Erland Bååth

The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil

The cell content of 12 bacterial phospholipid fatty acids (PLFA) was determined in bacteria extracted from soil by homogenization/centrifugation. The bacteria were enumerated using acridine orange direct counts. An average of 1.40×10-17 mol bacterial PLFA cell-1 was found in bacteria extracted from 15 soils covering a wide range of pH and organic matter contents. With this factor, the bacterial biomass based on PLFA analyses of whole soil samples was calculated as 1.0–4.8 mg bacterial C g-1 soil C. The corresponding range based on microscopical counts was 0.3–3.0 mg bacterial C g-1 soil C. The recovery of bacteria from the soils using homogenization/centrifugation was 2.6–16% (mean 8.7%) measured by PLFA analysis, and 12–61% (mean 26%) measured as microscopical counts. The soil content of the PLFA 18:2ω6 was correlated with the ergosterol content (r=0.92), which supports the use of this PLFA as an indicator of fungal biomass. The ratio 18:2ω6 to bacterial PLFA is therefore suggested as an index of the fungal:bacterial biomass ratio in soil. An advantage with the method based on PLFA analyses is that the same technique and even the same sample is used to determine both fungi and bacteria. The fungal:bacterial biomass ratio calculated in this way was positively correlated with the organic matter content of the soils (r=0.94).

Soil bacterial and fungal communities across a pH gradient in an arable soil.

Soils collected across a long-term liming experiment (pH 4.0–8.3), in which variation in factors other than pH have been minimized, were used to investigate the direct influence of pH on the abundance and composition of the two major soil microbial taxa, fungi and bacteria. We hypothesized that bacterial communities would be more strongly influenced by pH than fungal communities. To determine the relative abundance of bacteria and fungi, we used quantitative PCR (qPCR), and to analyze the composition and diversity of the bacterial and fungal communities, we used a bar-coded pyrosequencing technique. Both the relative abundance and diversity of bacteria were positively related to pH, the latter nearly doubling between pH 4 and 8. In contrast, the relative abundance of fungi was unaffected by pH and fungal diversity was only weakly related with pH. The composition of the bacterial communities was closely defined by soil pH; there was as much variability in bacterial community composition across the 180-m distance of this liming experiment as across soils collected from a wide range of biomes in North and South America, emphasizing the dominance of pH in structuring bacterial communities. The apparent direct influence of pH on bacterial community composition is probably due to the narrow pH ranges for optimal growth of bacteria. Fungal community composition was less strongly affected by pH, which is consistent with pure culture studies, demonstrating that fungi generally exhibit wider pH ranges for optimal growth.

Shifts in the structure of soil microbial communities in limed forests as revealed by phospholipid fatty acid analysis

Abstract The effects of lime and wood-ash on the microbial community structure were evaluated by analyzing the phospholipid fatty acid (PLFA) composition of soils from two areas in the south of Sweden. A pine forest was amended with lime or ash at two concentrations, and a spruce forest was limed at one concentration. The treatments were carried out 5–6 years before sampling and raised the pH from approx. 4.0 to values between 4.8 and 7.0. At both sites there was a difference in the PLFA composition between the treated plots and the controls. The changes found were similar at both sites and correlated to the pH changes. No difference was found between limed plots and those treated with wood-ash. The methyl-branched fatty acids i15:0, i16:0 and 10Me16:0, the monounsaturated fatty acids 16: 1ω 7t and 18: 1ω 9, the cyclopropane fatty acid cy 19:0, and the saturated fatty acid 20:0 were more abundant in the control plots. In the plots with the highest pH there was a three-fold increase in the fatty acid 16: lω 5. An increase was also found for the fatty acids i14:0, 16:lω9, 16:lω 7c, cy17:0, 18:lω 7 and 10Me18:0. No effect on 18:2ω6 was found. The changes in PLFA pattern indicated that the increased pH caused a shift in the bacterial community to more Gram-negative and fewer Gram-positive bacteria, while the amount of fungi was unaffected. The increase in 10Me18:0 in limed soils indicated an increase in actinomycetes.

