Substrate-induced Respiration Technique Research Articles

Proximal sensing of soil biological activity for precision agriculture

There is growing interest in monitoring soil biological health to complement the traditional evaluation of soil physical and chemical characteristics in agricultural fields. Activity of soil microorganisms mediates many essential soil processes that affect fertility, and, therefore, essential to the successful adoption of precision agriculture. However, there are technical limitations to cost-effective monitoring of spatial and temporal dynamics of soil biological activity across agricultural landscapes. This paper summarizes three consecutive studies on in situ measurement of soil biological activity. The first study reveals spatial heterogeneity of microbial population growth in three agricultural fields using bio-films. In the second study, microbiological activity was analyzed using a substrate-induced respiration technique. This technique was evaluated through a series of soil toxicity experiments that involved a comparison of fresh and autoclaved soil samples. Finally, the aim of the third study was to develop a portable instrumented system to evaluate carbon dioxide concentrations in soil by extracting air stored within the soil pores. This instrument was tested under various conditions to quantify the effects of soil moisture, compaction and presence of glucose (artificially increased microbial respiration). Optimization of the discussed techniques will allow for detailed mapping of these indices of soil biological health and their interactions with the physical and chemical environment at any specific point in time.

An Overview of Different Techniques on the Microbial Community Structure, and Functional Diversity of Plant Growth Promoting Bacteria

Soil is a dynamic biological system, in which it is difficult to determine the composition of microbial communities. Knowledge of microbial diversity and function in soils are limited because of the taxonomic and methodological limitations associated with studying the organisms. In this review, approaches to measure microbial diversity in soil were discussed. Research on soil microbes can be categorized as structural diversity, functional diversity and genetic diversity studies, and these include cultivation based and cultivation independent methods. Cultivation independent technique to evaluate soil structural diversity include different techniques such as Phospholipid Fatty Acids (PLFA) and Fatty Acid Methyl Ester (FAME) analysis. Carbon source utilization pattern of soil microorganisms by Community Level Physiological Profiling (CLPP), catabolic responses by Substrate Induced Respiration technique (SIR) and soil microbial enzyme activities are discussed. Genetic diversity of soil microorganisms using molecular techniques such as 16S rDNA analysis Denaturing Gradient Gel Electrophoresis (DGGE) / Temperature Gradient Gel Electrophoresis (TGGE), Terminal Restriction Fragment Length Polymorphism (T-RFLP), Single Strand Conformation Polymorphism (SSCP), Restriction Fragment Length Polymorphism (RFLP) / Amplified Ribosomal DNA Restriction Analysis (ARDRA) and Ribosomal Intergenic Spacer Analysis (RISA) are also discussed. The chapter ends with a final conclusion on the advantages and disadvantages of different techniques and advances in molecular techniques to study the soil microbial diversity.

Vegetation feedbacks of nutrient addition lead to a weaker carbon sink in an ombrotrophic bog

To study vegetation feedbacks of nutrient addition on carbon sequestration capacity, we investigated vegetation and ecosystem CO2 exchange at Mer Bleue Bog, Canada in plots that had been fertilized with nitrogen (N) or with N plus phosphorus (P) and potassium (K) for 7-12 years. Gross photosynthesis, ecosystem respiration, and net CO2 exchange were measured weekly during May-September 2011 using climate-controlled chambers. A substrate-induced respiration technique was used to determine the functional ability of the microbial community. The highest N and NPK additions were associated with 40% less net CO2 uptake than the control. In the NPK additions, a diminished C sink potential was due to a 20-30% increase in ecosystem respiration, while gross photosynthesis rates did not change as greater vascular plant biomass compensated for the decrease in Sphagnum mosses. In the highest N-only treatment, small reductions in gross photosynthesis and no change in ecosystem respiration led to the reduced C sink. Substrate-induced microbial respiration was significantly higher in all levels of NPK additions compared with control. The temperature sensitivity of respiration in the plots was lower with increasing cumulative N load, suggesting more labile sources of respired CO2 . The weaker C sink potential could be explained by changes in nutrient availability, higher woody : foliar ratio, moss loss, and enhanced decomposition. Stronger responses to NPK fertilization than to N-only fertilization for both shrub biomass production and decomposition suggest that the bog ecosystem is N-P/K colimited rather than N-limited. Negative effects of further N-only deposition were indicated by delayed spring CO2 uptake. In contrast to forests, increased wood formation and surface litter accumulation in bogs seem to reduce the C sink potential owing to the loss of peat-forming Sphagnum.

