Phospholipid Fatty Acid Composition and Heavy Metal Tolerance of Soil Microbial Communities along Two Heavy Metal-Polluted Gradients in Coniferous Forests

The effects of long-term heavy metal deposition on microbial community structure and the level of bacterial community tolerance were studied along two different gradients in Scandinavian coniferous forest soils. One was near the Harjavalta smelter in Finland, and one was at Ronnskar in Sweden. Phospholipid fatty acid (PLFA) analysis revealed a gradual change in soil microbial communities along both pollution gradients, and most of the individual PLFAs changed similarly to metal pollution at both sites. The relative quantities of the PLFAs br18:0, br17:0, i16:0, and i16:1 increased with increasing heavy metal concentration, while those of 20:4 and 18:2(omega)6, which is a predominant PLFA in many fungi, decreased. The fungal part of the microbial biomass was found to be more sensitive to heavy metals. This resulted in a decreased fungal/bacterial biomass ratio along the pollution gradient towards the smelters. The thymidine incorporation technique was used to study the heavy metal tolerance of the bacteria. The bacterial community at the Harjavalta smelter, exposed mainly to Cu deposition, exhibited an increased tolerance to Cu but not to Cd, Ni, and Zn. At the Ronnskar smelter the deposition consisting of a mixture of metals increased the bacterial community tolerance to all tested metals. Both the PLFA pattern and the bacterial community tolerance were affected at lower soil metal concentrations than were bacterial counts and bacterial activities. At Harjavalta the increased Cu tolerance of the bacteria and the change in the PLFA pattern of the microbial community were found at the same soil Cu concentrations. This indicated that the altered PLFA pattern was at least partly due to an altered, more metal-tolerant bacterial community. At Ronnskar, where the PLFA data varied more, a correlation between bacterial community tolerance and an altered PLFA pattern was found up to 10 to 15 km from the smelter. Farther away changes in the PLFA pattern could not be explained by an increased community tolerance to metals.     

Determinants of Soil Microbial Communities: Effects of Agricultural Management, Season, and Soil Type on Phospholipid Fatty Acid Profiles

Abstract Phospholipid fatty acid (PLFA) profiles were measured in soils from organic, low-input, and conventional farming systems that are part of the long term Sustainable Agriculture Farming Systems (SAFS) Project. The farming systems differ in whether their source of fertilizer is mineral or organic, and in whether a winter cover crop is grown. Sustained increases in microbial biomass resulting from high organic matter inputs have been observed in the organic and low-input systems. PLFA profiles were compared to ascertain whether previously observed changes in biomass were accompanied by a change in the composition of the microbial community. In addition, the relative importance of environmental variables on PLFA profiles was determined. Redundancy analysis ordination showed that PLFA profiles from organic and conventional systems were significantly different from April to July. On ordination plots, PLFA profiles from the low-input system fell between organic and conventional systems on most sample dates. A group of fatty acids (i14:0, a15:0, 16:1ω7c, 16:1ω5c, 14:0, and 18:2ω6c) was enriched in the organic plots throughout the sampling period, and another group (10Me16:0, 2OH 16:1 and 10Me17:0) was consistently lower in relative abundance in the organic system. In addition, another group (15:0, a17:0, i16:0, 17:0, and 10Me18:0) was enriched over the short term in the organic plots after compost incorporation. The relative importance of various environmental variables in governing the composition of microbial communities could be ranked in the order: soil type > time > specific farming operation (e.g., cover crop incorporation or sidedressing with mineral fertilizer) > management system > spatial variation in the field. Measures of the microbial community and soil properties (including microbial biomass carbon and nitrogen, substrate induced respiration, basal respiration, potentially mineralizable nitrogen, soil nitrate and ammonium, and soil moisture) were seldom associated with the variation in the PLFA profiles. Received: 3 February 1997; Accepted: 7 August 1997     

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 (Resp_POM), 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.     