Comparison of soil fungal/bacterial ratios in a pH gradient using physiological and PLFA-based techniques

We have compared the total microbial biomass and the fungal/bacterial ratio estimated using substrate-induced respiration (SIR) in combination with the selective inhibition technique and using the phospholipid fatty acid (PLFA) technique in a pH gradient (3.0-7.2) consisting of 53 mature broad-leaved forest soils. A fungal/bacterial biomass index using the PLFA technique was calculated using the PLFA 18:2w6,9 as an indicator of fungal biomass and the sum of 13 bacterial specific PLFAs as indicator of the bacterial biomass. Good linear correlation (p 0.8); for example the PLFAs 16:1w5 and 16:1w7c increased with increasing soil pH, while i16:0 and cy19:0 decreased. A principal component analysis of the total PLFA pattern gave a first component that was strongly correlated to soil pH (R-2 = 0.85, p < 0.001) indicating that the microbial community composition in these beech/beech-oak forest soils was to a large extent determined by soil pH. (Less)

Effects of heavy metals in soil on microbial processes and populations (a review)

The effects of Cd, Cu, Zn, and Pb on soil microorganisms and microbially mediated soil processes are reviewed. The emphasis is placed on temperate forest soils. The sensitivity of different measurements is discussed, and data compiled to compare relative toxicity of different metals. On the whole the relative toxicity of the metals (on a μg g−1 soil basis) decreased in the order Cd > Cu > Zn > Pb, but differences between different investigations were found. The influence of abiotic factors on metal toxicity is briefly discussed and especially examplified by different soil organic matter content. Evidence of tolerance and adaptation in the soil environment and the time scale involved in the evolution of a metal-tolerant microbial community after metal exposure are also considered.

Microbial biomass measured as total lipid phosphate in soils of different organic content

The use of total lipid phosphate as a measure of biomass was evaluated in soils with different organic matter content. Lipids were extracted with a one-phase mixture of chloroform, methanol, and a buffer, and digested with either persulfate or perchloric acid to liberate lipid-bound phosphate. This procedure was evaluated by varying the extraction buffer, the extraction and digestion times, the amount of soil extracted, and the amount of lipid material digested. An extraction period of 2 h was sufficient to yield maximum lipid phosphate. Neither sonication, vigorous mixing, nor longer extraction periods increased the amount of lipid phosphate extracted. However, the amount of lipid phosphate recovered was dependent on the choice of buffer. When organic soil was used, citrate buffer in the extraction mixture gave higher amounts of lipid phosphate than acetate buffer, Tris, H2O or phosphate buffer. In a sandy loam with low organic matter content, citrate or phosphate buffers performed equally well. When 13 soils of different organic matter content were examined, the two digestion methods showed a good linear correlation (r2 = 0.991). Substrate-induced respiration (SIR) and ATP contents of the different soils correlated well with the total lipid phosphate. Based on these measurements, a conversion factor of 500 μmol lipid phosphate·g−1 biomass-C (perchloric acid digestion) was calculated.

Contrasting Soil pH Effects on Fungal and Bacterial Growth Suggest Functional Redundancy in Carbon Mineralization

ABSTRACT The influence of pH on the relative importance of the two principal decomposer groups in soil, fungi and bacteria, was investigated along a continuous soil pH gradient at Hoosfield acid strip at Rothamsted Research in the United Kingdom. This experimental location provides a uniform pH gradient, ranging from pH 8.3 to 4.0, within 180 m in a silty loam soil on which barley has been continuously grown for more than 100 years. We estimated the importance of fungi and bacteria directly by measuring acetate incorporation into ergosterol to measure fungal growth and leucine and thymidine incorporation to measure bacterial growth. The growth-based measurements revealed a fivefold decrease in bacterial growth and a fivefold increase in fungal growth with lower pH. This resulted in an approximately 30-fold increase in fungal importance, as indicated by the fungal growth/bacterial growth ratio, from pH 8.3 to pH 4.5. In contrast, corresponding effects on biomass markers for fungi (ergosterol and phospholipid fatty acid [PLFA] 18:2ω6,9) and bacteria (bacterial PLFAs) showed only a two- to threefold difference in fungal importance in the same pH interval. The shift in fungal and bacterial importance along the pH gradient decreased the total carbon mineralization, measured as basal respiration, by only about one-third, possibly suggesting functional redundancy. Below pH 4.5 there was universal inhibition of all microbial variables, probably derived from increased inhibitory effects due to release of free aluminum or decreasing plant productivity. To investigate decomposer group importance, growth measurements provided significantly increased sensitivity compared with biomass-based measurements.

