which also resulted in the down-regulation of its elicitin genes over time when expression profiles were monitored using real time Reverse Transcription Polymerase Chain Reaction (RT-PCR). The down-regulation of elicitin genes in response to the two sterols also coincided with a reduction in the amount of elicitins detected in spent filtrates. Our study is the first to show the influence of sterols on elicitin gene expression in Phytophthora, which is important with respect to the ecology of elicitin secretion as sterol carrier proteins in the environment.

PLFA profiling of microbial community structure and seasonal shifts in soils of a Douglas-fir chronosequence.

The impact and frequency of forest harvesting could significantly affect soil microbial community (SMC) structure and functioning. The ability of soil microorganisms to perform biogeochemical processes is critical for sustaining forest productivity and has a direct impact on decomposition dynamics and carbon storage potential. The Wind River Canopy Crane Research Forest in SW, WA, provided a unique opportunity to study a forest chronosequence and the residual effects of harvesting on the SMC in comparison to old-growth forests. The objective of this study was to determine the effect of clear-cutting and stand age on temporal dynamics of SMC and physiological stress markers using phospholipid fatty acid (PLFA) profiling. Soil microbial PLFA profiles were determined seven times over 22 months (Nov. 02 to Sep. 04) in old-growth coniferous forest stands (300-500 years) and 8 (CC8)- or 25 (CC25)-year-old replanted clear-cuts. PLFA patterns of the SMC shifted because of clear-cutting, but seasonal temporal changes had greater shifts than differences among stand age. The microbial biomass (total PLFA) and bacterial, fungal, and selected other PLFAs were significantly reduced in CC8 but not in CC25 sites relative to the old-growth sites. An increase in stress indicators [PLFA ratios of saturated/monsaturated and (cy17:0 + cy19:0)/(16:1omega7 + 18:1omega7)] in late summer was related to water stress. Although the canopy and litter input are quite different for a 25-year clear-cut compared to virgin old-growth forest, we conclude that the composition of the microbial communities, 25 years after clear-cutting, has recovered sufficiently to be much more similar to old-growth forests than a recent clear-cut at this Pacific Northwest forest site. The study shows the potential of PLFA analysis for profiling microbial communities and their stress status under field conditions, but wide temporal shifts emphasize the need for sampling over seasons to fully interpret ecosystem management impacts on microbial populations.

Seasonal variations in sugar contents and microbial community in a ryegrass soil.

The relationship among sugar concentrations, microbial community and physical variables (precipitation and soil temperature) was investigated in a ryegrass soil from January 2004 to January 2005. Mono- and disaccharide sugars were extracted using a mixture of dichloromethane and methanol and analyzed as their TMS derivatives by GC-MS. Changes in microbial community were assessed using phospholipid and neutral lipid fatty acids (PLFA and NLFA, respectively) analysis. The results of a one-year study showed that the seasonal variability of sugar contents found in the soil samples is mainly related to biomass or nutritional status of the fungal community. The increase in sucrose and fructose exudation by plant roots in the beginning of the growing season (early spring) may be responsible for the highest fungal biomass amount (PLFAs) observed in this study. Fungal storage lipid abundances (NLFAs) peaked in summer, during the same period that the highest concentrations of mannitol and trehalose were detected. This is consistent with these two sugars being stress-induced fungal metabolites, produced due to the low soil moisture observed during this season. In contrast, bacterial community growth seems to be more dependent on plant substrate than on physical variables, since the strongest decrease in bacterial biomass amounts (PLFAs) was found after cutting of the ryegrass field in early July.

Microbial response to heavy metal-polluted soils: community analysis from phospholipid-linked fatty acids and ester-linked fatty acids extracts.

Heavy metal pollution of soil is of concern for human health and ecosystem function. The soil microbial community should be a sensitive indicator of metal contamination effects on bioavailability and biogeochemical processes. Simple methods are needed to determine the degree of in situ pollution and effectiveness of remediating metal-contaminated soils. Currently, phospholipid-linked fatty acids (PLFAs) are preferred for microbial profiling but this method is time consuming, whereas direct soil extraction and transesterification of total ester-linked fatty acids (ELFAs) is attractive because of its simplicity. The 1998 mining acid-metal spill of >4000 ha in the Guadiamar watershed (southwestern Spain) provided a unique opportunity to study these two microbial lipid profiling methods. Replicated treatments were set up as nonpolluted, heavy metal polluted and reclaimed, and polluted soils. Inferences from whole community-diversity analysis and correlations of individual fatty acids with metals suggested Cu, Cd, and Zn were the most important in affecting microbial community structure, along with pH. The microbial stress marker, monounsaturated fatty acids, was significantly lower for reclaimed and polluted soil over nonpolluted soils for both PLFA and ELFA extraction. Another stress marker, the monounsaturated to saturated fatty acids ratio, only showed this for the PLFA. The general fungal marker (18:2omega6c), the arbuscule mycorrhizae marker (16:1omega5c), and iso- and anteiso-branched PLFAs (gram positive bacteria) were suppressed with increasing pollution whereas 17:0cy (gram negative bacteria) increased with metal pollution. For both extraction methods, richness and diversity were greater in nonpolluted soils and lowest in polluted soils. The ELFA method was sensitive for reflecting metal pollution on microbial communities and could be suitable for routine use in ecological monitoring and risk assessment programs because of its simplicity and reproducibility.