Soil microbial biomass and its activity estimated by kinetic respiration analysis – Statistical guidelines

The use of kinetic respiration analysis to determine soil microbial biomass its active part and maximum specific growth rate has recently increased. With this method, the increase in soil respiration rate initiated by application of carbon growth substrate, e.g. glucose, and mineral nutrients is used to estimate parameters describing microbial growth in soil. This study refines the method by developing statistical guidelines for the data analysis and processing. The kinetic respiration analysis assumes that microbial growth is not limited by substrate and energy. That is why it is critically important to identify the time period corresponding to the unlimited growth. In this work, we studied how the unlimited growth phase can be identified in less subjective ways by examining 121 datasets of respiration time series of 44 different soil samples taken from field plots. Deflection of the respiratory curve from the exponential pattern indicates growth limitation. Subjective selection of the part of respiratory curve which fits to the exponential pattern resulted in a 30% bias in specific microbial growth rates. We propose rules that are based on inspecting the patterns in a series of plots of residuals of fitted respiration rate. By comparing those rules with a set of statistical criteria we find that the weighted-coefficient of determination (r²) can be used to objectively constrain the unlimited growth phase in those cases where double-limitation does not occur. Furthermore, we discuss how the uncertainty of estimated microbial parameters is influenced by a) measurement uncertainty, b) biased measurement at the beginning of the experiment, and c) the number and timing of respiration measurements. We recommend checking plots of fits and residuals as well as reporting uncertainty bounds together with the estimated microbial parameters. A free statistical package is provided to easily deal with those aspects.     

Estimating the active and total soil microbial biomass by kinetic respiration analysis

 A model describing the respiration curves of glucose-amended soils was applied to the characterization of microbial biomass. Both lag and exponential growth phases were simulated. Fitted parameters were used for the determination of the growing and sustaining fractions of the microbial biomass as well as its specific growth rate (μ _max). These microbial biomass characteristics were measured periodically in a loamy silt and a sandy loam soil incubated under laboratory conditions. Less than 1% of the biomass oxidizing glucose was able to grow immediately due to the chronic starvation of the microbial populations in situ. Glucose applied at a rate of 0.5 mg C g^–1 increased that portion to 4–10%. Both soils showed similar dynamics with a peak in the growing biomass at day 3 after initial glucose amendment, while the total (sustaining plus growing) biomass was maximum at day 7. The microorganisms in the loamy silt soil showed a larger growth potential, with the growing biomass increasing 16-fold after glucose application compared to a sevenfold increase in the sandy loam soil. The results gained by the applied kinetic approach were compared to those obtained by the substrate-induced respiration (SIR) technique for soil microbial biomass estimation, and with results from a simple exponential model used to describe the growth response. SIR proved to be only suitable for soils that contain a sustaining microbial biomass and no growing microbial biomass. The exponential model was unsuitable for situations where a growing microbial biomass was associated with a sustaining biomass. The kinetic model tested in this study (Panikov and Sizova 1996) proved to describe all situations in a meaningful, quantitative and statistically reliable way. Received: 19 July 1999     

Determination of microbial mineralization activity in soil by modified Wright and Hobbie method

Summary The Wright and Hobbie (1966) procedure, originally proposed for measurements of microbial activity in water, was adapted for soil studies. It was critically assessed, both for technical details and for the theoretical background of the isotope dilution principle. The heterogeneity of the soil microbial community was taken into account by introducing a double Michaelis-Menten equation instead of the simple Michaelis-Menten form. More complicated models were unsuitable because the kinetic parameters were not sufficiently stable in view of fluctuations of the experimental data within the measurement error. An integrated form of kinetic equation was preferable to a differential one, and a non-linear regression was better than a linear analysis of transformed data. In grey forest soil the heterotrophic potential of microorganisms was estimated to be 1.31 and 4.26 mg C h^–1 kg^–1 of soil for oligotrophic and copiotrophic components, respectively. The turnover time of indigenous, readily available, soil organic matter was 2.82 h.     

