Enzymatic Activity as a Measure of Total Microbial Activity on Historical Stone

Stones of historical monuments exposed to the open air deteriorate over the course of time depending on physical, chemical, and biological factors acting in co-association. Among the biological factors, microorganisms play a key role in the deterioration process of stones. Detecting the level of microbial activity on stones is an essential step in diagnostic and monitoring studies of stone biodeterioration, and aids in controlling the performance of treatments applied to the stones. Therefore, this study aimed to develop a practical and rapid method for the determination of microbial activity on historical stones and use this method on the Mount Nemrut monuments (MNMs) (Adiyaman, Turkey). For that purpose, the fluorescein diacetate (FDA) hydrolysis method, frequently employed for soil environments, was adapted for the estimation and assessment of total microbial activity to understand whether microorganisms posed a potential risk for the biodeterioration of the limestones and sandstones of the MNMs. The traditional plate count method was also applied simultaneously to the same stone samples to compare and assist in the interpretation of the results of the FDA hydrolysis method, which relies on the quantitative determination of bacterial and fungal colonies in nutrient agar and malt extract agar medium, respectively. The results of the FDA hydrolysis and plate count methods showed consistency. The total microbial activity determined by the FDA hydrolysis method was low for both types of stone samples. In addition, the plate count method showed low bacterial and fungal counts on all of the samples. This revealed that microbial activity did not play an important role in the stone deterioration process on the MNMs, although different lichen species were frequently observed on both the sandstones and the limestones. Hence, further investigation must be undertaken for determination of their long-term behavior and effects on the stones of the MNMs. On the other hand, the results of the FDA hydrolysis and plate count methods showed correlation. Lower bacterial counts were observed when lower enzymatic activity was observed in the stone samples, and likewise, higher bacterial counts were observed when higher enzymatic activity was observed. Consequently, the application of the FDA hydrolysis method was determined to be reliable for the estimation of total microbial activity on historical stones. The method had obvious advantages in terms of its rapid measurement rate and sensitivity, even on small samples.

Impact of weed management practices on soil biological activity in corn and soybean field crops in Québec (Canada)

Repeated applications and combination of glyphosate-containing herbicides (GCH) with other herbicides are two weed management practices (WMP) used to compensate for GCH decreasing efficiency impacts in field crops. These practices may have serious impacts on soil functions because GCH affect soil biota and soil biological activity (SBA). Two field experiments, one with corn and one with soybean crops, were conducted during one growing season. SBA indicators, soil respiration (SR) and fluorescein diacetate (FDA) hydrolysis, were measured at two sampling times following six WMP. These WMP included one or two GCH applications (GCH alone or combined with other herbicides), applications of other herbicides only and mechanical weeding. WMP did not affect FDA neither for corn or soybean at either sampling times. In contrast, WMP affected SR in corn fields at both sampling times and SR in soybean field at the first sampling time. Repeating GCH applications and combining different herbicides led to lower SR, suggesting that these practices decreased SBA, whilst one single GCH application presented higher SR, suggesting that this practice stimulated SBA. Our study demonstrates that using GCH in combination with other herbicides or in multiple applications affects SBA in field conditions. Affecting soil functions and carbon cycle do bear serious weed management implications, and the choice of WMP should be taken into consideration to minimize their impacts on SBA for field crops sustainability.

Activity of Antibiotics against Pseudomonas aeruginosa in an In Vitro Model of Biofilms in the Context of Cystic Fibrosis: Influence of the Culture Medium