Composition of Toluene-Degrading Microbial Communities from Soil at Different Concentrations of Toluene

Toluene-degrading bacteria were isolated from hydrocarbon-contaminated soil by incubating liquid enrichment cultures and agar plate cultures in desiccators in which the vapor pressure of toluene was controlled by dilution with vacuum pump oil. Incubation in desiccators equilibrated with either 100, 10, or 1% (wt/wt) toluene in vacuum pump oil and testing for genomic cross-hybridization resulted in four genomically distinct strains (standards) capable of growth on toluene (strains Cstd1, Cstd2, Cstd5, and Cstd7). The optimal toluene concentrations for growth of these standards on plating media differed considerably. Cstd1 grew best in an atmosphere equilibrated with 0.1% (wt/wt) toluene, but Cstd5 failed to grow in this atmosphere. Conversely, Cstd5 grew well in the presence of 10% (wt/wt) toluene, which inhibited growth of Cstd1. 16S ribosomal DNA sequencing and cross-hybridization analysis indicated that both Cstd1 and Cstd5 are members of the genus Pseudomonas. An analysis of the microbial communities in soil samples that were incubated with 10% (wt/wt) toluene with reverse sample genome probing indicated that Pseudomonas strain Cstd5 was the dominant community member. However, incubation of soil samples with 0.1% (wt/wt) toluene resulted in a community that was dominated by Pseudomonas strain Q7, a toluene degrader that has been described previously (Y. Shen, L. G. Stehmeier, and G. Voordouw, Appl. Environ. Microbiol. 64:637–645, 1998). Q7 was not able to grow by itself in an atmosphere equilibrated with 0.1% (wt/wt) toluene but grew efficiently in coculture with Cstd1, suggesting that toluene or metabolic derivatives of toluene were transferred from Cstd1 to Q7.     

Soil Bacterial Biomass, Activity, Phospholipid Fatty Acid Pattern, and pH Tolerance in an Area Polluted with Alkaline Dust Deposition

Soil bacterial biomass, phospholipid fatty acid pattern, pH tolerance, and growth rate were studied in a forest area in Finland that is polluted with alkaline dust from an iron and steel works. The pollution raised the pH of the humus layer from 4.1 to 6.6. Total bacterial numbers and the total amounts of bacterial phospholipid fatty acids in the humus layer did not differ between the unpolluted control sites and the polluted ones. The number of CFU increased by a factor of 6.4 in the polluted sites compared with the controls, while the bacterial growth rate, measured by the thymidine incorporation technique, increased about 1.8-fold in the polluted sites. A shift in the pattern of phospholipid fatty acids indicated a shift in the bacterial species composition. The largest proportional increase was found for the fatty acid 10Me18:0, which indicated an increase in the number of actinomycetes in the polluted sites. The levels of the fatty acids i14:0, 16:1ω5, cy17:0, 18:1ω7, and 19:1 also increased in the polluted sites while those of fatty acids 15:0, i15:0, 10Me16:0, 16:1ω7t, 18:1ω9, and cy19:0 decreased compared with the unpolluted sites. An altered pH tolerance of the bacterial assemblage was detected either as a decrease in acid-tolerant CFU in the polluted sites or as altered bacterial growth rates at different pHs. The latter was estimated by measuring the thymidine incorporation rate of bacteria extracted from soil by homogenization-centrifugation at different pHs.     

The Use of Phospholipid Fatty Acid Analysis to Measure Impact of Acid Rock Drainage on Microbial Communities in Sediments