Model of apparent and real priming effects: Linking microbial activity with soil organic matter decomposition

The most frequently used models simulating soil organic matter (SOM) dynamics are based on first-order kinetics. These models fail to describe and predict such interactions as priming effects (PEs), which are short-term changes in SOM decomposition induced by easily available C or N sources. We hypothesized that if decomposition rate depends not only on size of the SOM pool, but also on microbial biomass and its activity, then PE can be simulated. A simple model that included these interactions and that consisted of three C pools - SOM, microbial biomass, and easily available C - was developed. The model was parameterized and evaluated using results of ¹²C-CO₂ and ¹⁴C-CO₂ efflux after adding ¹⁴C-labeled glucose to a loamy Haplic Luvisol. Experimentally measured PE, i.e., changes in SOM decomposition induced by glucose, was compared with simulated PE. The best agreement between measured and simulated CO₂ efflux was achieved by considering both the total amount of microbial biomass and its activity. Because it separately described microbial turnover and SOM decomposition, the model successfully simulated apparent and real PE.The proposed PE model was compared with three alternative approaches with similar complexity but lacking interactions between the pools and neglecting the activity of microbial biomass. The comparison showed that proposed new model best described typical PE dynamics in which the first peak of apparent PE lasted for 1 day and the subsequent real PE gradually increased during 60 days. This sequential decomposition scheme of the new model, with immediate microbial consumption only of soluble substrate, was superior to the parallel decomposition scheme with simultaneous microbial consumption of two substrates with different decomposability. Incorporating microbial activity function in the model improved the fit of simulation results with experimental data, by providing the flexibility necessary to properly describe PE dynamics. We conclude that microbial biomass should be considered in models of C and N dynamics in soil not only as a pool but also as an active driver of C and N turnover.     

Kinetics of the respiratory response of the soil and rhizosphere microbial communities in a field experiment with an elevated concentration of atmospheric CO2

The effect of an elevated concentration of atmospheric CO₂ and the application rate of nitrogen fertilizers on the microbial biomass and maximum specific growth rate of microorganisms in the soil and rhizosphere was studied in a long-term field experiment involving the growing of sugar beets and winter wheat. It was shown that the treatment of field plots with carbon dioxide at a concentration higher than that in the atmosphere (550 ppm) for three-four years resulted in the formation of a microbial community with a higher maximum specific growth rate and a larger share of R-strategy microorganisms as compared to the soil under the control plants. No reliable differences in the total microbial biomass in the soil under the winter wheat were revealed between the treatments with the ambient and elevated CO₂ concentrations; in the soil under the beet plants, a reliable increase in the total microbial biomass at the elevated CO₂ concentration was noted only in the close vicinity of the plant roots.     

Comments on the paper by Kemmitt et al. (2008) ‘Mineralization of native soil organic matter is not regulated by the size, activity or composition of the soil microbial biomass - A new perspective’ [Soil Biology & Biochemistry 40, 61-73]: The biology of the Regulatory Gate

Kemmitt et al. (Kemmitt, S.J., Lanyon, C.V., Waite, I.S., Wen, Q., Addiscott, T.M., Bird, N.R.A., O'Donnell, A.G., Brookes, P.C., 2008. Mineralization of native soil organic matter is not regulated by the size, activity or composition of the soil microbial biomass - a new perspective. Soil Biology & Biochemistry 40, 61-73) recently proposed the “Regulatory Gate” hypothesis, which states that decomposition of soil organic matter (SOM) is regulated solely by abiotic factors. Without studying the mechanisms of such regulation, Kemmitt with coauthors challenged the classical Winogradsky theory of soil microbiology and questioned the concept of autochtonous and zymogenous microbial populations. In this letter, we revive the significance of microbial activity for SOM decomposition especially for the short-term (hours to weeks) processes and show that the “Regulatory Gate” is (micro)biologically driven.We explain the results of the three experiments in Kemmitt et al. (2008) from a microbiological point of view and suggest that SOM decomposition is mainly regulated by exoenzymes. We criticize the abiotic Regulatory Gate hypothesis based on bottleneck processes and pools limiting the SOM decomposition rate, comparison of constant and changing environmental conditions, as well as the connection between community structure and functions. We explain the results of Kemmitt et al. (2008) according to the properties of soil microbial community: functional redundancy and inconsistency between the excessive (but largely inactive) pool of total microbial biomass and the real mineralization activity. Finally, we suggest that to gain new perspectives on SOM decomposition and many other biochemical processes, future studies should focus on hot spots of (micro)biological activity (i.e., the rhizosphere, drillosphere, detritosphere, biopores, etc.) rather than on the bulk soil.     

