The review discusses origin, state and function of extracellular DNA in soils and sediments. Extracellular DNA can be released from prokaryotic and eukaryotic cells and can be protected against ...nuclease degradation by its adsorption on soil colloids and sand particles. Laboratory experiments have shown that DNA adsorbed by colloids and sand particles can be taken up by prokaryotic competent cells and be involved in natural transformation. Most of these experiments have been carried out under artificial conditions with pure DNA molecules and pure adsorbing matrices, but in soils and sediments, pure surface-reactive colloids are not present and DNA is present with other cellular components (wall debris, proteins, lipids, RNA, etc.) especially if released after cell lysis. The presence of inorganic compounds and organic molecules on both soil particles and DNA molecules can influence the DNA adsorption, degradation and transformation of competent cells. Extracellular DNA can be used as C, N and P sources by heterotrophic microorganisms and plays a significant role in bacterial biofilm formation. The nucleotides and nucleosides originated from the degradation of extracellular DNA can be re-assimilated by soil microorganisms. Extracellular DNA in soil can be leached and moved by water through the soil profile by capillarity. In this way, the extracellular DNA secreted by a cell can reach a competent bacterial cell far from the donor cell. Finally, the characterisation of extracellular DNA can integrate information on the composition of the microbial community of soil and sediments obtained by analysing intracellular DNA.
Microbial diversity and soil functions Nannipieri, P.; Ascher, J.; Ceccherini, M. T. ...
European journal of soil science,
January 2017, 2017-01-00, 20170101, Letnik:
68, Številka:
1
Journal Article
Recenzirano
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.
•We set up a culture-independent assessment of soil microbial biomass.•PicoGreen quantification of crude dsDNA extracts provides reliable data.•The method is simple, high-throughput, and does not ...require expensive equipment.
We set up a simple, culture independent, low-cost and high-throughput method for DNA-based quantitative assessment of soil microbial biomass using eight soils covering a wide range of physico-chemical properties. DNA was extracted with a 0.12M, pH 8 Na2HPO4 buffer using bead beating; double stranded DNA (dsDNA) was quantified in a crude (not purified) extract using PicoGreen reagent. In contrast to yields obtained by using a commercial standard method (FastDNA Kit for soil, MP-Biomedicals), our yields of dsDNA were generally higher, most probably because any purification method for obtaining highly pure DNA for downstream analyses leads to DNA loss. These results suggest the new method provides more reliable quantitative data; thus it is a good environmental indicator, as an underestimation of the soil microbial biomass due to DNA loss during purification can be excluded. The ratio between microbial C (Cmic) obtained by the traditional, widely used fumigation-extraction method and dsDNA ranged from 12.0 to 63.5μg Cmic per μg dsDNA. Crude DNA obtained by the new method as well as purified DNA obtained by using the commercial kit were compared in terms of quantity (fluorometry; spectrophotometry) and quality (purity indices: A260/A280, A260/A230; PCR compatibility; gel electrophoresis: molecular weight and molecular integrity). Our results suggest that the new method provides a high-throughput estimator of microbial biomass (expressed as μgdsDNAg−1 soil) in soils having widely different properties without the need for high-cost commercial extraction kits and/or cumbersome individual methods. Due to its simplicity, speed and low-cost, our method is capable for routine quantitative assessments of soil microbial biomass, assessable also for soil scientists with laboratories that are otherwise not equipped for molecular analyses.
Microbial diversity and soil functions Nannipieri, P.; Ascher, J.; Ceccherini, M. T. ...
European journal of soil science,
December 2003, Letnik:
54, Številka:
4
Journal Article
Recenzirano
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.
We studied the distribution of the indigenous bacterial and fungal communities in a forest soil profile. The composition of bacterial and fungal communities was assessed by denaturing gradient gel ...electrophoresis (DGGE) of total and extracellular DNA extracted from all the soil horizons. Microbial biomass C and basal respiration were also measured to assess changes in both microbial biomass and activity throughout the soil profile. The 16S rDNA-DGGE revealed composite banding patterns reflecting the high bacterial diversity as expected for a forest soil, whereas 18S rDNA-DGGE analysis showed a certain stability and a lower diversity in the fungal communities. The banding patterns of the different horizons reflected changes in the microbial community structure with increasing depth. In particular, the DGGE analysis evidenced complex banding patterns for the upper A1 and A2 horizons, and a less diverse microflora in the deeper horizons. The low diversity and the presence of specific microbial communities in the B horizons, and in particular in the deeper ones, can be attributed to the selective environment represented by this portion of the soil profile. The eubacterial profiles obtained from the extracellular DNA revealed the presence of some bands not present in the total DNA patterns. This could be interpreted as the remainders of bacteria not any more present in the soil because of changes of edaphic conditions and consequent shifting in the microbial composition. These characteristic bands, present in all the horizons with the exception of the A1, should support the concept that the extracellular DNA is able to persist within the soil. Furthermore, the comparison between the total and extracellular 16S rDNA-DGGE profiles suggested a downwards movement of the extracellular DNA.
