Application of the selective inhibition method to determine bacterial: fungal ratios in three beechwood soils rich in carbon — Optimization of inhibitor concentrations

Bacterial and fungal contributions to microbial respiration in three beechwood soils rich in C (two basalt soils and one limestone soil) were investigated by using streptomycin and cycloheximide to inhibit substrate-induced respiration after glucose (8000 g g^-1), N, and P addition to soil samples. The inhibitors were added as solutions (2000, 8000, and 16000 g g^-1) and the reduction in substrate-induced respiration after separate and combined inhibitor addition was measured in an automated electrolytic microrespirometer. Bacterial and fungal contributions to microbial respiration were calculated using the interval 6–10 h after inhibitor application. The microbial biomas was smaller in the two basalt soils (Oberhang and Mittelhang) than in the limestone soil (Unterhang). In the presence of both inhibitors, microbial respiration was inhibited by a maximum of 45, 45, and 25% in the two basalt soils and the limestone soil, respectively. Inhibition of microbial respiration was at a maximum at streptomycin and cycloheximide concentrations of 16000 g g^-1. The inhibitor additivity ratio approached 1.0 even at high inhibitor concentrations, indicating high inhibitor selectivity. Calculated prokaryote: eukaryote ratios indicated lower bacterial contributions to the microbial biomass in the Mettelhang (0.74) and Unterhang (0.73) than in the Oberhang (0.88) soil.     

Novel antibiotics as inhibitors for the selective respiratory inhibition method of measuring fungal:bacterial ratios in soil

The use of the selective inhibition (SI) method for measuring fungal:bacterial ratios may be limited due to biocide selectivity and the overlap of antibiotic activity. This study evaluated novel pairs of antibiotics for their specificity in soils of different origins and their potential reduction in inhibition of non-target organisms. Four soils selected for this study were from a semi-arid shrub-steppe, a loblolly pine forest and two grassland sites (restored and farmed prairie plots). Three bactericides were tested: oxytetracycline hydrochloride, streptomycin sulphate, and bronopol. Three fungicides were tested: captan, ketoconazole, and nystatin. The inhibitor additivity ratio and fungal:bacterial ratios were calculated from control and treated soils where inhibition was measured as CO₂ respiration reduction with biocides. We were able to minimize non-target inhibition by the antibiotics to <5% and thus calculate reliable fungal:bacterial ratios using captan to inhibit fungi in all four soils, and bronopol to inhibit bacteria in three of the four soils. The most successful bactericide in the restored prairie was oxytetracycline-HCl. Our results demonstrate that application of novel antibiotics is not uniformly successful in soils of different origin and that the SI technique requires more than just optimization of antibiotic concentration; it also requires optimization of antibiotic selection.     

Changes in soil fungal:bacterial biomass ratios following reductions in the intensity of management of an upland grassland

In this study we examined the effect on soil fungal:bacterial biomass ratios of withholding fertiliser, lime, and sheep-grazing from reseeded upland grassland. The cessation of fertiliser applications on limed and grazed grassland resulted in a reduction in soil pH from 5.4 to 5.1. The cessation of fertiliser applications and liming on grazed grassland resulted in a fall in pH from 5.4 to 4.7, whereas withholding fertiliser and lime and the removal of grazing resulted in a further reduction to pH 4.5. Substrate-induced respiration was reduced in the unfertilised grazed (21%; P<0.01) and unfertilised ungrazed (36%; P<0.001) treatments. Bacterial substrate-induced respiration and bacterial fatty acids were unaffected by the treatments. The relative abundance of the fungal fatty acid 18:26 increased by 39 and 72% (P<0.05) in the limed grazed and unfertilised grazed treatments, respectively. Fungal substrate-induced respiration increased in the limed grazed (18%) and unfertilised grazed (65%; P<0.05) treatments. The ratio of 18:26: bacterial fatty acids was correlated with the ratio of fungal:bacterial substrate-induced respiration (r=0.69; P<0.001).     