DNA determinations during growth of soil microbial biomasses

We attempted to quantify microbial growth in soil by means of DNA determination after glucose amendment. An FDNA conversion factor of 5.0 was used to convert μg DNA g−1 soil to μg Cmic g−1 soil during the growth phase. The conversion factor acquired rested on a regression analysis between soil microbial biomass-C (Cmic) estimated by the substrate-induced respiration technique (SIR) and dsDNA using a modified, miniaturized dsDNA extraction procedure which included 44 field and forest soils with a coefficient of determination of r2 = 0.95. Verification of this conversion factor was tested on eight arable soils where Cmic was determined by substrate-induced respiration (SIR)-, chloroform fumigation-incubation (CFI)-, chloroform fumigation-extraction (CFE)-, and application of the FDNA conversion factor. The congruency between the Cmic values obtained through these different techniques was satisfactory since five of eight soils gave similar Cmic values which were not statistically significantly different. The soils were thereafter amended with glucose and microbial growth followed by Cmic determinations with CFI, CFE, and DNA conversion over a period of up to 264 h at 22 °C. Concomitant CO2 analyses gave clues to two kinds of growth processes with respect to speed. Based on DNA conversion the calculated traditional growth parameters such as the specific growth rate (μ) lay in the range between 0.0046 and 0.022 h−1 which is several fold slower than μ values based on CO2 conversion but are in accordance with data in the earlier literature on growth rates for bacteria and fungi in soil done with traditional plate counts. These results suggest that DNA determinations can be applied as an alternative index for growth studies in situ.

Temporal changes in soil microbial biomass carbon in an arable soil. Consequences for soil sampling

Sugar beet, winter wheat and winter barley were planted within a crop rotation on an arable soil with conventional soil management. Soil samples were taken monthly from different depths of the whole plough layer (0–10, 10–20 and 20–30 cm) during a 56 month period. The samples were analysed for microbial biomass carbon using the substrate-induced respiration technique. Temporal changes in the amount of microbial biomass carbon were observed. Within a year, microbial biomass-C varied from low values (−15% of total mean) in winter to high values (+15% of total mean) in summer. Relative deviations from the annual means were calculated for each month in the year to demonstrate these fluctuations. Temporal changes in microbial biomass-C depended on the sources of sample variation (5 years, 3 crops, 3 sampling depths). The highest relative deviation from the annual mean microbial biomass-C was attributable to the factor “year”. Less variations were caused by “crops” and “sampling depth”. Soil microbial biomass-C remained constant during frost periods. From the observed temporal changes, recommendations for a suitable date for soil sampling are given, which allows a representative estimation of the mean annual microbial biomass-C content in arable soils.

Growth rate and response of bacterial communities to pH in limed and ash treated forest soils

Culturable and total bacterial counts, bacterial growth rate and tolerance to pH, as well as microbial biomass, were studied in two coniferous forest soils. The pH had been changed by addition of lime and wood-ash from 4.3–4.4 to 7.0 in one soil and from 3.9–4.4 to 6.1 in the other. Higher total microbial activities and higher bacterial growth rates, measured as soil respiration rate and thymidine incorporation rate, respectively, were found in the treatments with increased pH. Similar effects of soil pH on the thymidine incorporation rate were found using two different methods, by measuring rates in either a soil slurry with all bacteria present or using a subsample of bacteria extracted from soil after homogenization-centrifugation. The number of culturable bacteria was up to 5.1 times higher in the high pH soils, while the acridine orange direct counts were unaffected by the treatments. Thus, the proportions of culturable bacteria increased in the limed and ash-treated soils compared to the untreated controls. An altered bacterial community composition due to liming was indicated by an altered response to pH, where the pH of the soil was correlated to the optimum pH for growth of the bacterial community. The ATP content of the soil was unaffected or increased in treatments with high pH compared to the control, while microbial biomass estimated by the substrate induced respiration technique (SIR) was always higher in limed and ash-treated plots.

