Key factors controlling microbial community responses after a fire: Importance of severity and recurrence.

Wildfires are a major problem in Mediterranean forest ecosystems, affecting the same area year after year. Their severity is increasing, partly due to climate change and hence, every now and then, virulent fires of high severity spread ravage this region. The aim of this study was to evaluate the influence of fire regime (recurrence, severity) in soil microbial community structure analyzing the phospholipid fatty acid (PLFA) and the microbial functional diversity assessing the level physiological profiling technique (CLPP). Samples of a soil affected by a high severities wildfire and a soil affected by a low severity experimental fire were heated under laboratory conditions at different temperatures to simulate different fire severity. To simulate fire recurrence, the heating treatment was repeated after one month of incubation. The fire severity was estimated as the amount of heat supplied to samples by degree-hour methodology. A marked impact of fire regime on soil microorganisms was detected; the microbial community response varied depending on previous history of fire and the magnitude of changes in PLFA pattern and CLPP, was related to the amount of heat supplied to the samples. Wildfires had a greater impact on microbial community structure than subsequent soil heating in the laboratory. The total biomass and the biomass of specific groups of microorganisms decreased notably as a consequence of wildfire and minor changes were detected due to the experimental fire and soil heating under laboratory conditions. The results clearly showed the usefulness of PLFA pattern to study the effect of fire regimes and associated direct and indirect changes in soil microorganisms and in soil quality. The data also indicated that the degree-hour methodology rather than maximum temperature is adequate to simulate fire severity and evaluate the impact of thermal shock on soil ecosystems.

Use of biomass ash to reduce toxicity affecting soil bacterial community growth due to tetracycline antibiotics.

Tetracycline antibiotics (TA) used in veterinary medicine reach terrestrial ecosystems mostly via the repeated applications of animal manures and slurries on agricultural soils, where they may cause toxic effects on bacterial communities. In the current work, we studied the efficacy of adding doses of 0, 6, 24 and 48 g kg-1 of biomass ash (BA) to four different soils to reduce potential negative effects of tetracycline antibiotics. Specifically, soil samples were polluted with different concentrations of tetracycline, oxytetracycline or chlortetracycline, and the bacterial community growth was estimated using the 3H leucine incorporation technique. Soil amendment with BA increased soil pH (1.3-4.8 units), total carbon (0.7-5.8 g kg-1) and Fe and Al oxides concentrations (0.25-3.98 g kg-1), as well as bacterial activity (1-9 times compared to the control). In addition, BA amendment at high doses (24 or 48 g kg-1) resulted in a similar toxicity decrease for the three antibiotics, but with variations among soils. The reductions in antibiotics toxicity were very variable, ranging between 5% and 100% (total recovery). In view of that, the spreading of BA could be interesting as management practice to reduce risks of soil pollution and subsequent toxicity on bacterial communities due to tetracycline antibiotics.

Degradation of sulfadiazine, sulfachloropyridazine and sulfamethazine in aqueous media.

Antibiotics discharged to the environment constitute a main concern for which different treatment alternatives are being studied, some of them based on antibiotics removal or inactivation using by-products with adsorbent capacity, or which can act as catalyst for photo-degradation. But a preliminary step is to determine the general characteristics and magnitude of the degradation process effectively acting on antibiotics. A specific case is that of sulfonamides (SAs), one of the antibiotic groups most widely used in veterinary medicine, and which are considered the most mobile antibiotics, causing that they are frequently detected in both surface- and ground-waters, facilitating their entry in the food chain and causing public health hazards. In this work we investigated abiotic and biotic degradation of three sulfonamides (sulfadiazine -SDZ-, sulfachloropyridazine -SCP-, and sulfamethazine -SMT-) in aqueous media. The results indicated that, in filtered milliQ water and under simulated sunlight, the degradation sequence was: SCP > SDZ ≈ SMT. Furthermore, the rate of degradation clearly increased with the raise of pH: at pH 4.0, half-lives were 1.2, 70.5 and 84.4 h for SCP, SDZ and SMT, respectively, while at pH 7.2 they were 2.3, 9.4 and 13.2 h for SCP, SMT and SDZ. The addition of a culture medium hardly caused any change in degradation rates as compared to experiments performed in milliQ water at the same pH value (7.2), suggesting that in this case sulfonamides degradation rate was not affected by the presence of some chemical elements and compounds, such as sodium, chloride and phosphate. However, the addition of bacterial suspensions extracted from a soil and from poultry manure increased the rate of degradation of these antibiotics. This increase in degradation cannot be attributed to biodegradation, since there was no degradation in the dark during the time of the experiment (72 h). This indicates that photo-degradation constitutes the main removal mechanism for SAs in aqueous media, a mechanism that in this case was favored by humic acids supplied with the extracts from soil and manure. The overall results could contribute to the understanding of the environmental fate of the three sulfonamides studied, aiding to program actions that could favor their inactivation, which is especially relevant since its dissemination can involve serious environmental and public health risks.