The impact of acid rock drainage (ARD) and eutrophication on microbial communities in stream sediments above and below an abandoned mine site in the Adelaide Hills, South Australia, was quantified by PLFA analysis. Multivariate analysis of water quality parameters, including anions, soluble heavy metals, pH, and conductivity, as well as total extractable metal concentrations in sediments, produced clustering of sample sites into three distinct groups. These groups corresponded with levels of nutrient enrichment and/or concentration of pollutants associated with ARD. Total PLFA concentration, which is indicative of microbial biomass, was reduced by >70% at sites along the stream between the mine site and as far as 18 km downstream. Further downstream, however, recovery of the microbial abundance was apparent, possibly reflecting dilution effect by downstream tributaries. Total PLFA was >40% higher at, and immediately below, the mine site (0–0.1 km), compared with sites further downstream (2.5–18 km), even after accounting for differences in specific surface area of different sediment samples. The increased microbial population in the proximity of the mine source may be associated with the presence of a thriving iron-oxidizing bacteria community as a consequence of optimal conditions for these organisms while the lower microbial population further downstream corresponded with greater sediments metal concentrations. PCA of relative abundance revealed a number of PLFAs which were most influential in discriminating between ARD-polluted sites and the rest of the sites. These PLFA included the hydroxy fatty acids: 2OH12:0, 3OH12:0, 2OH16:0; the fungal marker: 18:26; the sulfate-reducing bacteria marker 10Me16:17; and the saturated fatty acids 12:0, 16:0, 18:0. Partial constrained ordination revealed that the environmental parameters with the greatest bearing on the PLFA profiles included pH, soluble aluminum, total extractable iron, and zinc. The study demonstrated the successful application of PLFA analysis to rapidly assess the toxicity of ARD-affected waters and sediments and to differentiate this response from the effects of other pollutants, such as increased nutrients and salinity.     

Microbial Diversity and Community Structure in Two Different Agricultural Soil Communities

Abstract In this study, two different agricultural soils were investigated: one organic soil and one sandy soil, from Stend (south of Bergen), Norway. The sandy soil was a field frequently tilled and subjected to crop rotations. The organic soil was permanent grazing land, infrequently tilled. Our objective was to compare the diversity of the cultivable bacteria with the diversity of the total bacterial population in soil. About 200 bacteria, randomly isolated by standard procedures, were investigated. The diversity of the cultivable bacteria was described at phenotypic, phylogenetic, and genetic levels by applying phenotypical testing (Biolog) and molecular methods, such as amplified rDNA restriction analysis (ARDRA); hybridization to oligonucleotide probes; and REP-PCR. The total bacterial diversity was determined by reassociation analysis of DNA isolated from the bacterial fraction of environmental samples, combined with ARDRA and DGGE analysis. The relationship between the diversity of cultivated bacteria and the total bacteria was elucidated. Organic soil exhibited a higher diversity for all analyses performed than the sandy soil. Analysis of cultivable bacteria resulted in different resolution levels and revealed a high biodiversity within the population of cultured isolates. The difference between the two agricultural soils was significantly higher when the total bacterial population was analyzed than when the cultivable population was. Thus, analysis of microbial diversity must ultimately embrace the entire microbial community DNA, rather than DNA from cultivable bacteria.     

Phospholipid Distribution and Fatty Acid Composition in Different Brain Regions During Chick Embryo Development

Abstract: The phospholipid profile of different chick embryo brain regions was studied from 11 to 21 days of development, revealing interesting changes in content and distribution. Total phospholipid phosphorus (P), in micrograms of P per microgram of DNA, increases significantly during development of cerebral hemispheres (CHs), optic lobes (OLs), and brainstem (BS). Compared with CH and OL, the BS shows at all stages a significantly higher concentration of phospholipid P, which in contrast decreases in the cerebellum (CB) during development. Moreover, the data show interesting differences between the right and the left portion of the brain. The distribution of phospholipid P and the fatty acid composition of phospholipids were asymmetric between left and right OL and CH, as were the concentrations of DNA and cholesterol, demonstrating lateralized neurochemical development in these structures, i.e., left OL, right OL, left CH, and right CH. The data are discussed also in relation to the potential importance of neurochemical lateralization for determining lateralized embryonic and postnatal behavior of this species.     

