Summary
Mechanisms for C stabilization in soils have received much interest recently due to their relevance in the global C cycle. Here we review the mechanisms that are currently, but often contradictorily or inconsistently, considered to contribute to organic matter (OM) protection against decomposition in temperate soils: (i) selective preservation due to recalcitrance of OM, including plant litter, rhizodeposits, microbial products, humic polymers, and charred OM; (ii) spatial inaccessibility of OM against decomposer organisms due to occlusion, intercalation, hydrophobicity and encapsulation; and (iii) stabilization by interaction with mineral surfaces (Fe-, Al-, Mn-oxides, phyllosilicates) and metal ions. Our goal is to assess the relevance of these mechanisms to the formation of soil OM during different stages of decomposition and under different soil conditions. The view that OM stabilization is dominated by the selective preservation of recalcitrant organic components that accumulate in proportion to their chemical properties can no longer be accepted. In contrast, our analysis of mechanisms shows that: (i) the soil biotic community is able to disintegrate any OM of natural origin; (ii) molecular recalcitrance of OM is relative, rather than absolute; (iii) recalcitrance is only important during early decomposition and in active surface soils; while (iv) during late decomposition and in the subsoil, the relevance of spatial inaccessibility and organo-mineral interactions for SOM stabilization increases. We conclude that major difficulties in the understanding and prediction of SOM dynamics originate from the simultaneous operation of several mechanisms. We discuss knowledge gaps and promising directions of future research.

Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions – a review

Summary
Soil is a complex and dynamic biological system, and still in 2003 it is difficult to determine the composition of microbial communities in soil. We are also limited in the determination of microbially mediated reactions because present assays for determining the overall rate of entire metabolic processes (such as respiration) or specific enzyme activities (such as urease, protease and phosphomonoesterase activity) do not allow any identification of the microbial species directly involved in the measured processes. The central problem posed by the link between microbial diversity and soil function is to understand the relations between genetic diversity and community structure and between community structure and function. A better understanding of the relations between microbial diversity and soil functions requires not only the use of more accurate assays for taxonomically and functionally characterizing DNA and RNA extracted from soil, but also high-resolution techniques with which to detect inactive and active microbial cells in the soil matrix.

Soil seems to be characterized by a redundancy of functions; for example, no relationship has been shown to exist between microbial diversity and decomposition of organic matter. Generally, a reduction in any group of species has little effect on overall processes in soil because other microorganisms can take on its function.

The determination of the composition of microbial communities in soil is not necessary for a better quantification of nutrient transformations. The holistic approach, based on the division of the systems in pools and the measurement of fluxes linking these pools, is the most efficient. The determination of microbial C, N, P and S contents by fumigation techniques has allowed a better quantification of nutrient dynamics in soil. However, further advances require determining new pools, such as active microbial biomass, also with molecular techniques. Recently investigators have separated 13C- and 12C-DNA, both extracted from soil treated with a 13C source, by density-gradient centrifugation. This technique should allow us to calculate the active microbial C pool by multiplying the ratio between labelled and total DNA by the microbial biomass C content of soil. In addition, the taxonomic and functional characterization of 13C-DNA allows us to understand more precisely the changes in the composition of microbial communities affected by the C-substrate added to soil.

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Microbial diversity and soil functions

Microorganisms have a variety of evolutionary adaptations and physiological acclimation mechanisms that allow them to survive and remain active in the face of environmental stress. Physiological responses to stress have costs at the organismal level that can result in altered ecosystem-level C, energy, and nutrient flows. These large-scale impacts result from direct effects on active microbes' physiology and by controlling the composition of the active microbial community. We first consider some general aspects of how microbes experience environmental stresses and how they respond to them. We then discuss the impacts of two important ecosystem-level stressors, drought and freezing, on microbial physiology and community composition. Even when microbial community response to stress is limited, the physiological costs imposed on soil microbes are large enough that they may cause large shifts in the allocation and fate of C and N. For example, for microbes to synthesize the osmolytes they need to survive a single drought episode they may consume up to 5% of total annual net primary production in grassland ecosystems, while acclimating to freezing conditions switches Arctic tundra soils from immobilizing N during the growing season to mineralizing it during the winter. We suggest that more effectively integrating microbial ecology into ecosystem ecology will require a more complete integration of microbial physiological ecology, population biology, and process ecology.