Considering fungal:bacterial dominance in soils - Methods, controls, and ecosystem implications

An expectation in soil ecology is that a microbial communities’ fungal:bacterial dominance indicates both its response to environmental change and its impact on ecosystem function. We review a selection of the increasing body of literature on this subject and assess the relevance of its expectations by examining the methods used to determine, the impact of environmental factors on, and the expected ecosystem consequences of fungal:bacterial dominance. Considering methods, we observe that fungal:bacterial dominance is contingent on the actual measure used to estimate it. This has not been carefully considered; fungal:bacterial dominance of growth, biomass, and residue indicate different, and not directly relatable aspects, of the microbial community’s influence on soil functioning. Considering relationships to environmental factors, we found that shifts in fungal:bacterial dominance were not always in line with the general expectation, in many instances even being opposite to them. This is likely because the traits expected to differentiate bacteria from fungi are often not distinct. Considering the impact of fungal:bacterial dominance on ecosystem function, we similarly found that expectations were not always upheld and this too could be due to trait overlap between these two groups. We explore many of the potential reasons why expectations related to fungal:bacterial dominance were not met, highlighting areas where future research, especially furthering a basic understanding of the ecology of bacteria and fungi, is needed.     

Do growth yield efficiencies differ between soil microbial communities differing in fungal:bacterial ratios? Reality check and methodological issues

Soil communities dominated by fungi such as those of no-tillage (NT) agroecosystems are often associated with greater soil organic matter (SOM) storage. This has been attributed in part to fungi having a higher growth yield efficiency (GYE) compared to bacteria. That is, for each unit of substrate C utilized, fungi invest a greater proportion into biomass and metabolite production than do bacteria. The assumption of higher fungal efficiency may be unfounded because results from studies in which fungal and bacterial efficiencies have been characterized are equivocal and because few studies have measured microbial GYE directly. In this study, we measured microbial GYE in agricultural soils by following ¹³C-labeled glucose loss, total CO₂-C, and ¹³CO₂-C evolution at 2 h intervals for 20 h in two experiments (differing in N amendment levels) in which the fungal:bacterial biomass ratios (F:B) were manipulated. No differences in efficiency were observed for communities with high versus low F:B in soils with or without added inorganic N. When calculated using ¹³CO₂-C (in contrast to total CO₂-C) evolution, growth yield efficiencies of soils having high and low F:B were 0.69±0.01 and 0.70±0.01, respectively. When soils were amended with N, soils with high and low F:B had growth yield efficiencies of 0.78±0.01 and 0.76±0.01, respectively. Our experiments do not support the widely held assumption that soil fungi have greater growth efficiency than soil bacteria. Thus, claims of greater fungal efficiency may be unsubstantiated and should be evoked cautiously when explaining the mechanisms underlying greater C storage and slower C turnover in fungal-dominated soils.     

A simple method for detection of lipolytic microorganisms in soils

Media selective for the isolation of bacteria, actinomycetes and fungi were amended with 0.1% sunflower oil emulsified with 0.01% Tween 80. Lipase-producing microorganisms produced clear zones on these media. When lipase-producing bacteria were cultured on a polycarbonate membrane laid on the selective medium for bacteria, clear zones were produced on the medium when the membrane along with bacteria was removed. The agar disc cut from the clear zone also produced a clear zone when placed on the fresh medium, indicating that clear zone formation is the result of the activity of extracellular lipases. The largest population of lipase-producing microorganisms in an agricultural soil was actinomycetes followed by bacteria and fungi. Ranging from 12 to 75% of bacteria, actinomycetes and fungi isolates from soils collected from three different locations were capable of producing lipases. In general, relatively small percentages of soil bacteria were lipase producers, and lipase producers were more common among soil actinomycetes and fungi. These three groups of microorganisms appear to be all important in decomposition of oils in organic matters in soils.     

Differences in the activity and bacterial community structure of drained grassland and forest peat soils

The microbial activity and bacterial community structure were investigated in two types of peat soil in a temperate marsh. The first, a drained grassland fen soil, has a neutral pH with partially degraded peat in the upper oxic soil horizons (16% soil organic carbon). The second, a bog soil, was sampled in a swampy forest and has a very high soil organic carbon content (45%), a low pH (4.5), and has occasional anoxic conditions in the upper soil horizons due to the high water table level. The microbial activity in the two soils was measured as the basal and substrate-induced respiration (SIR). Unexpectedly, the SIR (μl CO₂ g⁻¹ dry soil) was higher in the bog than in the fen soil, but lower when CO₂ production was expressed per volume of soil. This may be explained by the notable difference in the bulk densities of the two soils. The bacterial communities were assessed by terminal restriction fragment length polymorphism (T-RFLP) profiling of 16S rRNA genes and indicated differences between the two soils. The differences were determined by the soil characteristics rather than the season in which the soil was sampled. The 16S rRNA gene libraries, constructed from the two soils, revealed high proportions of sequences assigned to the Acidobacteria phylum. Each library contained a distinct set of phylogenetic subgroups of this important group of bacteria.     