Estimating High-Affinity Methanotrophic Bacterial Biomass, Growth, and Turnover in Soil by Phospholipid Fatty Acid 13C Labeling

A time series phospholipid fatty acid (PLFA) ¹³C-labeling study was undertaken to determine methanotrophic taxon, calculate methanotrophic biomass, and assess carbon recycling in an upland brown earth soil from Bronydd Mawr (Wales, United Kingdom). Laboratory incubations of soils were performed at ambient CH₄ concentrations using synthetic air containing 2 parts per million of volume of ¹³CH₄. Flowthrough chambers maintained a stable CH₄ concentration throughout the 11-week incubation. Soils were analyzed at weekly intervals by gas chromatography (GC), GC-mass spectrometry, and GC-combustion-isotope ratio mass spectrometry to identify and quantify individual PLFAs and trace the incorporation of ¹³C label into the microbial biomass. Incorporation of the ¹³C label was seen throughout the experiment, with the rate of incorporation decreasing after 9 weeks. The δ¹³C values of individual PLFAs showed that ¹³C label was incorporated into different components to various extents and at various rates, reflecting the diversity of PLFA sources. Quantitative assessments of ¹³C-labeled PLFAs showed that the methanotrophic population was of constant structure throughout the experiment. The dominant ¹³C-labeled PLFA was 18:1ω7c, with 16:1ω5 present at lower abundance, suggesting the presence of novel type II methanotrophs. The biomass of methane-oxidizing bacteria at optimum labeling was estimated to be about 7.2 × 10⁶ cells g⁻¹ of soil (dry weight). While recycling of ¹³C label from the methanotrophic biomass must occur, it is a slower process than initial ¹³CH₄ incorporation, with only about 5 to 10% of ¹³C-labeled PLFAs reflecting this process. Thus, ¹³C-labeled PLFA distributions determined at any time point during ¹³CH₄ incubation can be used for chemotaxonomic assessments, although extended incubations are required to achieve optimum ¹³C labeling for methanotrophic biomass determinations.     

Development of Metal Tolerance in Soil Bacterial Communities Exposed to Experimentally Increased Metal Levels

The development of metal tolerance in soil bacterial communities exposed to different heavy metals was examined under laboratory conditions. An agricultural soil amended with different Zn concentrations was studied most intensively, and measurements were made over a 28-month incubation period by means of the thymidine incorporation technique. Tolerance levels were not affected by metal concentrations lower than 2 mmol of Zn kg (dry weight) of soil(sup-1), but above this value, the level of Zn tolerance increased exponentially with the logarithm of the soil Zn concentration. An increased metal tolerance was detected after only 2 days of Zn exposure. Thereafter, stable tolerance values were observed at different sampling times for bacterial communities exposed to up to 8 mmol of Zn kg (dry weight)(sup-1), indicating no changes in tolerance with time. The tolerance of bacterial communities exposed to 32 mmol of Zn kg (dry weight)(sup-1) increased rapidly within the second week of incubation, but then the values remained unchanged until the end of the experiment. Bacterial communities from soil contaminated with 16 mmol of Zn kg (dry weight)(sup-1) showed an increase of the same magnitude, but the increase started later, after 4 months of incubation, and took place for a much longer period (more than 1 year). Cd, Cu, and Ni addition also resulted in metal-tolerant communities, and the level of tolerance increased with prolonged incubations of the soils. The bacterial community at the end of the incubation period also exhibited a lower pH optimum and an increased tolerance to low osmotic potential. The results suggest that the increase in metal tolerance of the community after adding metals can be attributed to an immediate effect due to the death of sensitive species and a later effect due to different competitive abilities and adaptation of surviving bacteria.     