Microbial stress‐response physiology and its implications for ecosystem function

Despite their low carbon (C) content, most subsoil horizons contribute to more than half of the total soil C stocks, and therefore need to be considered in the global C cycle. Until recently, the properties and dynamics of C in deep soils was largely ignored. The aim of this review is to synthesize literature concerning the sources, composition, mechanisms of stabilisation and destabilization of soil organic matter (SOM) stored in subsoil horizons. Organic C input into subsoils occurs in dissolved form (DOC) following preferential flow pathways, as aboveground or root litter and exudates along root channels and/or through bioturbation. The relative importance of these inputs for subsoil C distribution and dynamics still needs to be evaluated. Generally, C in deep soil horizons is characterized by high mean residence times of up to several thousand years. With few exceptions, the carbon-to-nitrogen (C/N) ratio is decreasing with soil depth, while the stable C and N isotope ratios of SOM are increasing, indicating that organic matter (OM) in deep soil horizons is highly processed. Several studies suggest that SOM in subsoils is enriched in microbial-derived C compounds and depleted in energy-rich plant material compared to topsoil SOM. However, the chemical composition of SOM in subsoils is soil-type specific and greatly influenced by pedological processes. Interaction with the mineral phase, in particular amorphous iron (Fe) and aluminum (Al) oxides was reported to be the main stabilization mechanism in acid and near neutral soils. In addition, occlusion within soil aggregates has been identified to account for a great proportion of SOM preserved in subsoils. Laboratory studies have shown that the decomposition of subsoil C with high residence times could be stimulated by addition of labile C. Other mechanisms leading to destabilisation of SOM in subsoils include disruption of the physical structure and nutrient supply to soil microorganisms. One of the most important factors leading to protection of SOM in subsoils may be the spatial separation of SOM, microorganisms and extracellular enzyme activity possibly related to the heterogeneity of C input. As a result of the different processes, stabilized SOM in subsoils is horizontally stratified. In order to better understand deep SOM dynamics and to include them into soil C models, quantitative information about C fluxes resulting from C input, stabilization and destabilization processes at the field scale are necessary.

Deep soil organic matter—a key but poorly understood component of terrestrial C cycle

Soil organic matter (SOM) consists of various functional pools that are stabilized by specific mechanisms and have certain turnover rates. For the development of mechanistic models that predict changes in SOM storage, these pools have to be quantified and characterized. In the past, numerous fractionation schemes have been developed to separate and analyse such SOM fractions. In this review, the SOM fractions obtained with such operational fractionation procedures are described in terms of their pool sizes, chemical properties, and turnover rates. The main objective of this review is to evaluate these operationally defined fractions with respect to their suitability to describe functional SOM pools that could be used to parameterize SOM turnover models. Fractionation procedures include (1) physical separation of SOM into aggregate, particle size, and density fractions and fractions according to their magnetic susceptibility, and (2) various wet chemical procedures that fractionate SOM according to solubility, hydrolysability, and resistance to oxidation or by destruction of the mineral phase. Furthermore, combinations of fractionation methods are evaluated. The active SOM pool with turnover rates

SOM fractionation methods: Relevance to functional pools and to stabilization mechanisms

The objective of this study was to investigate whether preferential flow paths have higher microbial biomass and different microbial community structures than the rest of the soil. The organic C concentrations in the preferential flow paths were 10 to 70% higher than in the matrix. The organic N concentrations were also enriched in the preferential flow paths, as well as the effective cation exchange capacity and the base saturation. Microbial biomass was 9 to 92% higher in the preferential flow paths than in the matrix, probably due to the better nutrient and substrate supply. The DNA concentrations and direct cell counts showed a similar pattern, while domain-specific genetic fingerprints did not reflect the differences between flow regions. However, Pseudomonas displayed different population structures between preferential flow paths and soil matrix. This indicated that possibly only few populations with a broad acceptance for substrates and aerobic as well as anaerobic growth specifically profit from the favourable conditions in the preferential flow paths.

Preferential Flow Paths: Biological Hot Spots in Soils

A strategy for optimizing quality and quantity of DNA extracted from soil

Prior to registration of crop protection products (CPPs) their persistence in soil has to be determined under defined conditions. For this purpose, soils are collected in the field and stored for up to 3 months prior to the tests. During storage, stresses like drying may induce changes in microbiological soil characteristics (MSCs) and thus may influence CPP degradation rates. We investigated the influence of soil storage-related stress on the resistance and resilience of different MSCs by assessing the impact of a single severe drying-rewetting cycle and by monitoring recovery from this event for 34 days. The degradation and mineralization of the fungicide metalaxyl-M and the insecticide lufenuron were delayed by factors of 1.5 to 5.4 in the dried and rewetted soil compared to the degradation and mineralization in an undisturbed reference. The microbial biomass, as estimated by direct cell counting and from the soil DNA content, decreased on average by 51 and 24%, respectively. The bulk microbial activities, as determined by measuring substrate-induced respiration and fluorescein diacetate hydrolysis, increased after rewetting and recovered completely within 6 days after reequilibration. The effects on Bacteria, Archaea, and Pseudomonas were investigated by performing PCR amplification of 16S rRNA genes and reverse-transcribed 16S rRNA, followed by restriction fragment length polymorphism (RFLP) and terminal RFLP (T-RFLP) fingerprinting. Statistical analyses of RFLP and T-RFLP profiles indicated that specific groups in the microbial community were sensitive to the stress. In addition, evaluation of rRNA genes and rRNA as markers for monitoring the stress responses of microbial communities revealed overall similar sensitivities. We concluded that various structural and functional MSCs were not resistant to drying-rewetting stress and that resilience depended strongly on the parameter investigated.