Evaluation of the acetylene inhibition method for measuring denitrification in soilless plant culture systems

Denitrification measurements by means of the acetylene inhibition method require a continuous presence of acetylene to block the microbial reduction of N₂0 to N₂. To examine the effect of such steady exposures on the growth of plants, roots of cucumber and tomato seedlings were treated with different acetylene concentrations. Acetylene concentrations of 1 vol% in the gas phase, which were necessary for complete inhibition of N₂ formation, led to a significant retardation of root growth. This was partly due to trace amounts of ethylene contained in the acetylene gas which could not be removed with the usual prescrubbing through a sulfuric acid train. As a result of the growth impairment, oxygen consumption in the root zone decreased after 4 days of exposure. In order to avoid these side effects, the denitrification measurements in soilless cultures were performed on individual plants over a limited period of 2–3 days. The flow-through chamber method proved to be suitable for determining the gaseous N losses in a closed-loop system. It avoided greater air variations from the environmental conditions (substrate temperature, airflow and plant composition) and excluded errors in measurement caused by injury to roots and spatial variability of denitrification activity in the root medium. For exact estimation of the gaseous N losses, preceding 24-h acetylene fumigation was necessary. Subsequently at least three gas samples had to be taken throughout the day, because the N₂O+N₂ emissions were subject to a pronounced diurnal variability.     

Solution chemistry in acid forest soils: Are the BC : Al ratios as critical as expected in Switzerland?

The molar ratio of base nutrient cations to total dissolved aluminum (BC : Al_tot) in the soil solution was measured at six forest sites in Switzerland in acid mineral soils to determine whether the ratio measured in the field was lower than the critical value of 1, as predicted by the mapping of exceedances of critical loads of acidity. The soil chemistry was then related to the soil solution composition to characterize the typical effective base saturation (BS) and BC : Al ratio in soil leading to critical BC : Al_tot in the soil solution. The median BC : Al_tot ratio in the soil solution never reached the critical value in the root zone at any sites for the whole observation period (1999–2002), suggesting that the BC : Al_tot ratios measured in the field might be higher than those modeled for the determination of critical loads of acidity. The gibbsite model usually applied for the calculation of critical loads was a poor predictor of the Al³⁺ activity at the study sites. A curvilinear pH‐pAl³⁺ relationship was found over the whole range of pH (3.8–6.5). Above a pH of 5.5, the slope of the pH‐pAl³⁺ relation was close to 3, suggesting equilibrium with Al(OH)₃. It decreased to values smaller than 1.3 below a pH of 5.5, indicating complexation reactions with soil organic matter. The BS and the BC : Al ratios in the soils were significantly correlated to the BC : Al_tot ratios in the soil solution. The soil solutions with the lowest BC : Al_tot ratios (≤ 2) were typically found in mineral soils with a BS below 10 % and a BC : Al ratio in the soil lower than 0.2. In acid pseudogleyed horizons overlying a calcareous substrate, the soil solution chemistry was strongly influenced by the composition of the underlying soil layers. The soil solutions at 80 cm had pH values and BC : Al_tot ratios much higher than expected. This situation should be taken into account for the calculations of critical loads of acidity.     

Effect of an increasing carbon: nitrate-N ratio on the reliability of acetylene in blocking the N2O-reductase activity of denitrifying bacteria in soil