Seasonal variations of soil microbial biomass carbon within the plough layer

Over 31 months microbial biomass-C was estimated using the substrate induced respiration technique in fields with conventional soil management, where sugar beet-winter wheat-winter barley were grown in a crop rotation. Analysing the plough layer at different depths (0–10, 10–20 and 20–30 cm) seasonal variations were observed in the amount of microbial biomass-C due to soil and crop management. Although after ploughing microbial biomass-C distribution was homogeneous, a gradient, with decreasing amounts of biomass-C with depth, developed within the plough layer during the early growing season. This was due to a rapid increase of microbial biomass-C in the surface layer (0–10 cm). Throughout autumn and winter there was no change in microbial biomass-C distribution with depth. Within the crop rotation microbial biomass-C content increased in the order sugar beet, winter wheat and winter barley.

Measurement of microbial biomass in arctic tundra soils using fumigation-extraction and substrate-induced respiration procedures

The microbial biomass of seven arctic tundra soils was measured using both the chloroform fumigation-extraction procedure (biomass-C and -N) and the substrate-induced respiration method (biomass-C only) to test the suitability of these two methods for organic and mineral arctic soils. Results indicate that in general the two methods gave consistent microbial biomass-C measurements. Microbial biomass-C, estimated by the fumigation-extraction procedure (using K ec = 0.35) was highly correlated ( r = 0.831, P < 0.001) with the microbial biomass-C values measured by the substrate-induced respiration technique across all soil types. Microbial biomass-N of the seven arctic soils measured by the fumigation-extraction procedure showed a higher linear correlation with the biomass-C values produced by the substrate-induced respiration procedure ( r = 0.88) than those produced by the fumigation-extraction procedure ( r = 0.69). This result seems to suggest that the fumigation-extraction procedure works better for microbial biomass-N measurements than for biomass-C in these arctic soil conditions. The fumigation-extraction and substrate-induced respiration methods, developed to study mostly temperate agricultural soils, can be successfully applied to arctic tundra soils.

Are the soil microbial biomass and basal respiration governed by the climatic regime?

Soils in C equilibrium from various climatic regions were sampled to assess the influence of macroclimate on soil microbial biomass (C mic) and basal respiration (CO 2-evolution). C mic was measured using the substrate-induced respiration technique. C mic (μg C mic g −1 soil d.m.) was significantly correlated with several climatic variables, among them mean annual temperature (TEMP). At 20° and 5°C TEMP. 50 and 500 μg C mic g −1 soil were found, respectively. When C mic was calculated based on organic C (C mic-to-C org ratio), a very high correlation with precipitation/evaporation as the climatic variable was found. Of the variance 73% could be explained with the quadratic function y = 64.1− 109.5 x + 55.7 x 2, where y = C mic-to-C org ratio (mg C mic g −1 C org) and x = precipitation/evaporation. Soils from arid climates exhibited a high C mic-to-C org ratio (up to 50 mg C mic g −1 C org). in soils from climates with balanced precipitation and evaporation (P/E = 1), the C mic-to-C org ratio was lowest (15mg C mic g −1 C org). As P/E exceeds this, the C mic-to-C org ratio increased. Any deviation of the C mic-to-C org ratio from this regression line would indicate that a certain soil is not in C equilibrium but is losing or accumulating organic matter. In this study, for soils from a wide climatic range, the effects of pH, N or clay content on C mic and the C mic-to-C org ratio were small. For basal respiration, too, a significant relationship with climatic variables was found. Soils from warmer climates exhibited a basal respiration of 0.3 mg CO 2 g −1 soil h −1 compared to 0.1 mg for cooler climates. The metabolic quotient qCO 2 (μg respiratory CO 2-C g −1 C mic h −1) increased with temperature.