Biotic and abiotic dissipation of tetracyclines using simulated sunlight and in the dark.

Veterinary antibiotics reaching soils and water bodies are considered emerging pollutants deserving special attention. In this work, dissipation of tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC) is investigated. Dissipation experiments in filtered water, using simulated sunlight, resulted in the following degradation sequence: TC < OTC ≈ CTC, with half-life values of 229, 101 and 104 min, respectively; however, no dissipation took place in the dark. Dissipation of the three tetracyclines in culture medium and with simulated sunlight was much higher, giving the sequence TC ≈ OTC < CTC, with half-lives of 9, 10 and 7 min, respectively; in the dark, TC and OTC did not suffer dissipation, but it was around 28% for CTC at the end of the experiment (480 min). The variable explaining a higher dissipation in culture medium and with light was pH, as this parameter caused changes in the distribution of species of tetracyclines, affecting degradation. Adding bacterial suspensions extracted from soil and poultry manure increased dissipation, giving the sequence: TC ≈ OTC < CTC, which is attributed to the presence of humic acids, which adsorb these antibiotics. These results could facilitate understanding the fate of antibiotics reaching environmental compartments and causing public health hazards.

Response of soil microbial communities to fire and fire-fighting chemicals.

Worldwide, fire-fighting chemicals are rapidly gaining acceptance as an effective and efficient tool in wildfires control and in prescribed burns for habitat management. However, despite its widespread use as water additives to control and/or slow the spread of fire, information concerning the impact of these compounds on soil ecosystems is scarce. In the present work we examine, under field conditions, the response of the microbial communities to three different fire-chemicals at normal doses of application. The study was performed with a Humic Cambisol over granite under heath, located in the temperate humid zone (Galicia, NW Spain) with the following treatments: unburned soil (US) and burned soil added with water alone (BS) or mixed with the foaming agent Auxquímica RFC-88 at 1% (BS+Fo), Firesorb at 1.5% (BS+Fi) and FR Cross ammonium polyphosphate at 20% (BS+Ap). The microbial mass (microbial C), activity (ß-glucosidase, urease) and community structure [phospholipids fatty acids (PLFA) pattern] were measured on soil samples collected at different sampling times during a 5year period after a prescribed fire. The results showed a negative short-term effect of the fire on the microbial properties. The microbial biomass and activity levels tended to recover with time; however, changes in the microbial community structure (PLFA pattern) were still detected 5years after the prescribed fire. Compared to the burned soil added with water, the ammonium polyphosphate and the Firesorb treatments were the fire-fighting chemicals that showed a higher influence on the microbial communities over the whole study period. Our data indicated the usefulness of the PLFAs analysis to detect the long-term impact of both fire and fire-fighting chemicals on the soil microbial communities and hence on the soil quality of forest ecosystems.

Extractable atrazine and its metabolites in agricultural soils from the temperate humid zone.