Extractability of microbial N as influenced by C : N ratio in the flush after drying or fumigation

 Extractability of microbial N was estimated using in situ labelling of the microbial population with ¹⁵N. Four arable soils (one grey forest soil and three chernozems with different long-term fertilization) were amended with (NH₄)₂SO₄ (unlabelled or labelled with ¹⁵N) and d-glucose with a C : N ratio of 10 : 1 or 20 : 1 for the grey forest soil and 50 : 1 for the chernozems. d-glucose and labelled N with a C : N ratio of 20 : 1 did not cause microbial immobilization of unlabelled N. The use of substrates with a C : N ratio of 50 : 1 led to a pronounced priming action on soil N and decreased the extractability of immobilized ¹⁵N. Values of the extractable biomass N fraction (k _EN ) assessed for the fumigation-extraction and rehydration procedures were similar and varied in inverse proportion to the C : N ratio of the flush. The k _EN factor was calculated using values of the C : N ratio in flushes and the fixed C : N ratio of structural cell components, with the assumption that the C : N ratio of the extractable cytoplasmic cell fraction is variable. The ratio between the extractable and non-extractable biomass N fraction (k _EC ) and the C : N ratio of non-extractable cell components were assessed as equation parameters optimized for the measured k _EN and C : N ratio of flush data. Received: 31 October 1997     

Soil respiration and carbon balance of gray forest soils as affected by land use

 Soil respiration was measured by closed chamber and gradient methods in soils under forest, sown meadow and crops. Annual total soil respiration determined with the closed chamber method ranged from 180 to 642 g CO₂-C m^–2 year^–1 and from 145 to 382 g CO₂-C m^–2 year^–1 determined with the CO₂ profile method. Soil respiration increased in the order: cropland<sown meadow<forest. The C balance calculated as the difference between net primary production (sink) and respiration of heterotrophs (source) suggested an equilibrium between the input and output of C in the cropland, and sequestration of 135 and 387 g CO₂-C m^–2 year^–1 in the forest and meadow, respectively. Received: 1 December 1997     

Temperature dependence of the activity of polyphenol peroxidases and polyphenol oxidases in modern and buried soils

Under conditions of the global climate warming, the changes in the reserves of soil humus depend on the temperature sensitivities of polyphenol peroxidases (PPPOs) and polyphenol oxidases (PPOs). They play an important role in lignin decomposition, mineralization, and humus formation. The temperature dependence of the potential enzyme activity in modern and buried soils has been studied during incubation at 10 or 20°C. The experimental results indicate that it depends on the availability of the substrate and the presence of oxygen. The activity of PPOs during incubation in the absence of oxygen for two months decreases by 2–2.5 times, which is balanced by an increase in the activity of PPPOs by 2–3 times. The increase in the incubation temperature to 20°C and the addition of glucose accelerates this transition due to the more abrupt decrease in the activity of PPOs. The preincubation of the soil with glucose doubles the activity of PPPOs but has no significant effect on the activity of PPOs. The different effects of temperature on two groups of the studied oxidases and the possibility of substituting enzymes by those of another type under changing aeration conditions should be taken into consideration in predicting the effect of the climate warming on the mineralization of the soil organic matter. The absence of statistically significant differences in the enzymatic activity between the buried and modern soil horizons indicates the retention by the buried soil of some of its properties (soil memory) and the rapid restoration of high enzymatic activity during the preincubation.     

Oxygen and substrate availability interactively control the temperature sensitivity of CO2 and N2O emission from soil

Biology and Fertility of Soils - We investigated how oxygen availability, substrate amount, and quality affect the temperature dependency of enzymatic processes involved in the production of carbon...