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Impact of Soil Drying-Rewetting Stress on Microbial Communities and Activities and on Degradation of Two Crop Protection Products

Prior to registration and commercialization, crop protection products (CPP) need to be tested for their environmental impact and fate. For fate studies, laboratory microcosm experiments are performed with defined natural soils. Quality control of these soils represents a challenge, since it is often difficult to obtain fresh soil and soil storage may affect soil characteristics. According to guidelines of the Organization for Economic Cooperation and Development (OECD) frozen storage is recommended for periods exceeding three months. The present study was performed to investigate the influence of soil frozen storage on microbiological soil characteristics (MSCs) and CPP mineralization rates. For this purpose an agricultural soil was frozen for four days, thawed, and subsequently equilibrated and monitored during 42 days under controlled conditions. Substrate-induced respiration (SIR) rates increased shortly after thawing and returned to reference levels within ten days. Degradation of the CPP methidathion appeared unaffected in the soils that had been frozen. Conversely, we found that soil DNA contents and direct cell counts decreased by 24 and 22%, respectively, in the frozen stored soil. Impacts on soil microbial population structures were analyzed by use of domain-specific PCR of small subunit ribosomal RNA genes for Eucarya, Bacteria, and Archaea in conjunction with restriction fragment length polymorphism (RFLP) analysis. Since the Archaea community appeared most sensitive to the freezing stress, cloning and sequencing was performed to identify marker genes for freezing stress sensitive and resistant groups. Novel specific PCR detections were developed for a freezing stress resistant and a freezing stress sensitive group. These new tools allowed monitoring and comparison of archaeal population shifts without using RFLP analysis. Over all, the molecular biological approach revealed that the initial MSCs were not restored after 42 days equilibration of the frozen soil, even though SIR rates and mineralization of one specific CPP appeared unaffected. Our results demonstrate that specific molecular tools can identify sensitive indicators of altered MSCs and may be applied for the description of stress effects that can occur during soil storage.

Effects of freeze–thaw stress during soil storage on microbial communities and methidathion degradation

Methanogenic activity was investigated in a petroleum hydrocarbon-contaminated aquifer by using a series of four push-pull tests with acetate, formate, H2 plus CO2, or methanol to target different groups of methanogenic Archaea. Furthermore, the community composition of methanogens in water and aquifer material was explored by molecular analyses, i.e., fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes amplified with the Archaea-specific primer set ARCH915 and UNI-b-rev, and sequencing of DNA from dominant DGGE bands. Molecular analyses were subsequently compared with push-pull test data. Methane was produced in all tests except for a separate test where 2-bromoethanesulfonate, a specific inhibitor of methanogens, was added. Substrate consumption rates were 0.11 mM day−1 for methanol, 0.38 mM day−1 for acetate, 0.90 mM day−1 for H2, and 1.85 mM day−1 for formate. Substrate consumption and CH4 production during all tests suggested that at least three different physiologic types of methanogens were present: H2 plus CO2 or formate, acetate, and methanol utilizers. The presence of 15 to 20 bands in DGGE profiles indicated a diverse archaeal population. High H2 and formate consumption rates agreed with a high diversity of methanogenic Archaea consuming these substrates (16S rRNA gene sequences related to several members of the Methanomicrobiaceae) and the detection of Methanomicrobiaceae by using FISH (1.4% of total DAPI [4′,6-diamidino-2-phenylindole]-stained microorganisms in one water sample; probe MG1200). Considerable acetate consumption agreed with the presence of sequences related to the obligate acetate degrader Methanosaeata concilii and the detection of this species by FISH (5 to 22% of total microorganisms; probe Rotcl1). The results suggest that both aceticlastic and CO2-type substrate-consuming methanogens are likely involved in the terminal step of hydrocarbon degradation, while methanogenesis from methanol plays a minor role. DGGE profiles further indicate similar archaeal community compositions in water and aquifer material. The combination of hydrogeological and molecular methods employed in this study provide improved information on the community and the potential activity of methanogens in a petroleum hydrocarbon-contaminated aquifer.

Manuel Pesaro

Papers

Preferential Flow Paths: Biological Hot Spots in Soils

A strategy for optimizing quality and quantity of DNA extracted from soil

Impact of Soil Drying-Rewetting Stress on Microbial Communities and Activities and on Degradation of Two Crop Protection Products

Effects of freeze–thaw stress during soil storage on microbial communities and methidathion degradation

Activity and Diversity of Methanogens in a Petroleum Hydrocarbon-Contaminated Aquifer