Changes in carbon content, respiration rate, ATP content, and microbial biomass in nitrogen-fertilized pine forest soils in Sweden

Carbon content and indices of microbial biomass and activity were determined in 1985 in different soil horizons of two nitrogen-fertilized pine forests in Sweden. The Kroksbo site was fertilized in 1974 with 150 and 600 kg N•ha−1 using NH4NO3 or urea, while the Nissafors site was fertilized in 1977 and 1984 with 150 kg NH4NO3-N•ha−1 The absolute amount of C per square metre of forest floor increased in fertilizer treatments compared with the control (by 10–26%, depending on the site or fertilizer treatment). Respiration rate, ATP content, and microbial biomass C, measured with the substrate-induced respiration technique, decreased in all horizons when expressed per gram of C. The decrease was most evident with NH4NO3, and at the highest level of fertilization. However, on an area basis there were no differences between fertilizer and control treatments. A calculated increase in litter fall in the fertilized plots could not explain all the increase in the amount of C per square metre compared with the control. Decreased microbial activity per gram of C therefore appeared to be an important factor in the increase in C content in fertilized plots.

Influence of macroclimate on soil microbial biomass

Soils from 12 agricultural long-term experimental fields located in contrasting climatic regions were used to assess the influence of macroclimate on soil microbial biomass (C micr). C micr was measured using the substrate-induced respiration technique. When C micr was calculated on the basis of soil dry mass (mg C micr g −1 soil d.m.), only poor correlations with climatic variables were found. However, when C micr was calculated based on organic carbon (mg C micr g −1 C org), close relationships with climatic variables were found. Especially, integrative climatic variables which reflected not only the temperature regime but also the moisture conditions, were found to be good predictors of the C micr-to-C org ratio. The best predictor was the precipitation-evaporation quotient which accounted for 68% of the variance. With a stepwise, multiple-linear regression procedure, the clay content and pH of the soils were found to account for another 2 and 3% of the variance, respectively. Parts of the remaining variance could be accounted for by differences in fertilization, crops, tillage practices or residue returns. The C micr-to-C org ratio of monocultures was significantly lower than that of crop rotations, and so was that of mineral fertilized compared to organically manured plots. The organic matter content of the soils studied were at or near the equilibrium level. An equilibrium function for the C micr-to-C org ratio was calculated: y = 18.18+ 108.3-e −6.728 x , where x = precipitation/ evaporation. Deviation from this equilibrium line would indicate that a certain soil is losing or accumulating organic matter.

A simple method for measuring co 2 in a continuous air-flow system: Modifications to the substrate-induced respiration technique

A simple, inexpensive method for measurement of respiration (CO 2 evolution) under a continuous air-flow was calibrated against an i.r. gas analyzer. Regression equations were developed to apply the method to general soil respiration measurement in the laboratory. The substrate-induced respiration technique was modified. By using 120% FWHC as a standard soil water condition during incubation, initial substrate-induced maximal respiration was measured in an optimal fashion. The problem of high experimental errors associated with soils with high pH values (pH > 6.5) can be avoided by using the simple method of CO 2 measurement with this continuous air-flow system.

Changes in microbial biomass C, ATP content, soil phospho-monoesterase and phospho-diesterase activity following air-drying of soils

The microbial biomass C in 20 grassland soils from New Zealand was estimated using the CHCl 3 fumigation method and from the rate of respiration using a modified substrate-induced respiration technique (SIR). Estimates were made before and after air-drying. The ATP contents and the activities of the enzymes phospho-monoesterase and phospho-diesterase of moist and air-dried soils were also measured. The fumigation method gave erratic results on dried soils and poor agreement with biomass C estimated by the SIR method. The SIR biomass C, ATP content, phospho-monoesterase and phosphodiesterase activities all declined after air-drying, the average decrease being by 39, 68, 38 and 29%, respectively. In general, biomass C determined by either method, ATP content and enzyme activities were positively correlated to the C content of the soil, but the relationships between the various indices were variable, and changed considerably after air-drying. The relationships were not sufficiently consistent for useful conversion factors to be derived. The modified SIR technique appears to have potential to estimate the biomass C of drier soils, particularly if the biomass at the time of sampling is required and rewetting of the soil is not desirable.