The effect of metal oxide nanoparticles on functional bacteria and metabolic profiles in agricultural soil.

A significant knowledge gap in nanotechnology is the absence of standardized protocols for examining the effect of engineered nanoparticles on soil microorganisms. In this study, agricultural soil was exposed to ZnO, SiO2, TiO2 and CeO2 nanoparticles at 1 mg g(-1). The toxicity effect was evaluated by thermal metabolism, the abundance of functional bacteria and enzymatic activity. ZnO and CeO2 nanoparticles were observed to hinder thermogenic metabolism, reduce numbers of soil Azotobacter, P-solubilizing and K-solubilizing bacteria and inhibit enzymatic activities. TiO2 nanoparticles reduced the abundance of functional bacteria and enzymatic activity. SiO2 nanoparticles slightly boosted the soil microbial activity. Pearson's correlation analysis showed that thermodynamic parameters had a strong correlation with abundance of functional bacteria and enzymatic activity. These findings demonstrated that the combined approach of monitoring thermal metabolism, functional bacteria and enzymatic activity is feasible for testing the ecotoxicity of nanoparticles on agricultural soil.

Polycyclic aromatic hydrocarbons degrading microflora in a tropical oil-production well.

The surrounding environment near Dagang oil-production well suffers polycyclic aromatic hydrocarbons (PAHs) pollution. In the present study, indigenous microorganisms capable of degrading PAHs were isolated and the efficiency of PAHs removal was investigated. Seven PAH-degrading strains were isolated with the ability to grow on naphthalene, phenanthrene, pyrene and fluorene. They belonged to the genus Pseudomonas, Bacillus and Rhodococcus. The strain having the highest degrading capacity for each PAH was selected by the screening test. The removal efficiency of PAHs was found to be in the order of naphthalene > fluorene > phenanthrene > pyrene. The kinetics of PAHs degradation was then followed by liquid chromatography determination and the results showed it conforms to a first-order reaction kinetic model. This study would be highly important for investigating the ability of microorganisms to utilize PAHs as growth substrates.

Functional gene expression of oil-degrading bacteria resistant to hexadecane toxicity.

Contamination with oil poses a threat to the environment and to human health worldwide. Biological methodologies have proved to be economical, versatile and efficient for the remediation of pollutants. In this paper, a highly efficient oil-degrading bacterial strain USTB-2 was isolated from an oil production well of Dagang oil field in Tianjin, China. The 16S rRNA sequence of USTB-2 showed 100% similarity with that of Bacillus subtilis BSn5. Hexadecane is one of the most important components in petroleum. The half inhibitory ratio (IC50) of hexadecane inhibited organisms, determined by microcalorimetry, was lower in USTB-2 than in B. BSn5. The results indicate that the strain USTB-2 degrades hexadecane to make it less toxic compared with the normal strain. RT-PCR was used to evaluate the expression of oil-degrading enzymes, specifically 4-hydroxyphenylacetate 3-monooxygenase genes (HPMO). A sharp increase in the expression of HPMO genes was observed for USTB-2, while the expression of HPMO genes in reference strain B. BSn5 remained relatively stable. These methods can be used to study the metabolic potential of microorganisms for in situ oil decontamination.

A real-scale soil phytoremediation.

In the present investigation, a phytoremediation process with a combination of different plant species (Populus nigra (var.italica), Paulownia tomentosa and Cytisus scoparius), and natural growing vegetation has been proposed at real-scale (10.000 m(2)) to bioremediate and functionally recover a soil historically contaminated by heavy metals and hydrocarbons. In the attempts to assess both effectiveness and evolution of the remediation system towards a natural soil ecosystem, besides the pollution parameters, also parameters describing the efficiency of the microbiological components (enzyme activities), were investigated. In 3 years, the total content of hydrocarbons and heavy metals in soil decreased with time (40 % and 20-40 %, respectively), reaching concentrations under the limit of National legislation and making the site suitable for environmental reusing. The reduction in pollutants was probably the reason of the increase in dehydrogenase (indicator of overall microbial activity), ß-glucosidase and phosphatase activities, enzymes related to C and P cycles, respectively. However, this trend was obviously due also to the increase of chemical nutrients, acting as substrate of these enzymes. Moreover, a phytotest carried out with Raphanus sativus, showed, after 3 years, a significant increase in percentage of plant growth, confirming a reduction in soil toxicity and an improvement in soil nutritional state. Therefore, this phytoremediation system seems very promising to perform both decontamination and functional recovery of a polluted soil at real-scale level.

Bioremediation of polluted soil through the combined application of plants, earthworms and organic matter.