Extractable atrazine and its metabolites (hydroxyatrazine, deethylatrazine and deisopropylatrazine) were evaluated in agricultural soils from the temperate humid zone (Galicia, NW Spain) under laboratory conditions. The experiment was performed with five soils with different properties (organic C, soil texture and atrazine application history), both unamended and treated with atrazine at field application rate. Measurements of the atrazine compounds were made at different time intervals (1, 3, 6, 9 and 12 weeks) during a 3-month incubation period. Results showed that only hydroxyatrazine was detected in the extractable fraction of the unamended soils, with values remaining relatively constant throughout the incubation period. Atrazine addition notably increased the concentration of the parent compound and its degradation products; deisopropylatrazine and hydroxyatrazine were the main metabolites detected in the extractable fraction of the treated soils, whereas deethylatrazine was not detected. After 7 days incubation, values of total extractable residues, expressed as percentage of initially added atrazine, ranged from 75 to 86% (25-68% of atrazine, 7-11% of hydroxyatrazine and 9-57% of deisopropylatrazine). The values decreased rapidly during the first 3 weeks of incubation, showing values of 2-8% in soils with higher atrazine application and from 28 to 30% in soils with lower application history. At the end of the incubation, 2-8% of total extractable residues were still detected (0-4% of atrazine, 2-3% of hydroxyatrazine and 0-2% of deisopropylatrazine), indicating a residual effect of atrazine addition. These variations in the extractable fraction indicated that most added atrazine was rapidly degraded, especially in soils with higher application history.

Tolerance (PICT) of the bacterial communities to copper in vineyards soils from Spain.

To detect effects of Cu pollution, the Cu tolerance of soil bacterial communities extracted from several vineyards located in NW Spain was measured. Bacterial community tolerance was estimated by means of the thymidine (TdR) and leucine (Leu) incorporation techniques using either IC(50) values (the log of the metal concentration that reduced incorporation to 50%) or the percentage of activity at one specific Cu concentration (10(-6) mol L(-1)). The tolerance measurements by the TdR incorporation technique were similar to those obtained by the Leu incorporation method, indicating that the two methods were equivalent in terms of suitability for detecting the toxicity of Cu to soil bacterial communities. The two tolerance indices considered (IC50 values and percentage of activity) were closely correlated (r = 0.975, P < 0.001), showing that both were equally good in measuring Cu tolerance of the bacterial community. An increased bacterial community tolerance to Cu, indicating a pollution effect, was observed in vineyard soils with more than 100 mg Cu kg(-1) soil. Thus, the long-term use of Cu in vineyards has a toxic effect on the soil bacterial community, resulting in an increased tolerance. An effect of increased levels of Cu could not be detected when measuring bacterial community activity, pointing to the increased sensitivity to detect toxicity in field studies using tolerance measurements.

Atrazine degradation and residues distribution in two acid soils from temperate humid zone.

Mineralization of atrazine and formation of extractable and non-extractable "bound" residues were followed under laboratory conditions in two contrasting soils (organic C, texture, and atrazine application history) from northern Spain. The soils, a Humic Cambisol (MP) and a Gleyic Cambisol (G) were incubated with labeled atrazine (ring-13C atrazine) at field application dose and measurements were made at different time intervals during 3 mo. Fate and behavior of atrazine along the incubation showed different patterns between the two soils, the time taken for degradation of 50% (DT50) being 9 and 44 d for MP and G soils, respectively. In MP soil, with 40 yr of atrazine application and lower organic C and clay content, more than 89% of U-13C-atrazine added was mineralized after 12 wk, with most mineralization occurring within the first 2 wk. G soil, with 10 yr of atrazine application, exhibited a more progressive U-13C-atrazine mineralization, reaching 54% of initially added atrazine at 12 wk. Hydroxyatrazine and deisopropylatrazine were the metabolites founded in the extractable fraction, demonstrating that both chemical and biological processes are involved in atrazine degradation. Soil G showed during all the incubation times an extractable residues fraction greater than that in MP soil, indicating a high potential risk of soil and water contamination. Rapid microbial degradation through s-triazine ring cleavage was proposed to be the main decomposition pathway of atrazine for the two soils studied. Bound residues pool also differed notably between soils accounting for 9 and 41% of initially added atrazine, the higher values shown by soil with higher organic matter and clay content (G soil).