Effects of Sieving, Storage, and Incubation Temperature on the Phospholipid Fatty Acid Profile of a Soil Microbial Community

Disturbances typically associated with the study of soil microbial communities, i.e., sieving, storage, and subsequent incubation at elevated temperatures, were investigated with phospholipid fatty acid (PLFA) analyses. Treatment effects were quantified by statistical analyses of the mole percentage distribution of the individual fatty acids. Changes in the concentrations of individual fatty acids over a 7-week storage period at 4.5°C were generally not statistically significant. Sieving effects (mesh size, 4 or 2 mm) on CO₂ evolution and the PLFA profile were monitored over 3 weeks; the physical disturbance had only minor effects, although some damage to fungal hyphae by the first sieving (<4 mm) was suggested by a decrease in the signature fatty acid 18:2 ω6c. Temperature effects were investigated by incubating soil for up to 3 weeks at 4.5, 10, or 25°C. Principal component analyses demonstrated a significant shift in the PLFA composition at 25°C over the first 2 weeks, while changes at the other two temperatures were minor. Several of the changes observed at 25°C could be explained with reference to mechanisms of temperature adaptation or as a response to conditions of stress, including a decrease in the degree of unsaturation, an increased production of cyclopropyl fatty acids, and increased ratios of the branched-chain fatty acids iso-15:0 and iso-17:0 over anteiso-15:0 and anteiso-17:0, respectively. A decrease in the total amount of PLFA was also indicated.     

Organic Amendments to Avocado Crops Induce Suppressiveness and Influence the Composition and Activity of Soil Microbial Communities

One of the main avocado diseases in southern Spain is white root rot caused by the fungus Rosellinia necatrix Prill. The use of organic soil amendments to enhance the suppressiveness of natural soil is an inviting approach that has successfully controlled other soilborne pathogens. This study tested the suppressive capacity of different organic amendments against R. necatrix and analyzed their effects on soil microbial communities and enzymatic activities. Two-year-old avocado trees were grown in soil treated with composted organic amendments and then used for inoculation assays. All of the organic treatments reduced disease development in comparison to unamended control soil, especially yard waste (YW) and almond shells (AS). The YW had a strong effect on microbial communities in bulk soil and produced larger population levels and diversity, higher hydrolytic activity and strong changes in the bacterial community composition of bulk soil, suggesting a mechanism of general suppression. Amendment with AS induced more subtle changes in bacterial community composition and specific enzymatic activities, with the strongest effects observed in the rhizosphere. Even if the effect was not strong, the changes caused by AS in bulk soil microbiota were related to the direct inhibition of R. necatrix by this amendment, most likely being connected to specific populations able to recolonize conducive soil after pasteurization. All of the organic amendments assayed in this study were able to suppress white root rot, although their suppressiveness appears to be mediated differentially.     

Soil Microbial Biomass,Basal Respiration and Enzyme Activity of Main Forest Types in the Qinling Mountains

Different forest types exert essential impacts on soil physical-chemical characteristics by dominant tree species producing diverse litters and root exudates, thereby further regulating size and activity of soil microbial communities. However, the study accuracy is usually restricted by differences in climate, soil type and forest age. Our objective is to precisely quantify soil microbial biomass, basal respiration and enzyme activity of five natural secondary forest (NSF) types with the same stand age and soil type in a small climate region and to evaluate relationship between soil microbial and physical-chemical characters. We determined soil physical-chemical indices and used the chloroform fumigation-extraction method, alkali absorption method and titration or colorimetry to obtain the microbial data. Our results showed that soil physical-chemical characters remarkably differed among the NSFs. Microbial biomass carbon (Cmic) was the highest in wilson spruce soils, while microbial biomass nitrogen (Nmic) was the highest in sharptooth oak soils. Moreover, the highest basal respiration was found in the spruce soils, but mixed, Chinese pine and spruce stands exhibited a higher soil qCO₂. The spruce soils had the highest Cmic/Nmic ratio, the greatest Nmic/TN and Cmic/Corg ratios were found in the oak soils. Additionally, the spruce soils had the maximum invertase activity and the minimum urease and catalase activities, but the maximum urease and catalase activities were found in the mixed stand. The Pearson correlation and principle component analyses revealed that the soils of spruce and oak stands obviously discriminated from other NSFs, whereas the others were similar. This suggested that the forest types affected soil microbial properties significantly due to differences in soil physical-chemical features.     