ABSTRACT Pseudomonas aeruginosa is a major cause of respiratory biofilm-related infections in patients with cystic fibrosis. We developed an in vitro pharmacodynamic model to study the activity of antipseudomonal antibiotics against PAO1 biofilms grown in artificial sputum medium with agar [ASM(+)] versus that against biofilms grown in Trypticase soy broth supplemented with glucose and NaCl (TGN). We measured bacterial counts, metabolic activity (fluorescein diacetate [FDA] hydrolysis), and biomass (crystal violet absorbance). Biofilms grew slower in ASM(+) than in TGN but reached the same CFU counts and metabolic activity in both media and a slightly higher biomass after 48 h in ASM(+) than in TGN. The concentration-response curves of the antibiotics after 24 h of incubation with mature biofilms showed maximal effects ranging from a 3 (ciprofloxacin)- to a 1.5 (ceftazidime, meropenem)-log10-CFU decrease, with tobramycin and colistin showing intermediate values. These maximal reductions in the numbers of CFU were similar in both media for ciprofloxacin and β-lactams but lower in ASM(+) than in TGN for tobramycin and colistin; they were reached at concentrations lower than the human maximum concentration in plasma for ciprofloxacin and β-lactams only. The reductions in metabolic activity and in biomass were low in both media. Small-colony variants were selected by tobramycin in ASM(+) and by ciprofloxacin in both media. The model was then successfully applied to 4 isolates from patients with cystic fibrosis. These biofilms showed CFU counts similar to those of PAO1 biofilms in ASM(+) but a higher biomass than PAO1 biofilms in ASM(+) and moderate differences in their susceptibility to antibiotics from that of PAO1 biofilms grown in this medium. This model proved useful to establish the pharmacodynamic profile of drugs against P. aeruginosa biofilms in the context of cystic fibrosis.

Microbial activity in soil with onion grown in a no-tillage system with single or intercropped cover crops

ABSTRACT: Microbial biomass is a driving force in the dynamics of soil organic matter, and microbial activity is an indicator of soil quality in agroecosystems, reflecting changes in management practices and environmental conditions. We evaluated the effect of monoculture and intercropped winter cover crops on soil chemical attributes, microbial biomass carbon (MBC), basal respiration (BR), metabolic quotient (qCO2), urease, β-glucosidase, and fluorescein diacetate (FDA) hydrolysis activity, as well as onion yield in a no-tillage system. Soil is a Typic Humudept, and treatments were control with spontaneous vegetation, barley, rye, oilseed radish (OR), OR + rye, and OR + barley. The soil was sampled (0-10 cm) five times between June and December. There were no differences among treatments for MBC and BR, and the highest values for those attributes occurred in June, when cover plants were in their initial stage. Although, qCO2 was not affected by any treatment, it varied among sampling periods, ranging from 0.62 to 10 µg C-CO2 mg-1 MBC h-1, indicating a low- or no stress environment. Cover crops had little influence on enzyme activity, but FDA was lowered in areas with single crops of barley and rye. Average onion yield in cover crops treatments was 13.01 (Mg ha-1), 30-40% higher than in the control treatment.

Fluorescein Diacetate Hydrolysis Using the Whole Biofilm as a Sensitive Tool to Evaluate the Physiological State of Immobilized Bacterial Cells

Due to the increasing interest and the use of immobilized biocatalysts in bioremediation studies, there is a need for the development of an assay for quick and reliable measurements of their overall enzymatic activity. Fluorescein diacetate (FDA) hydrolysis is a widely used assay for measuring total enzymatic activity (TEA) in various environmental samples or in monoculture researches. However, standard FDA assays for TEA measurements in immobilized samples include performing an assay on cells detached from the carrier. This causes an error, because it is not possible to release all cells from the carrier without affecting their metabolic activity. In this study, we developed and optimized a procedure for TEA quantification in the whole biofilm formed on the carrier without disturbing it. The optimized method involves pre-incubation of immobilized carrier in phosphate buffer (pH 7.6) on the orbital shaker for 15 min, slow injection of FDA directly into the middle of the immobilized carrier, and incubation on the orbital shaker (130 rpm, 30 °C) for 1 h. Biofilm dry mass was obtained by comparing the dried weight of the immobilized carrier with that of the unimmobilized carrier. The improved protocol provides a simple, quick, and more reliable quantification of TEA during the development of immobilized biocatalysts compared to the original method.