Microbial biomass, community structure and metal tolerance of a naturally Pb-enriched forest soil.

The effect of long-term elevated soil Pb levels on soil microbiota was studied at a forest site in Norway, where the soil has been severely contaminated with Pb since the last period of glaciation (several thousand years). Up to 10% Pb (total amount, w/w) has been found in the top layer. The microbial community was drastically affected, as judged from changes in the phospholipid fatty acid (PLFA) pattern. Specific PLFAs that were high in Pb-enriched soil were branched (especially br17:0 and br18:0), whereas PLFAs common in eukaryotic organisms such as fungi (18:2omega6,9 and 20:4) were low compared with levels at adjacent, uncontaminated sites. Congruent changes in the PLFA pattern were found upon analyzing the culturable part of the bacterial community. The high Pb concentrations in the soil resulted in increased tolerance to Pb of the bacterial community, measured using both thymidine incorporation and plate counts. Furthermore, changes in tolerance were correlated to changes in the community structure. The bacterial community of the most contaminated soils showed higher specific activity (thymidine and leucine incorporation rates) and higher culturability than that of control soils. Fungal colony forming units (CFUs) were 10 times lower in the most Pb-enriched soils, the species composition was widely different from that in control soils, and the isolated fungi had high Pb tolerance. The most commonly isolated fungus in Pb-enriched soils was Tolypocladium inflatum. Comparison of isolates from Pb-enriched soil and isolates from unpolluted soils showed that T. inflatum was intrinsically Pb-tolerant, and that the prolonged conditions with high Pb had not selected for any increased tolerance.

Recycling of organic wastes in burnt soils: combined application of poultry manure and plant cultivation.

A pot experiment was conducted to investigate the efficacy of a post-fire land management practice, including plant cultivation (Lolium perenne) combined with poultry manure addition, for restoring the protective vegetation cover in soils degraded by high intensity wildfires. The greenhouse experiment was performed with three burnt pine forest soils with added poultry manure at two doses of application and comparing the data with those obtained using NPK fertilizer. A significant effect of the amendment, soil properties and the interaction between amendment and soil properties on vegetation cover (phytomass production, nutrient content) was detected, but often the amendment treatment explained most of the variance. Changes induced by the organic amendment were more marked than those induced by inorganic fertilization. The increase of phytomass and nutrient uptake with poultry manure addition indicated the beneficial effects of this soil management practice. These findings can serve to develop field experiments and burnt soils reclamation technology.

Co-composting of chestnut burr and leaf litter with solid poultry manure.

A co-composting of chestnut burr and leaf litter mixed with solid poultry manure was assessed by comparison of several chemical, physicochemical and biological parameters. The final pH of the co-compost was 8.89 and the C/N ratio was 13. The germination index (GI) obtained using the co-compost varied with the seeds used. It was 155.35% for ryegrass seeds, 56.56% for wheat seeds and 100% for barley seeds. The co-compost was mature in 103 days from a biological point of view.

Effect of metal-rich sludge amendments on the soil microbial community.

The effects of heavy-metal-containing sewage sludge on the soil microbial community were studied in two agricultural soils of different textures, which had been contaminated separately with three predominantly single metals (Cu, Zn, and Ni) at two different levels more than 20 years ago. We compared three community-based microbiological measurements, namely, phospholipid fatty acid (PLFA) analysis to reveal changes in species composition, the Biolog system to indicate metabolic fingerprints of microbial communities, and the thymidine incorporation technique to measure bacterial community tolerance. In the Luddington soil, bacterial community tolerance increased in all metal treatments compared to an unpolluted-sludge-treated control soil. Community tolerance to specific metals increased the most when the same metal was added to the soil; for example, tolerance to Cu increased most in Cu-polluted treatments. A dose-response effect was also evident. There were also indications of cotolerance to metals whose concentration had not been elevated by the sludge treatment. The PLFA pattern changed in all metal treatments, but the interpretation was complicated by the soil moisture content, which also affected the results. The Biolog measurements indicated similar effects of metals and moisture to the PLFA measurements, but due to high variation between replicates, no significant differences compared to the uncontaminated control were found. In the Lee Valley soil, significant increases in community tolerance were found for the high levels of Cu and Zn, while the PLFA pattern was significantly altered for the soils with high levels of Cu, Ni, and Zn. No effects on the Biolog measurements were found in this soil.