Differences of Soil Microbial Biomass and Nitrogen Transformation under Two Forest Types in Central Germany

In order to observe the tree species effect on soil N status, soil microbial biomass C and N (C_mic, N_mic), potential N mineralization and potential nitrification (under laboratory incubation conditions, 22 °C) in different subhorizons (LOf₁, Of₂, Oh and mineral soil at 0–10 cm depth) were determined at three forest sites in central Germany. At each site, two contrasting stands (Beech, Norway spruce or Scots pine) were selected, where the initial soil conditions were similar. Three sampling dates that represented different stages of tree growth were selected: growing season - August, dormant season - November, after budbreak – April. In organic layers, C_mic-to-total C (C_t) ratios under beech and under conifer were 0.72–4.74% and 0.34–2.11%, respectively. N_mic-to-total N (N_t) ratios were 2.47–11.61% and 0.71–5.77%, respectively. Both concentrations of C_mic and N_mic were significantly affected by the stand type and sampling time. Potential N mineralization rates, ranging from 3.7 to 19.7 mg N kg⁻¹ d⁻¹, showed no clear pattern in relation to stand type. However, potential nitrification rates were mostly significantly higher under beech than under contrasting conifer. In mineral soils, concentrations of C_mic and N_mic showed a clear temporal pattern in the order: August>November>April. The average N_mic and N_mic-to-N_t were higher in soils from beech than conifer, while C_mic and C_mic-to-C_t ratios were similar between the two forest types. In organic layers, the highest values of C_mic-to-N_mic ratio and C_mic were found in November samples, especially under beech. By contrast, in mineral soils the highest value of C_mic-to-N_mic ratios were found in April samples, and at that time the C_mic concentrations were the lowest, especially under conifer. These results revealed the differences in microbial growth form and survival strategy associated with different tree species and soil layers.     

Evaluation of Microbial Biomass and Activity in Different Soils Exposed to Increasing Level of Arsenic Pollution: A Laboratory Study

Soil contamination with arsenic is a widespread phenomenon in many parts of the world and it is well known that it may affect the soil microbial community. A laboratory experiment was performed to evaluate the acute toxicity effect of different As concentrations (0, 0.1, 1.0, 10, 100, and 200 mg/kg) on soil biological parameters like microbial biomass carbon (MBC), active microbial biomass carbon (AMBC), basal soil respiration (BSR), fluorescein diacetate hydrolase activity (FDA), and dehydrogenase activity (DHA) under three different soil types (Inceptisol, Vertisol, and Entisol). Soil enzyme activities, microbial biomass, and respiration were significantly decreased by arsenic exposure. The soil metabolic quotient increased with increasing As concentration, the maximum being observed for Entisol followed by Vertisol and Inceptisol. Except for soil enzyme activities, the majority of which started to decrease after the addition of 10 mg/kg arsenic, all other parameters decreased at arsenic dose ≥0.1 mg/kg. The decrease of AMBC/MBC ratio showed that acute arsenic stress affected the active microorganisms more than the dormant component of total microbial biomass. Among the three soils studied, Vertisol was comparatively more tolerant to As exposure in terms of soil enzymes and biomass, followed by Inceptisol and Entisol. MBC has evolved as a distinct parameter in cluster analysis, while some others, such as DHA and AMBC, and FDA and BSR, appeared essentially redundant.     

Phylogenetic Diversity of Microbial Communities from the Surface of Polyethylene Terephthalate Materials Exposed to Different Water Environments

Microbiology - The goal of the present work was to investigate the composition of microbial communities developing on polyethylene terephthalate (PET) samples immersed in aqueous media of various...     

Effects of Pesticides on the Fatty Acid and Phospholipid Composition of Escherichia coli

[This corrects the article on p. 232 in vol. 40.].