Two plant species (Paulownia tomentosa and Cytisus scoparius), earthworms (Eisenia fetida), and organic matter (horse manure) were used as an ecological approach to bioremediate a soil historically contaminated by heavy metals and hydrocarbons. The experiment was carried out for six months at a mesoscale level using pots containing 90 kg of polluted soil. Three different treatments were performed for each plant: (i) untreated planted soil as a control (C); (ii) planted soil + horse manure (20:1 w/w) (M); (iii) planted soil + horse manure + 15 earthworms (ME). Both the plant species were able to grow in the polluted soil and to improve the soil's bio-chemical conditions, especially when organic matter and earthworms were applied. By comparing the two plant species, few significant differences were observed in the soil characteristics; Cytisus scoparius improved soil nutrient content more than Paulownia tomentosa, which instead stimulated more soil microbial metabolism. Regarding the pollutants, Paulownia tomentosa was more efficient in reducing the heavy metal (Pb, Cr, Cd, Zn, Cu, Ni) content, while earthworms were particularly able to stimulate the processes involved in the decontamination of organic pollutants (hydrocarbons). This ecological approach, validated at a mesoscale level, has recently been transferred to a real scale situation to carry out the bioremediation of polluted soil in San Giuliano Terme Municipality (Pisa, Italy).

Effects of petroleum contamination on soil microbial numbers, metabolic activity and urease activity.

The influence of petroleum contamination on soil microbial activities was investigated in 13 soil samples from sites around an injection water well (Iw-1, 2, 3, 4) (total petroleum hydrocarbons (TPH): 7.5-78 mg kg(-1)), an oil production well (Op-1, 2, 3, 4, 5) (TPH: 149-1110 mg kg(-1)), and an oil spill accident well (Os-1, 2, 3, 4) (TPH: 4500-34600 mg kg(-1)). The growth rate constant (µ) of glucose stimulated organisms, determined by microcalorimetry, was higher in Iw soil samples than in Op and Os samples. Total cultivable bacteria and fungi and urease activity also decreased with increasing concentration of TPH. Total heat produced demonstrated that TPH at concentrations less than about 1 g kg(-1) soil stimulated anaerobic respiration. A positive correlation between TPH and soil organic matter (OM) and stimulation of fungi-bacteria-urease at low TPH doses suggested that TPH is bound to soil OM and slowly metabolized in Iw soils during OM consumption. These methods can be used to evaluate the potential of polluted soils to carry out self-bioremediation by metabolizing TPH.

Almond tree and organic fertilization for soil quality improvement in southern Italy.

The semi-arid Mediterranean region, characterized by long dry periods followed by heavy bursts of rainfall, is particularly prone to soil erosion. The main goal of this study is to evaluate the soil quality under different practices of bio-physical amelioration which involve the soil-plant system (almond trees) and microorganism-manure. This study, carried out in the South of Italy (Basilicata Region- Pantanello farm), considered two types of fertilization (mineral and organic) and three slope gradients (0, 2 and 6%), in order to evaluate the effects of management practices in resisting soil erosion. Chemical (organic carbon and nitrogen), physical (soil shrinkage and bulk density) and biochemical (dehydrogenase activity and hydrolytic enzyme activities) parameters were selected as markers to follow agro-ecological changes with time. The organic treatment affected soil microbiological and physico-chemical properties by increasing soil nutrient availability, microbial activity, and improving soil structure. The consistently higher values of the hydrolytic enzyme activities (ß-glucosidase, phosphatase, urease and protease) often observed in the presence of plants and on the 0 and 2% slopes, suggested the stimulation of nutrient cycles by tree roots, which improve the conditions for soil microorganisms in carrying out their metabolic activity. In the 6% slope and, in particular, in the mineral fertilizer treatment, soil metabolism was lower as suggested by the dehydrogenase activity which was 50% lower than that found in the 0 and 2% slopes, this seemed to be related to a slowdown in the nutrient cycling and organic carbon metabolism. However, on this slope, in both mineral and organic treatments, a significant stimulation of hydrolytic enzyme activities and an improvement of soil structure (reduction of bulk density of about 10% and increase in total shrinkage from 20 to 60%) were observed with plants compared to the control soil. The combination of organic fertilization and almond trees resulted effective, also in the highest slope, in mitigating the degradation processes through the improvement of chemico-nutritional, biochemical and physical soil properties.

A comparative cytotoxicity study of isomeric alkylphthalates to metabolically variant bacteria.

