Introducing bacterial community turnover times to elucidate temporal and spatial hotspots of biological instability in a large Austrian drinking water distribution network.

Ensuring biological stability in drinking water distribution systems (DWDSs) is important to reduce the risk of aesthetic, operational and hygienic impairments of the distributed water. Drinking water after treatment often changes in quality during transport due to interactions with pipe-associated biofilms, temperature increases and disinfectant residual decay leading to potential biological instability. To comprehensively assess the potential for biological instability in a large chlorinated DWDS, a tool-box of bacterial biomass and activity parameters was applied, introducing bacterial community turnover times (BaCTT) as a direct, sensitive and easy-to-interpret quantitative parameter based on the combination of 3H-leucine incorporation with bacterial biomass. Using BaCTT, hotspots and periods of bacterial growth and potential biological instability could be identified in the DWDS that is fed by water with high bacterial growth potential. A de-coupling of biomass from activity parameters was observed, suggesting that bacterial biomass parameters depict seasonally fluctuating raw water quality rather than processes related to biological stability of the finished water in the DWDS. BaCTT, on the other hand, were significantly correlated to water age, disinfectant residual, temperature and a seasonal factor, indicating a higher potential of biological instability at more distant sampling sites and later in the year. As demonstrated, BaCTT is suggested as a novel, sensitive and very useful parameter for assessing the biological instability potential. However, additional studies in other DWDSs are needed to investigate the general applicability of BaCTT depending on water source, applied treatment processes, biofilm growth potential on different pipe materials, or size, age and complexity of the DWDS.

Seasonal variation in temperature sensitivity of bacterial growth in a temperate soil and lake.

Faster bacterial biomass turnover is expected in water compared to soil, which would result in more rapid community adaption to changing environmental conditions, including temperature. Bacterial community adaptation for growth is therefore predicted to have larger seasonal amplitudes in lakes than in soil. To test this prediction, we compared the seasonal variation in temperature adaptation of bacterial community growth in a soil and lake in Southern Sweden (Tin situ 0-20°C, mean 10°C) during 1.5 years, based on monthly samplings including two winters and summers. An indicator of community adaptation, minimum temperature for growth (Tmin), was calculated from bacterial growth measurements (Leu incorporation) using the Ratkowsky model. The seasonal variation in Tmin (sinusoidal function, R2 = 0.71) was most pronounced for the lake bacterial community, with an amplitude for Tmin of 3.0°C (-4.5 to -10.5°C) compared to 0.6°C (-7 to -8°C) for the soil. Thus, Tmin in water increased by 0.32°C/degree change of Tin situ. Similar differences were also found when comparing four lakes and soils in the winter and summer (amplitudes 2.9°C and 0.9°C for lakes and soils, respectively). Thus, seasonal variation in temperature adaptation has to be taken into account in lakes, while for soils a constant Tmin can be used.

Effects of ciprofloxacin, trimethoprim, and amoxicillin on microbial structure and growth as emerging pollutants reaching crop soils.

The presence of emerging pollutants, and specifically antibiotics, in agricultural soils has increased notably in recent decades, causing growing concern as regards potential environmental and health issues. With this in mind, the current study focuses on evaluating the toxicity exerted by three antibiotics (amoxicillin, trimethoprim, and ciprofloxacin) on the growth of soil bacterial communities, when these pollutants are present at different doses, and considered in the short, medium, and long terms (1, 8 and 42 days of incubation). Specifically, the research was carried out in 12 agricultural soils having different physicochemical characteristics and was performed by means of the leucine (3H) incorporation method. In addition, changes in the structure of soil microbial communities at 8 and 42 days were studied in four of these soils, using the phospholipids of fatty acids method for this. The main results indicate that the most toxic antibiotic was amoxicillin, followed by trimethoprim and ciprofloxacin. The results also show that the toxicity of amoxicillin decreases with time, with values of Log IC50 ranging from 0.07 ± 0.05 to 3.43 ± 0.08 for day 1, from 0.95 ± 0.07 to 3.97 ± 0.15 for day 8, and from 2.05 ± 0.03 to 3.18 ± 0.04 for day 42, during the incubation period. Regarding trimethoprim, 3 different behaviors were observed: for some soils the growth of soil bacterial communities was not affected, for a second group of soils trimethoprim toxicity showed dose-response effects that remained persistent over time, and, finally, for a third group of soils the toxicity of trimethoprim increased over time, being greater for longer incubation times (42 days). As regards ciprofloxacin, this antibiotic did not show a toxicity effect on the growth of soil bacterial communities for any of the soils or incubation times studied. Furthermore, the principal component analysis performed with the phospholipids of fatty acids results demonstrated that the microbial community structure of these agricultural soils, which persisted after 42 days of incubation, depended mainly on soil characteristics and, to a lesser extent, on the dose and type of antibiotic (amoxicillin, trimethoprim or ciprofloxacin). In addition, it was found that, in this research, the application of the three antibiotics to soils usually favored the presence of fungi and Gram-positive bacteria.