Summary In model experiments with a silty loam soil the effect of different C : NO _inf3 ^sup- -N ratios on the reliability of C₂H₂ (1% v/v) in blocking N₂O-reductase activity was examined. The soil was carefully mixed with different amounts of powdered lime leaves (Tilia vulgaris) to obtain organic C contents of about 1.8, 2.3, and 2.8%, and of NO _inf3 ^sup- solution to give C : NO _inf3 ^sup- -N ratios of 84, 107, 130, 156, 200, and 243. The soil samples were incubated in specially modified anaerobic jars (22 days, 25°C, 80% water-holding capacity, He atmosphere) and the atmosphere was analysed for N₂, N₂O, CO₂, and C₂H₂ by gas chromatography at regular intervals. Destruction jars were used to analyse soil NO _inf3 ^sup- , NH ₄ ⁺ and C. The results clearly showed that N₂O-reductase activity was completely blocked by 1% (v/v) C₂H₂ only as long as NO _inf3 ^sup- was present. In the presence of C₂H₂, NO _inf3 ^sup- was apparently entirely converted into N₂O. The C₂H₂ blockage of N₂O-reductase activity ceased earlier in soils with a wide C : NO _inf3 ^sup- -N ratio (156, 200, and 243) than in those with closer C : NO _inf3 ^sup- -N ratios (84, 107, and 130). As soon as NO _inf3 ^sup- was exhausted, N₂O was reduced to N₂ in spite of C₂H₂. The wider the C : NO _inf3 ^sup- -N ratio, the earlier the production of N₂ and the less the reliability of the C₂H₂ blockage. In the untreated control complete inhibition of N₂O-reductase activity by C₂H₂ lasted for 7–12 days. In the field, estimates of total denitrification losses by the C₂H₂ inhibition technique should be considered reliable only as long as NO _inf3 ^sup- is present. Consequently, NO _inf3 ^sup- monitoring in the field is essential, particularly in soils supplied with easily decomposable organic matter.     

Evaluation of an optimal extraction method for measuring d-ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) in agricultural soils and its association with soil microbial CO2 assimilation

Assimilating atmospheric carbon (C) into terrestrial ecosystems is recognized as a primary measure to mitigate global warming. Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is the dominant enzyme by which terrestrial autotrophic bacteria and plants fix CO₂. To investigate the possibility of using RubisCO activity as an indicator of microbial CO₂ fixation potential, a valid and efficient method for extracting soil proteins is needed. We examined three methods commonly used for total soil protein extraction. A simple sonication method for extracting soil protein was more efficient than bead beating or freeze–thaw methods. Total soil protein, RubisCO activity, and microbial fixation of CO₂ in different agricultural soils were quantified in an incubation experiment using ¹⁴C-CO₂ as a tracer. The soil samples showed significant differences in protein content and RubisCO activity, defined as nmol CO₂ fixed g⁻¹ soil min⁻¹. RubisCO activities ranged from 10.68 to 68.07 nmol CO₂ kg⁻¹ soil min⁻¹, which were closely related to the abundance of cbbL genes (r = 0.900, P = 0.0140) and the rates of microbial CO₂ assimilation (r = 0.949, P = 0.0038). This suggests that RubisCO activity can be used as an indicator of soil microbial assimilation of atmospheric CO₂.     

Evaluation of three organic wastes for reclaiming burnt soils: Improvement in the recovery of vegetation cover and soil fertility in pot experiments

The efficacy of three abundant organic wastes: poultry manure (PM), cattle slurry (CS) and sewage sludge (SS) for the reclamation of burnt soils was evaluated. A forest soil, previously furnace-heated in order to simulate exposure to a high-intensity wildfire, was labelled with nitrogen-15 (¹⁵N) to evaluate the contribution of N derived from the organic waste to the burnt soil and vegetation. Four treatments were performed with the heated ¹⁵N-labelled soil: an unamended control soil (S) and three waste amended soils (S+PM, S+CS and S+SS) at a dose waste of 167mg total N kg^–1 soil. Lolium perenne was grown in all the pots for 3 months. In each treatment the phytomass produced and its N content decreased significantly in the following order of treatments: S+PM S+CS > S+SS S. The percentage of plant N derived from the waste was similar in the S+PM (22.8%) and S+CS (24.0%) treatments, but significantly lower in the S+SS treatment (16.5%). At the end of the 3 month experimental period, the available N reserves (phytomass N+soil inorganic N) in the control soil accounted for 51.5–71.5% of those in the S+PM, S+CS and S+SS treatments, whereas the yield of the plants was only 13.4–29.8% of that in the manured soils. These results demonstrated the importance of the addition of organic wastes, particularly PM, for the recovery of the vegetation cover and for the stabilization of the soil ash layer. They also showed that the level of N was not the main controlling factor of plant growth in the control soil, which, moreover, did not show evidence of a shortage of macronutrients, i.e. phosphorus, potassium, calcium or magnesium. It is hypothesized that, as occurs in heat-sterilized soils, phytomass production in the control-heated soil could have been inhibited by the heat-induced production of phytotoxic compounds, their negative effects being microbially or chemically suppressed by the addition of organic wastes. Received: 3 March 1997