MICROBIAL CHARACTERISTICS OF SOILS UNDER AN INTEGRATED CROP-LIVESTOCK SYSTEM

Integrated crop-livestock systems (ICLs) are a viable strategy for the recovery and maintenance of soil characteristics. In the present study, an ICL experiment was conducted by the Instituto Agronômico do Paraná in the municipality of Xambre, Parana (PR), Brazil, to evaluate the effects of various grazing intensities. The objective of the present study was to quantify the levels of microbial biomass carbon (MBC) and soil enzymatic activity in an ICL of soybean (summer) and Brachiaria ruziziensis (winter), with B. ruziziensis subjected to various grazing intensities. Treatments consisted of varying pasture heights and grazing intensities (GI): 10, 20, 30, and 40 cm (GI-10, GI-20, GI-30, and GI-40, respectively) and a no grazing (NG) control. The microbial characteristics analysed were MBC, microbial respiration (MR), metabolic quotient (qCO2), the activities of acid phosphatase, β-glucosidase, arylsuphatase, and cellulase, and fluorescein diacetate (FDA) hydrolysis. Following the second grazing cycle, the GI-20 treatment (20-cm - moderate) grazing intensity) contained the highest MBC concentrations and lowest qCO2 concentrations. Following the second soybean cycle, the treatment with the highest grazing intensity (GI-10) contained the lowest MBC concentration. Soil MBC concentrations in the pasture were favoured by the introduction of animals to the system. High grazing intensity (10-cm pasture height) during the pasture cycle may cause a decrease in soil MBC and have a negative effect on the microbial biomass during the succeeding crop. Of all the enzymes analyzed, only arylsuphatase and cellulase activities were altered by ICL management, with differences between the moderate grazing intensity (GI-20) and no grazing (NG) treatments.

Predictive Capacity of Visible-Near Infrared Spectroscopy for Quality Parameter Assessment of Compost

Screening tests are basic procedures commonly used to assess compost quality. Important parameters for quality assessment are the germination capacity and the suppression of plant pathogens which have to be measured by time-consuming laboratory methods. The objective was to test whether visible (vis) and near infrared (NIR) spectroscopy (vis-NIR) is useful to analyse parameters important for compost quality. Ninety seven compost samples from Switzerland were analysed by conventional methods and by vis-NIR. The content of organic (Corg) and inorganic C (Cinorg), total N (Ntot), mineralisable N after 56 days (Nmin_d56), total P (Ptot), K, Ca and salt, the C/N ratio, pH and microbiological characteristics [hydrolysis of fluorescein diacetate (FDA-hydrolysis) as indicator of total enzyme activity and cellulase activity] were determined. Furthermore, plant tolerance and the suppression of pathogens were tested using germination tests with salad, cress, ryegrass and bean or a Rhizoctonia solani bioassay, respectively. The samples were scanned in the range of 400–2500 nm (visible light and NIR) using a Foss NIRSystems spectrometer 6500. A modified partial least squares regression method and the whole spectrum were used to develop cross-validation equations for all constituents. For this, the first to third derivative was calculated. The prediction accuracy was evaluated as excellent for Corg and good for N, and the C/N ratio based on the RSC values (ratio of standard deviation of laboratory results to standard error of cross-validation) and the coefficients of determination ( r2). Approximate quantitative predictions were possible for the contents of Ptot, K, Ca and salt, whereas for the constituents Cinorg, Nmin_d56, FDA-hydrolysis and the germination tests with cress and salad only between high and low values could be discriminated. Unsuccessful predictions as indicated by RSC values lower than 1.5 and r2 values below 0.50 were obtained for pH, cellulase activity, germination tests with ryegrass and bean and the disease suppression test using R. solani. Overall the results of the present study indicate that vis-NIR spectroscopy has the potential to be used for quality assessment of composts and to replace time-consuming methods such as germination tests using salad and cress. However, the use for monitoring purposes requires further research to clarify whether other complex quality parameters such as disease suppression indicators may also be predicted successfully.