Use of waste materials to prevent tetracycline antibiotics toxicity on the growth of soil bacterial communities.

The increase of concentrations of tetracycline antibiotics in agricultural soils worldwide is of special concern, due to its potential toxic effects on soil bacterial communities. In the present work, the reuse of two waste/by-product materials as soil amendments was tested as a preventive practice for reducing tetracycline antibiotics toxicity in soils. Pine bark (PB), with high percentage of organic carbon, and crushed mussel shell (CMS), a frequent natural liming material, were added to 4 soils in doses 0, 6, 12 and 48 g of by-product per kg-1 of soil (dry weight) of each one (separately). The soils and soil-waste mixtures were then spiked with tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC). After one day of incubation, the bacterial growth was estimated in soils and soil-mixtures using the leucine incorporation technique. The addition of PB to the soils showed two different behaviors, depending on the antibiotics. The toxicity of TC and OTC decreased with the addition of PB (toxicities going from 6 to 25% and from 5 to 36%, respectively). However, CTC toxicity did not change, or even increased in response to the PB amendment. Regarding soil amendment with CMS, it was not effective to prevent the toxicity of any of the three antibiotics studied.

The Toxicity Exerted by the Antibiotic Sulfadiazine on the Growth of Soil Bacterial Communities May Increase over Time.

The toxicity exerted by the antibiotic sulfadiazine on the growth of soil bacterial communities was studied in two agricultural soils for a period of 100 days. In the short-term (2 days of incubation), the effect of sulfadiazine on bacterial growth was low (no inhibition or inhibition <32% for a dose of 2000 mg·kg-1). However, sulfadiazine toxicity increased with time, achieving values of 40% inhibition, affecting bacterial growth in both soils after 100 days of incubation. These results, which were here observed for the first time for any antibiotic in soil samples, suggest that long-term experiments would be required for performing an adequate antibiotics risk assessment, as short-term experiments may underestimate toxicity effects.

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.

INT Toxicity over Natural Bacterial Assemblages from Surface Oligotrophic Waters: Implications for the Assessment of Respiratory Activity.

Plankton community respiration (R) is a major component of the carbon flux in aquatic ecosystems. However, current methods to measure actual respiration from oxygen consumption at relevant spatial scales are not sensitive enough in oligotrophic environments where respiration rates are very low. To overcome this drawback, more sensitive indirect enzymatic approaches are commonly used as R proxies. The in vivo electron transport system (ETSvivo) assay, which measures the reduction of (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl tetrazolium chloride salt, INT) to INT-formazan in the presence of natural substrate levels, was recently proposed as an indirect reliable estimation of R for natural plankton communities. However, under in vivo conditions, formazan salts could be toxic to the cells. Here, we test the toxicity of 0.2 mM of final INT concentration, widely used for ETSvivo assays, on natural bacterial assemblages collected in coastal and oceanic waters off Gran Canaria (Canary Islands, subtropical North Atlantic), in eight independent experiments. After 0.5 h of incubation, a significant but variable decline in cell viability (14-49%) was observed in all samples inoculated with INT. Moreover, INT also inhibited leucine uptake in less than 90 min of incubation. In the light of these results, we argue that enzymatic respiratory rates obtained with the ETSvivo method need to be interpreted with caution to derive R in oceanic regions where bacteria largely contribute to community respiration. Moreover, the variable toxicity on bacterial assemblages observed in our experiments questions the use of a single R/ETSvivo relationship as a universal proxy for regional studies.

Evaluation of dimethyl sulfoxide (DMSO) as a co-solvent for toxicity testing of hydrophobic organic compounds.