Development of metal tolerance in soil bacterial communities exposed to experimentally increased metal levels.

The development of metal tolerance in soil bacterial communities exposed to different heavy metals was examined under laboratory conditions. An agricultural soil amended with different Zn concentrations was studied most intensively, and measurements were made over a 28-month incubation period by means of the thymidine incorporation technique. Tolerance levels were not affected by metal concentrations lower than 2 mmol of Zn kg (dry weight) of soil(sup-1), but above this value, the level of Zn tolerance increased exponentially with the logarithm of the soil Zn concentration. An increased metal tolerance was detected after only 2 days of Zn exposure. Thereafter, stable tolerance values were observed at different sampling times for bacterial communities exposed to up to 8 mmol of Zn kg (dry weight)(sup-1), indicating no changes in tolerance with time. The tolerance of bacterial communities exposed to 32 mmol of Zn kg (dry weight)(sup-1) increased rapidly within the second week of incubation, but then the values remained unchanged until the end of the experiment. Bacterial communities from soil contaminated with 16 mmol of Zn kg (dry weight)(sup-1) showed an increase of the same magnitude, but the increase started later, after 4 months of incubation, and took place for a much longer period (more than 1 year). Cd, Cu, and Ni addition also resulted in metal-tolerant communities, and the level of tolerance increased with prolonged incubations of the soils. The bacterial community at the end of the incubation period also exhibited a lower pH optimum and an increased tolerance to low osmotic potential. The results suggest that the increase in metal tolerance of the community after adding metals can be attributed to an immediate effect due to the death of sensitive species and a later effect due to different competitive abilities and adaptation of surviving bacteria.

Multiple heavy metal tolerance of soil bacterial communities and its measurement by a thymidine incorporation technique.

A thymidine incorporation technique was used to determine the tolerance of a soil bacterial community to Cu, Cd, Zn, Ni, and Pb. An agricultural soil was artificially contaminated in our laboratory with individual metals at three different concentrations, and the results were compared with the results obtained by using the plate count technique. Thymidine incorporation was found to be a simple and rapid method for measuring tolerance. Data obtained by this technique were very reproducible. A linear relationship was found between changes in community tolerance levels obtained by the thymidine incorporation and plate count techniques (r = 0.732, P < 0.001). An increase in tolerance to the metal added to soil was observed for the bacterial community obtained from each polluted soil compared with the community obtained from unpolluted soil. The only exception was when Pb was added; no indication of Pb tolerance was found. An increase in the tolerance to metals other than the metal originally added to soil was also observed, indicating that there was multiple heavy metal tolerance at the community level. Thus, Cu pollution, in addition to increasing tolerance to Cu, also induced tolerance to Zn, Cd, and Ni. Zn and Cd pollution increased community tolerance to all five metals. Ni amendment increased tolerance to Ni the most but also increased community tolerance to Zn and, to lesser degrees, increased community tolerance to Pb and Cd. In soils polluted with Pb increased tolerance to other metals was found in the following order: Ni > Cd > Zn > Cu. We found significant positive relationships between changes in Cd, Zn, and Pb tolerance and, to a lesser degree, between changes in Pb and Ni tolerance when all metals and amendment levels were compared. The magnitude of the increase in heavy metal tolerance was found to be linearly related to the logarithm of the metal concentration added to the soil. Threshold tolerance concentrations were estimated from these linear relationships, and changes in tolerance could be detected at levels of soil contamination similar to those reported previously to result in changes in the phospholipid fatty acid pattern (A. Frostegård, A. Tunlid, and E. Bååth, Appl. Environ. Microbiol. 59: 3605-3617, 1993).