This work investigated the toxicity of two isomeric alkylphthalates, i.e., di-n-octyl phthalate (DOP) and di-2-ethylhexyl phthalate (DEHP) to two model bacteria, Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis), which have been previously used to study the toxicity of environmental pollutants. Microcalorimetry was used as the key analytical tool alongside scanning electron microscopy (SEM) and traditional microbiology techniques. The thermokinetic parameters from microcalorimetry showed that the phthalates had a biphasic effect on the metabolic activities of the bacteria; serving as energy sources for the bacteria thereby stimulating their growth at low dosages (< or = 150 microg/mL), but displaying inhibitory effects at higher dosages (> or = 300 microg/mL), indicated by a sharp decrease in growth rate constants at 450 microg/mL. The SEM revealed that the bacterial cells were morphological deformed, with shrunk cells and elongated strands at 600 microg/mL of both phthalates. The elongated strands inferred that the phthalates inhibited the reproductive processes of the bacteria by possibly impeding some stages of cell division. The half inhibitory concentrations of the phthalates showed that DEHP was more toxic than DOP. Additionally, E. coli, a facultative anaerobe, was more susceptible to the toxic effects of phthalates than B. subtilis, an obligate aerobe capable of forming endospores crucial for tolerating extreme environmental conditions.

Use of earthworms (Eisenia fetida) to reduce phytotoxicity and promote humification of pre-composted olive oil mill wastewater.

BACKGROUND: Olive mill wastewaters (OMWW) contain a high recalcitrant organic load and an associated toxicity that make their treatment necessary before environmental application. The aim of this study was to evaluate the feasibility of promoting the valorization and reducing the phytotoxicity of OMWW through a pre-composting process together with straw-chip bulking materials followed by the application of earthworms (Eisenia fetida) in the presence of oat seedlings (Avena sativa L.) seedlings. RESULTS: After 3 months, the pre-composted material showed properties similar to a partially digested compost with some significant amelioration of chemical-physical and biochemical properties. The application of earthworms permitted a significant decrease in chemical (total organic carbon, water-extractable organic carbon, total nitrogen) and biological parameters (dehydrogenase enzyme activity), and an increase in humic substances and available nitrogen forms. In the presence of plants a higher C/N ratio and a lower content of nitrates were observed. In addition, the reduction in phenolic compounds observed in treatments with earthworms caused a decrease in phytotoxicity by about 50% with respect to the pre-composted material, which results in an increase in germination index. CONCLUSION: The utilization of earthworms, in particular in the presence of plants, may be an ecologically sound and economically feasible technology to obtain a non-toxic, high-value product useful for agricultural purposes.

Microcalorimetric investigation of the toxic action of pyrene on the growth of PAH-degrading bacteria Acinetobacter junii.

A multi-channel thermal activity microcalorimeter was used to determine the pyrene-induced toxic effect on two polycyclic aromatic hydrocarbon (PAH)-degrading bacteria Acinetobacter junii (A. junii) and Bacillus subtilis (B. subtilis). Power-time curves were analyzed and calorimetric parameters including growth rate constant (k), half inhibitory concentration (IC50) and total thermal effect (QT) were obtained. A. junii and B. subtilis were completely inhibited when the concentration of pyrene reached 400 and 160 microg mL(-1), respectively. The relationships between the calorimetric parameters and concentration of pyrene were studied. The growth rate of A. junii decreased with the increase in pyrene concentration at 50-200 microg mL(-1). The growth of biomass for A. junii at various concentrations of pyrene was determined. The count of A. junii after 8 day's incubation reached maximum irrespective of the initial pyrene concentrations ranging from 50 to 200 microg mL(-1) and the smallest stimulative action of pyrene was at 200 microg mL(-1). The variations of biomass during the growth of A. junii were consistent with the microcalorimetric data, indicating that microcalorimetry can be an effective technique to investigate the effect of pyrene on microorganisms.

Development and analytical application of a glucose biosensor based on glucose oxidase/O-(2-hydroxyl)propyl-3-trimethylammonium chitosan chloride nanoparticle-immobilized onion inner epidermis.

