The Determination of Soil Microbial Biomass Carbon and Adenosine Triphosphate Concentrations at Different Temperatures ^†

Abstract

Freezing soil samples at subzero temperatures is a commonly employed preservation method in soil science to halt microbial activity and enzymatic processes, preserving the sample’s composition and structure. The concentration of adenosine triphosphate (ATP) and the soil microbial biomass carbon content were determined from soil samples stored at +4 °C, −21 °C and −80 °C for 24 h, 7 days, and 20 days. The results showed that the ATP in the soil was not significantly affected by temperature or storage time. Larger differences were observed in the carbon content of microbial biomass, where the amount of this parameter increased by 14.2% after 20 days of the experiment.

1. Introduction

To ensure the sustainability of agroecosystems, with an emphasis on soil quality and health, it is important to monitor changes in soil properties. Important factors for this assessment are physical, chemical, and biological characteristics; vegetation cover; and management practices [1]. In all microbiological studies, it is preferred to work with freshly collected soil samples; however, for practical reasons, this is not always possible. Freezing is the most frequently used method for preserving soil for microbiological analysis. Storage temperature and length can have different effects on the total number of monitored parameters in soil samples [2,3]. Soil microbial biomass is an essential component of soil and is responsible for nutrient cycling, energy flow, and the regulation of soil organic matter conversion [4]. To measure the biomass of microorganisms, the determination of soil adenosine triphosphate (ATP), which is a part of all living forms, is a universal form of energy in biological systems, and is rapidly degraded in dead cells, is required. ATP fulfills very important functions in the soil, as it is a storehouse and transports energy, participates in the synthesis of DNA and RNA, and in the biosynthesis of cells and the regulation of cell metabolism [2,5]. ATP is a critical biomolecule in soil microbiology and is indicative of soil microbial activity [6]. It serves as an energy currency for various soil microorganisms, facilitating essential biochemical processes [7]. The measurement of soil ATP can provide insights into soil health and fertility, as higher ATP levels often correlate with greater microbial activity and nutrient cycling [8]. Conversely, low ATP levels may indicate poor soil conditions or degradation [9]. The ATP content in soil can fluctuate seasonally, with higher levels typically observed during periods of increased microbial activity, such as in the spring and summer [10]. This variability underscores the dynamic nature of soil ecosystems [11]. Researchers commonly employ bioluminescence assays to quantify soil ATP levels, a method that relies on the light emission produced by ATP when it reacts with luciferase enzymes [12]. Soil management practices, such as organic matter additions and reduced tillage, can influence soil ATP levels by promoting microbial growth and activity [13]. Understanding soil ATP dynamics is essential for sustainable agriculture and soil conservation efforts, as it can guide decisions regarding nutrient management and soil health improvement [14]. However, it is crucial to note that while soil ATP is a valuable indicator, it should be used in conjunction with other soil health metrics for a comprehensive assessment [15].

The aim of this study was to monitor the amount of microbial biomass carbon and ATP concentration in soils stored at +4 °C, −21 °C and −80 °C for 24 h, 7 days, and 20 days.

2. Material and Methods

The collection of soil samples for the determination of ATP and microbial biomass carbon (C_mic) was carried out on research plots of Prešov University study field on a permanent grassland. The investigated location was characterized by good organic carbon content (4.1%) and a neutral soil reaction (pH 6.9). The dominant soil type within the research locality was Fluvisol, which is typical of alluvial plains, river fans, valleys, and tidal marshes on all continents and in all climate zones. The university study field focuses on the cultivation of medicinal plants used in research. The collected soil samples were manually freed of plant and animal residues, sieved (<2 mm), and adjusted to a 40% soil-holding capacity (WHC). Subsequently, these samples were incubated at three different temperature ranges (+4 °C, −21 °C and −80 °C) under aerobic conditions in the dark. Microbial biomass carbon and soil ATP concentrations were determined at all temperature ranges every time after 24 h, 7 days, and 21 days in triplicate. C_mic was determined by the fumigation extraction method [16], which uses chloroform vapor to completely kill microorganisms, and the released organic carbon was easily extractable with 0.5 M K₂SO₄. ATP in the soil was extracted using a TCA reagent, the main component of which was 0.5 M trichloroacetic acid, and the determination of ATP itself was carried out by scintillation using the enzyme luciferin-luciferase [17].

3. Results and Discussion

The results showed that ATP in the soil was not significantly affected by temperature or storage time. The concentration did not change significantly during incubation (Figure 1). Some studies [18,19] have found that soil ATP concentration tends to decrease with decreasing temperature, particularly in colder climates. This may be due to reduced microbial activity in cold soils. Those studies have shown that soil ATP levels can vary seasonally, with higher concentrations during the warmer months and lower concentrations during the winter. This is often linked to changes in microbial activity and plant root exudates. Some research [20,21] has demonstrated that the activity of soil microbes responsible for ATP production can be temperature dependent. Different types of microbes may respond differently to temperature changes, affecting the overall ATP levels in the soil, which was not shown in our study. Scientific investigations have examined the broader ecological consequences of temperature changes on soil ATP dynamics. Changes in temperature can influence not only ATP levels but also nutrient cycling, carbon storage, and overall ecosystem functioning in soil [22].

In our study, larger differences were observed in the carbon content of microbial biomass, where the amount of this parameter increased by 14.2% after 20 days of the experiment, which could probably be due to the decomposability of organic soil matter and the subsequent synthesis of new biomass during the one-day incubation before the actual determination (Figure 2).

Microbial biomass exhibited a pronounced response to temperature variations, with increased temperatures generally leading to higher microbial biomass production [23], while in temperate ecosystems, microbial biomass tended to peak during the spring and fall when temperatures are moderate and declined during hot summer and cold winter periods [24]. Studies focusing on arctic soils have shown that microbial biomass declines significantly as temperatures rise due to the sensitivity of cold-adapted microbial communities to warming [25]. Temperature increases in tropical ecosystems can stimulate microbial biomass growth, but this effect may be limited by nutrient availability [26]. However, at high temperatures exceeding 40 °C, microbial biomass in soils can decline sharply due to thermal stress and the denaturation of enzymes and cellular proteins [27]. Understanding the intricate relationship between temperature and microbial biomass is essential for predicting how climate change may impact ecosystem functioning and biogeochemical cycling [28].

4. Conclusions

In this study, we investigated the impact of storage conditions on soil ATP and microbial biomass, and our results consistently demonstrated that storage duration and temperature did not exert any significant effect on these parameters. Larger differences were observed in the carbon content of microbial biomass. Our findings suggest that the soil ATP content and microbial biomass remain remarkably stable over time, irrespective of storage conditions, indicating the resilience of these biological indicators to environmental variation. In summary, our research reinforces the notion that soil ATP and microbial biomass are robust and unaffected by storage conditions, confirming their utility as reliable indicators for assessing soil microbial activity and ecosystem health over time. These findings emphasize the stability and reliability of soil ATP and microbial biomass measurements under various storage conditions, which make them valuable tools for ecological and environmental research.