Microbial functioning refers to microbial activity because only the active microorganisms drive biogeochemical processes. Despite the importance of active microorganisms, most methods focus on estimating total microbial biomass and fail to evaluate its active fraction. At first, we have described the differences among the active , potentially active , and dormant microbial states in soil and suggested threshold values of parameters for their identification. Secondly, we critically reviewed the ability of a broad range of approaches to estimate and characterize the active and the potentially active microorganisms in soil. Following approaches were evaluated: plate count and microbial cultures; direct microscopy combined with cell staining; ATP, PLFA, DNA and RNA content; microarray analyses; PCR-based approaches; stable isotope probing; soil proteomics, enzymes activity; and various approaches based on respiration and substrate utilization. The “static” approaches, mainly based on the single-stage determination of cell components (ATP, DNA, RNA, and molecular biomarkers), detect well the presence of microorganisms and total biomass, but they fail to evaluate the active part and consequently the functions. In contrast, the dynamic approaches, estimating the changes of these parameters during microbial growth and based on process rates: substrate utilization and product formation, e.g., respiration, help to evaluate active microbial biomass and relate it to specific process rates. Based on a comparison of all approaches for their universality (possibility to analyze active, potentially active and dormant microorganisms), we concluded that 1) direct microscopy with complementary stains, 2) a combination of RNA-based FISH with staining of total microbial biomass, and 3) approaches based on microbial growth were the most advantageous and allowed simultaneous quantitative estimation of active , potentially active, and dormant microorganisms in soil. The active microorganisms compose only about 0.1–2% of the total microbial biomass and very seldom exceed 5% in soils without input of easily available substrates. Nonetheless, the fraction of potentially active microorganisms (ready to start utilization of available substrates within few hours) is much higher, contributing between 10 and 40% (up to 60%) of the total microbial biomass. Therefore, we emphasize the role of potentially active microorganisms with quick response to fluctuating substrate input in soil microhabitats and hotspots. The transition from the potentially active to the active state occurs in minutes to hours, but the shift from dormant to active state takes anywhere from hours to days. Despite very fast activation, the reverse process – fading to the potentially active and dormant stage – requires a much longer period and is very different for individual criteria: ATP, DNA, RNA, enzyme production, respiration rates. This leads to further difficulties in the estimation of the active part of microbial community by methods based on these parameters. Consequently, the standardization, further elaboration, and broad application of approaches focused on the portion of active microorganisms in soil and their functions are urgently needed. We conclude that because active microorganisms are the solely microbial drivers of main biogeochemical processes, analyses of the active and potentially active fractions are necessary in studies focused on soil functions.

Active microorganisms in soil: Critical review of estimation criteria and approaches

Application of microbial metabolic potential (bioremediation) is accepted as an environmentally benign and economical measure for decontamination of polluted environments. Bioremediation methods are generally categorized into ex situ and in situ bioremediation. Although in situ bioremediation methods have been in use for two to three decades, they have not yet yielded the expected results. Their limited success has been attributed to reduced ecological sustainability under environmental conditions. An important determinant of sustainability of in situ bioremediation is pollutant bioavailability. Microbial chemotaxis is postulated to improve pollutant bioavailability significantly; consequently, application of chemotactic microorganisms can considerably enhance the performance of in situ degradation. The environmental fate of degradative microorganisms and the ecological consequence of intervention constitute other important descriptors for the efficiency and sustainability of bioremediation processes. Integrative use of culture-dependent, culture-independent methods (e.g. amplified rDNA restriction analysis, terminal restriction fragment length polymorphism, denaturing/thermal gradient gel electrophoresis, phospholipid fatty acid, etc.), computational and statistical analyses has enabled successful monitoring of the above aspects. The present review provides a detailed insight into some of the key factors that affect the efficiency of in situ bioremediation along with a comprehensive account of the integrative approaches used for assessing the ecological sustainability of processes. The review also discusses the possibility of developing suicidal genetically engineered microorganisms for optimized and controlled in situ bioremediation.

Integrative approaches for assessing the ecological sustainability of in situ bioremediation

Recovery and quantification of bacterial cells associated with streambed sediments.

https://pure.mpg.de/pubman/item/item_2485599_2/component/file_3270361/Abed6.pdf

Bacterial diversity of a cyanobacterial mat degrading petroleum compounds at elevated salinities and temperatures

Routine fluorescence in situ hybridization in soil.

Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry.

Low average temperatures and temperature fluctuations in temperate soils challenge the efficacy of microbial strains used for clean up of pollutants In this study, we investigated the cold tolerance of Arthrobacter chlorophenolicus A6, a microorganism previously shown to degrade high concentrations of 4-chlorophenol at 28°C Luciferase activity from a luc-tagged derivative of the strain (A6L) was used to monitor the metabolic status of the population during 4-chlorophenol degradation The A6L strain could degrade 200–300 μg mL−1 4-chlorophenol in pure cultures incubated at 5°C, although rates of degradation, growth and the metabolic status of the cells were lower at 5°C compared to 28°C When subjected to temperature fluctuations between 5 and 28°C, A6L continued to degrade 4-chlorophenol and remained active In soil microcosm experiments, the degradation rates were significantly faster the first week at 28°C, compared to 5°C However, this difference was no longer seen after 7 days, and equally low 4-chlorophenol concentrations were reached after 17 days at both temperatures During 4-chlorophenol degradation in soil, CFU and luciferase activity values remained constant at both 5 and 28°C However, once most of the 4-chlorophenol was degraded, both values decreased by 1–15 logarithmic values at 28°C, whereas they remained constant at 5°C, indicating a high survival of the cells at low temperatures Because of the ability of A chlorophenolicus A6 to degrade high concentrations of 4-chlorophenol at 5°C, together with its tolerance to temperature fluctuations and stress conditions found in soil, this strain is a promising candidate for bioaugmentation of chlorophenol-contaminated soil in temperate climates

Degradation of 4-Chlorophenol at Low Temperature and during Extreme Temperature Fluctuations by Arthrobacter chlorophenolicus A6

Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28degreesC. In this study, we investigated the effects of temperature on the physiological status ...

https://aem.asm.org/content/aem/70/5/2952.full.pdf

Impact of Temperature on the Physiological Status of a Potential Bioremediation Inoculant, Arthrobacter chlorophenolicus A6

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Papers

Monitoring physiological status of GFP-tagged Pseudomonas fluorescens SBW25 under different nutrient conditions and in soil by flow cytometry.

Degradation of 4-Chlorophenol at Low Temperature and during Extreme Temperature Fluctuations by Arthrobacter chlorophenolicus A6

Impact of Temperature on the Physiological Status of a Potential Bioremediation Inoculant, Arthrobacter chlorophenolicus A6