Toxicity testing of hydrophobic compounds with low aqueous solubility remains challenging. Dimethyl sulfoxide (DMSO) is widely used as a co-solvent for toxicity testing of hydrophobic chemicals, but it may modulate chemical toxicity patterns. In this study, we critically evaluated the suitability of DMSO as a co-solvent for toxicity testing of hydrophobic organic compounds in aqueous solutions. As the toxicity measure, we used growth inhibition of a natural bacterial community, and the test toxicants included phenol, BTEX (benzene, toluene, ethylbenzene and xylene) and transformation products of polycyclic aromatic hydrocarbons (PAHs). We found that dose-response curves for phenol were unaffected by DMSO concentrations up to 10% (v/v) and that DMSO (5% v/v) did not affect the degree of bacterial growth inhibition for any of the other test compounds in short-term experiments (3.5 h). By contrast, marked co-solvent effects of DMSO were observed in the long-term assay (25 and 27 h). We therefore conclude that DMSO has excellent co-solvent properties for short-term (≤3.5 h) toxicity testing of sparingly water-soluble compounds and its application provides a simple, inexpensive approach for screening of various environmentally relevant hydrophobic chemicals. Importantly, the use of DMSO allows for generation of full dose-responses that may otherwise not be attained.

The Effect of Light on Bacterial Activity in a Seaweed Holobiont.

Holobionts are characterized by the relationship between host and their associated organisms such as the biofilm associated with macroalgae. Considering that light is essential to macroalgae survival, the aim of this study was to verify the effect of light on the heterotrophic activity in biofilms of the brown macroalgae Sargassum furcatum during its growth cycle. Measurements of heterotrophic activity were done under natural light levels at different times during a daily cycle and under an artificial extinction of natural light during the afternoon. We also measured Sargassum primary production under these light levels in the afternoon. Both measurements were done with and without photosynthesis inhibitor and antibiotics. Biofilm composition was mainly represented by bacteria but diatoms, cyanobacteria, and other organisms were also common. When a peak of diatom genera was recorded, the heterotrophic activity of the biofilm was higher. Heterotrophic activity was usually highest during the afternoon and the presence of a photosynthesis inhibitor caused an average reduction of 17% but there was no relationship with Sargassum primary production. These results indicate that autotrophic production in the biofilm was reduced by the inhibitor with consequences on bacterial activity. Heterotrophic activity was mainly bacterial and the antibiotics chloramphenicol and penicillin were more effective than streptomycin. We suggest primary producers in the biofilm are more important to increase bacterial activity than the macroalgae itself because of coherence of the peaks of heterotrophic and autotrophic activity in biofilm during the afternoon and the effects of autotrophic inhibitors on heterotrophic activity.

Physiological Ecology of Microorganisms in Subglacial Lake Whillans.

Subglacial microbial habitats are widespread in glaciated regions of our planet. Some of these environments have been isolated from the atmosphere and from sunlight for many thousands of years. Consequently, ecosystem processes must rely on energy gained from the oxidation of inorganic substrates or detrital organic matter. Subglacial Lake Whillans (SLW) is one of more than 400 subglacial lakes known to exist under the Antarctic ice sheet; however, little is known about microbial physiology and energetics in these systems. When it was sampled through its 800 m thick ice cover in 2013, the SLW water column was shallow (~2 m deep), oxygenated, and possessed sufficient concentrations of C, N, and P substrates to support microbial growth. Here, we use a combination of physiological assays and models to assess the energetics of microbial life in SLW. In general, SLW microorganisms grew slowly in this energy-limited environment. Heterotrophic cellular carbon turnover times, calculated from 3H-thymidine and 3H-leucine incorporation rates, were long (60 to 500 days) while cellular doubling times averaged 196 days. Inferred growth rates (average ~0.006 d-1) obtained from the same incubations were at least an order of magnitude lower than those measured in Antarctic surface lakes and oligotrophic areas of the ocean. Low growth efficiency (8%) indicated that heterotrophic populations in SLW partition a majority of their carbon demand to cellular maintenance rather than growth. Chemoautotrophic CO2-fixation exceeded heterotrophic organic C-demand by a factor of ~1.5. Aerobic respiratory activity associated with heterotrophic and chemoautotrophic metabolism surpassed the estimated supply of oxygen to SLW, implying that microbial activity could deplete the oxygenated waters, resulting in anoxia. We used thermodynamic calculations to examine the biogeochemical and energetic consequences of environmentally imposed switching between aerobic and anaerobic metabolisms in the SLW water column. Heterotrophic metabolisms utilizing acetate and formate as electron donors yielded less energy than chemolithotrophic metabolisms when calculated in terms of energy density, which supports experimental results that showed chemoautotrophic activity in excess of heterotrophic activity. The microbial communities of subglacial lake ecosystems provide important natural laboratories to study the physiological and biogeochemical behavior of microorganisms inhabiting cold, dark environments.

Original Research: Atorvastatin prevents rat cardiomyocyte hypertrophy induced by parathyroid hormone 1-34 associated with the Ras-ERK signaling.