Long-term effects of high Cd concentrations on enzyme activities, microbial biomass and respiration and bacterial community structure of soils were assessed in sandy soils where Cd was added between ...1988 and 1990 as Cd(NO
3)
2 to reach concentrations ranging from 0 to 0.36 mmol Cd
kg
−1 dry weight soil. Soils were mantained under maize and grass cultivation, or ‘set-aside’ regimes, for 1 year. Solubility of Cd and its bioavailability were measured by chemical extractions or by the BIOMET bacterial biosensor system. Cadmium solubility was very low, and Cd bioavailability was barely detectable even in soils polluted with 0.36 mmol Cd
kg
−1. Soil microbial biomass carbon (
B
C) was slightly decreased and respiration was increased significantly even at the lower Cd concentration and as a consequence the metabolic quotient (
qCO
2) was increased, indicating a stressful condition for soil microflora. However, Cd-contaminated soils also had a lower total organic C (TOC) content and thus the microbial biomass C-to-TOC ratio was unaffected by Cd. Alkaline phosphomonoesterase, arylsulphatase and protease activities were significantly reduced in all Cd-contaminated soils whereas acid phosphomonoesterase, β-glucosidase and urease activites were unaffected by Cd. Neither changes in physiological groups of bacteria, nor of Cd resistant bacteria could be detected in numbers of the culturable bacterial community. Denaturing gradient gel electrophoresis analysis of the bacterial community showed slight changes in maize cropped soils containing 0.18 and 0.36 mmol Cd
kg
−1 soil as compared to the control. It was concluded that high Cd concentrations induced mainly physiological adaptations rather than selection for metal-resistant culturable soil microflora, regardless of Cd concentration, and that some biochemical parameters were more sensitive to stress than others.
Beringite (B) and zerovalent iron grit (Z), singly and in combination (BZ), were added to a loamy sand soil contaminated by trace elements (Reppel, Belgium), mainly by arsenic (As), to reduce As ...labile fractions and phytoavailability. An uncontaminated sandy soil was studied for comparison. Soils were placed in large lysimeters cultivated with maize and vegetables for 6 years. pH, organic C and total N content increased in amended soils. The Z and BZ treatments reduced the Ca(NO
3)
2
− extractable soil As and As uptake by lettuce. The BZ lettuces had also the lowest foliar Pb, Cd, Zn, and Mn concentrations. All amendments had positive effects on the soil microbial biomass and reduced the qCO
2. Glucose mineralization was increased in Z and BZ amended soils. Acid phosphomonoesterase activity was higher in the untreated soil than in the other soils; the alkaline phosphomonoesterase, phosphodiesterase and protease activities were increased by Z and BZ treatments, whereas B amendment had less positive effects. Genetic fingerprinting using Denaturing Gradient Gel Electrophoresis (DGGE) revealed shifts in the composition of eubacterial and fungal communities of the amended soils. Microbial species richness decreased rather than increased in the treated soils, regardless of reduced trace element availability and increased soil microbial biomass and activity.
The soil DNA pool consists of an intracellular (iDNA) and extracellular fraction (eDNA). Challenging to improve the extraction efficiency of soil DNA, and to quantitatively and qualitatively ...characterize both DNA fractions, we set up a molecular approach consisting of sequential and comparative DNA extraction and microbial community fingerprinting. eDNA was extracted by alkaline soil washings (ASW); iDNA by mechanical chemical cell lysis (MCCL) of the residual soil pellet after the extraction of eDNA (ASW–MCCL). The molecular approach was compared in terms of quantity (fluorometer) and quality (agarose gel electrophoresis; small subunit rRNA-denaturing gradient gel electrophoresis) to directly extracted soil DNA comprising both eDNA and iDNA (tDNA; FastDNA Spin Kit for Soil, BIO101). The proposed method appeared to be a potential tool to separately extract and analyze eDNA (6.07
μg
g
−1
soil) and iDNA (11.46
μg
g
−1
soil) and to obtain a greater amount of DNA from soil with broader genetic information about eubacterial and fungal communities with respect to directly extracted tDNA (8.79
μg
g
−1
soil). Our results revealed the extracellular fraction to be quantitatively and qualitatively important of the soil metagenome. As the sequential DNA extraction method not only increased the total amount of extractable soil DNA (17.53
μg
g
−1
soil) but also that of iDNA, it is suggested to be suitable for extracting the soil metagenome.
The aim of this work was to study the synthesis and persistence of acid and alkaline phosphomonoesterases in three soils with different pH values amended with ryegrass residues. The organic input ...increased soil respiration, as estimated by CO
2–C evolution in all soils. The ATP content of the three soils showed a 3–7-fold increase between 7 and 10
d in the different soils since the amendment. The dsDNA content of the three amended soils also peaked between 7 and 10
d, increasing by 2.5–3.5 times in the different soils. The bacterial species richness increased in the amended as compared to the control soils during the early stages (7–10
d) of organic matter decomposition, as indicated by the decreasing values of the Sørensen similarity index between the treatments in this period. Soil amendment increased the alkaline phosphomonoesterase activity by 6, 8 and 15 times in the Vallombrosa acidic, Romola neutral and Vicarello alkaline soil, respectively, whereas the acid phosphomonoesterase activity showed a 6-, 2- and 10-fold increase in the Vallobrosa acidic, Romola neutral and Vicarello alkaline soil, respectively. Phosphatase activities peaked between 4 and 10
d, depending on the activity and the soil considered, but activity of alkaline phosphomonoesterase was higher in alkaline soils and persisted longer than the acid phosphomonoesterase activities; the opposite occurred in the acid soil. During a 180
d decomposition period, both acid and alkaline phosphomonoesterase activities were related to dsDNA and ATP contents in all soils. Peaks of phosphmonoesterase activity coincided with the changes in the composition of the bacterial microflora, as detected by 16S-rDNA analysis, although no relationship between bacterial community composition and persistence of the phosphomonoesterase activities could be shown. It was concluded that acid and alkaline phosphomonoesterases are produced in greater amounts during plant residue decomposition, and that in acid soils acid phosphomonoesterase activity predominates and in neutral and alkaline soils alkaline phosphomonoesterase activity predominates. However, the persistence of newly produced enzymes is determinated by other factors such as soil texture, organic matter content and formation of soil colloid–enzyme complexes.
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