MICROBIAL COMMUNITY STRUCTURE AND pH RESPONSE IN RELATION TO SOIL ORGANIC MATTER QUALITY IN WOOD-ASH FERTILIZED, CLEAR-CUT OR BURNED CONIFEROUS FOREST SOILS

Humus phospholipid fatty acid (PLFA) analysis was used in clear-cut, wood-ash fertilized (amounts applied: 1000, 2500, and 5000 kg ha−1), or prescribed burned (both in standing and clear-cut) coniferous forests to study the effects of treatments on microbial biomass and community structure. The microbial biomass (total PLFAs) decreased significantly due to the highest rate of wood-ash fertilization, clear-cutting, and the two different fire treatments when compared to control amounts. Fungi appeared more seriously reduced by these treatments than bacteria, as revealed by a decreased index of fungal:bacterial PLFAs when compared to the controls. The community structure was evaluated using the PLFA pattern. The largest treatment effect was due to burning in both areas studied, which resulted in increases in 16:1ω5 and proportional decreases in 18:2ω6. Clear-cutting and the different amounts of ash application resulted in similar changes in the PLFA pattern to the burning treatments, but these were less pronounced. Attempts to correlate the changes in the PLFA pattern to soil pH, bacterial pH response patterns (measured using thymidine incorporation), or substrate quality (measured using IR spectroscopy) were only partly successful. Instead, we hypothesize that the changes in the PLFA pattern of the soil organisms were related to an altered substrate quantity, that is the availability of substrates after the treatments.

The use of phospholipid and neutral lipid fatty acids to estimate biomass of arbuscular mycorrhizal fungi in soil

Cucumber seedlings associated with the arbuscular mycorrhizal (AM) fungi Glomus WUM10 or G. caledanium (BEG 15) were grown in PVC tubes with a lateral root-free compartment and an identical compartment containing both hyphae and roots. The amounts of specific fatty acids, in the neutral lipid and phospholipid fractions, were measured in both compartments and compared with controls without mycorrhiza. The phospholipid fatty acids (PLFAs) 16:1 omega 5, 18:1 omega 7c, 20:4 and 20:5 were present in higher amounts in soil with mycorrhizal hyphae than in soil without mycorrhizal hyphae. The largest relative difference was found in 20:5, but a good correlation existed between 16:1 omega 5 and 20:5 in soil with hyphae. Amounts of these fatty acids were correlated both with length of mycorrhizal hyphae and with amounts of ATP in soil. Conversion factors to calculate hyphal length and AM fungal biomass carbon using the phospholipid fatty acids could thus be estimated; 38 nmol PLFA 16:1 omega 5 mg(-1) AM fungal biomass C (Glomus WUM10) and 22 nmol PLFA 20:5 mg(-1) biomass C. The fatty acid 16:1 omega 5 from the neutral lipid fraction, containing triglycerides, dominated in soils with mycorrhizal hyphae. The amount of 16:1 omega 5 in the neutral lipid fraction decreased during storage of soils, indicating a decrease in storage lipids, while the proportion of 16:1 omega 5 in the phospholipid fraction was almost unaffected. (Less)

Changes in microbial community structure during long-term incubation in two soils experimentally contaminated with metals

The effects of Zn contamination on the microbial community structure of a forest humus and an arable soil, as estimated by phospholipid fatty acid (PLFA) analysis, were followed during 18 months. The soils were contaminated at 10 different metal concentrations and incubated in plastic jars at 22°C. In both soils effects of heavy metal contamination could be detected after 2 weeks. Qualitatively similar changes in the PLFA pattern were found at the later sampling occasions, although the changes became more pronounced with prolonged incubation. In the forest soil the double-unsaturated 18:2ω6, indicating fungi, increased proportionally due to the metal amendment, while there was a strong negative effect of incubation on the fungal biomass in all samples of this soil type. In the arable soil 18:2ω6 showed a strong increase in response to the Zn pollution. As in the forest soil, incubation decreased the mol% of 18:2ω6, although the effect was less pronounced than in the forest soil. The proportions of several individual bacterial PLFAs changed in both soils due to the treatments, indicating shifts within the bacterial community in the soils, but these shifts could not be interpreted in terms of changes in the proportional abundance of specific taxonomic groups of bacteria. The ratio of 16:1ω7t-to-16:1ω7c, which has been proposed as a starvation index, increased in the forest soil due to Zn contamination. In the high-metal samples this ratio decreased during incubation, while it remained unchanged in the uncontaminated control. In the arable soil no clear effect was found on the trans-to-cis ratio either in response to metal contamination or to incubation. The ATP content decreased during incubation. Little or no effect was found on the total amount of PLFAs or on the lipid phosphate content, except after 18 months when these biomass measurements decreased.