We investigated the effects of atorvastatin (Ator) on cardiomyocyte hypertrophy (CMH) induced by rat parathyroid hormone 1-34 (PTH1-34) and Ras-extracellular signal regulated protein kinases 1/2 (ERK1/2) signaling. Rat cardiomyocytes were randomly divided into seven groups: normal controls (NC), PTH1-34 (10(-7) mol/L), Ator (10(-5) mol/L), farnesyl transferase inhibitors-276 (FTI-276, 4 × 10(-5) mol/L), PTH1-34 + Ator, PTH1-34 + FTI-276 and PTH1-34 + Ator + mevalonic acid (MVA, 10(-4) mol/L). After treatment, the hypertrophic responses of cardiomyocytes were assessed by measuring cell diameter, detecting protein synthesis, and single-cell protein content. The concentrations of hypertrophic markers such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) were measured by ELISA. Protein expressions of ERK1/2, p-ERK1/2 and Ras were detected by western blotting. The results showed that compared with the PTH1-34 group, cellular diameter, 3H-leucine incorporation, single-cell protein content, ANP and BNP concentration decreased by 12.07 µm, 1622 cpm/well, 84.34 pg, 7.13 ng/L and 20.04 µg/L, respectively, and the expressions of Ras and p-ERK1/2 were downregulated in PTH1-34 + Ator group (P < 0.05). Compared to the PTH1-34 + Ator group, the corresponding hypertrophic responses and hypertrophic markers increased by 4.95 µm, 750 cpm/well, 49.08 pg, 3.12 ng/L and 9.35 µg/L, respectively, and the expressions of Ras and p-ERK1/2 were upregulated in the PTH1-34 + Ator + MVA group (P < 0.05). In conclusion, Ator prevents neonatal rat CMH induced by PTH1-34 and Ras-ERK signaling may be involved in this process.

Microbial food web components, bulk metabolism, and single-cell physiology of piconeuston in surface microlayers of high-altitude lakes.

Sharp boundaries in the physical environment are usually associated with abrupt shifts in organism abundance, activity, and diversity. Aquatic surface microlayers (SML) form a steep gradient between two contrasted environments, the atmosphere and surface waters, where they regulate the gas exchange between both environments. They usually harbor an abundant and active microbial life: the neuston. Few ecosystems are subjected to such a high UVR regime as high altitude lakes during summer. Here, we measured bulk estimates of heterotrophic activity, community structure and single-cell physiological properties by flow cytometry in 19 high-altitude remote Pyrenean lakes and compared the biological processes in the SML with those in the underlying surface waters. Phototrophic picoplankton (PPP) populations, were generally present in high abundances and in those lakes containing PPP populations with phycoerythrin (PE), total PPP abundance was higher at the SML. Heterotrophic nanoflagellates (HNF) were also more abundant in the SML. Bacteria in the SML had lower leucine incorporation rates, lower percentages of "live" cells, and higher numbers of highly-respiring cells, likely resulting in a lower growth efficiency. No simple and direct linear relationships could be found between microbial abundances or activities and environmental variables, but factor analysis revealed that, despite their physical proximity, microbial life in SML and underlying waters was governed by different and independent processes. Overall, we demonstrate that piconeuston in high altitude lakes has specific features different from those of the picoplankton, and that they are highly affected by potential stressful environmental factors, such as high UVR radiation.

Temperature adaptation of bacterial communities in experimentally warmed forest soils.

A detailed understanding of the influence of temperature on soil microbial activity is critical to predict future atmospheric CO2 concentrations and feedbacks to anthropogenic warming. We investigated soils exposed to 3-4 years of continuous 5 °C-warming in a field experiment in a temperate forest. We found that an index for the temperature adaptation of the microbial community, Tmin for bacterial growth, increased by 0.19 °C per 1 °C rise in temperature, showing a community shift towards one adapted to higher temperature with a higher temperature sensitivity (Q10(5-15 °C) increased by 0.08 units per 1 °C). Using continuously measured temperature data from the field experiment we modelled in situ bacterial growth. Assuming that warming did not affect resource availability, bacterial growth was modelled to become 60% higher in warmed compared to the control plots, with the effect of temperature adaptation of the community only having a small effect on overall bacterial growth (<5%). However, 3 years of warming decreased bacterial growth, most likely due to substrate depletion because of the initially higher growth in warmed plots. When this was factored in, the result was similar rates of modelled in situ bacterial growth in warmed and control plots after 3 years, despite the temperature difference. We conclude that although temperature adaptation for bacterial growth to higher temperatures was detectable, its influence on annual bacterial growth was minor, and overshadowed by the direct temperature effect on growth rates.