A glucose biosensor comprising a glucose oxidase/O-(2-hydroxyl)propyl-3-trimethylammonium chitosan chloride nanoparticle (O-HTCC NP)-immobilized onion inner membrane and a dissolved oxygen (O(2)) sensor has been successfully developed. The detection scheme is based on the depletion of dissolved O(2) content upon exposure to glucose. The decrease in O(2) level was monitored and related to the glucose concentration. The biosensor shows linear response to glucose from 0.0 to 0.60 mM with a detection limit of 50 microM (S/N=3). The effect of O-HTCC NP and enzyme loading, pH, temperature, and phosphate buffer concentration on the sensitivity of the biosensor was studied in detail. The biosensor exhibits fast response time (70s), good repeatability (3.2%, n=10) and storage stability (90% of initial sensitivity after 3-week storage). Common interferents including acetic acid, lactic acid, propionic acid, butyric acid, folic acid, methanol, glycine, DL-alpha-alanine and DL-cysteine do not cause significant interferences on the biosensor. The proposed biosensor method was successfully applied to determine the glucose content in real samples such as orange juice, red wine and tea drink and the results were comparable to that obtained from a spectrophotometric method. The glucose recovery test demonstrates that the proposed glucose biosensor offers an excellent, accurate and precise method for the determination of glucose in real samples.

Vermicomposting of olive oil mill wastewaters.

The disposal of olive oil mill wastewaters (OMW) represents a substantial environmental problem in Italy. A vermicompost process could be an alternative and valid method for the management of OMW. In a laboratory experiment, the OMW were absorbed onto a ligno-cellulosic solid matrix and 30 adult earthworms of Eisenia fetida specie were added. The experiment was carried out for 13 weeks. The number of earthworms increased throughout the experimental period and after 2 weeks about 90% of the earthworms had become sexually mature. The decrease in total organic carbon (about 35%), C : N ratio (from 31.2 to 12.3) and biochemical parameters (hydrolytic enzymes averagely 40% and dehydrogenase 23%), and the increase in humification rate (pyrophosphate extractable carbon (PEC) from 17.6 to 33.3 mg g(-1), and PEC : water-soluble carbon from 1.76 to 2.97) indicated the mineralization and the stabilization of organic matter at the end of the vermicomposting process. At the end of the experiment, the extracellular beta-glucosidase, phosphatase, urease and protease activities, measured in the pyrophosphate extract of the vermicompost, were found to be always higher or equal to that measured at the beginning of the vermicomposting process, suggesting that the enzymes bound to humic matter resisted biological attack and environmental stress. Moreover, the results obtained from the phyto-test showed that the OMW lose their toxicity and stimulate plant germination and growth.

Biological and microcalorimetric studies of the toxic effect of organoarsenic(V) compounds to wild strain of Bacillus thuringiensis.

Microcalorimetric and biological methods were carried out to determine the toxicity of dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA) to wild strain of Bacillus thuringiensis. Thermokinetic parameters were obtained from the power-time curves, showing that the peak-heat output power, total heat output, and number of colonies decreased with the increases in concentration of DMA and MMA. In addition, the generation time and peak maximal time increased with the increases in the dosage of DMA and MMA. The half inhibitory concentrations of DMA and MMA were 99.02 and 142.02 microg mL(-1), respectively for the wild strain of B. thuringiensis. DMA shows higher toxicity to bacteria than MMA. The toxicity resistance of B. thuringiensis against organoarsenic(V) is quite high for the wild strain. Our work demonstrates that microcalorimetry is a very sensitive, simple, and useful technique for in vitro investigation of the toxic effect of organoarsenic(V) on microbial activity.

Microcalorimetric measurements of the microbial activities of single- and mixed-species with trivalent iron in soil.

A microcalorimetric technique was applied to a series of experiments to follow the toxic effect caused by the trivalent iron on the single and mixed microbes in sterilized soil that was inoculated with the single Bacillus subtilis (B. subtilis) (prokaryotic bacterium), single Candida humicola (C. humicola) (eukaryotic fungus) and the mixed-species. The microbial activity was stimulated by the addition of 5.0mg glucose and 5.0mg ammonium sulfate under a 35% controlled humidity in the studied soil samples of 1.2g. The power-time curves from every experiment were analyzed, and from these analyses characteristic parameters, such as growth rate constant (k) and total thermal effect (Q) which can reflect the biochemical reactions were determined. The mixed-species have moderate tolerance to the iron overload, comparing with single species, and exhibit synergistic interaction in exponential growth phase (0-400.0 microg mL(-1)). Meanwhile, there is no much difference in the thermal effect (Q) per gram soil sample for the single and mixed culture. This also validates that the nutrient substances in natural environment determine the organisms' metabolic activities. Ultraviolet-visible spectrophotometry and dissolved oxygen sensor also were successfully applied to reflect the activities of B. subtilis and C. humicola in the pure culture. The investigation could provide insight into the microbial ecology of bacteria and fungi in ecological niches.

The toxic effect of cadmium on pure microbes using a microcalorimetric method and a biosensor technique.

A microcalorimetric technique based on microbes heat-output was explored to evaluate the effect of Cd (II) on Bacillus subtilis and Candida humicola. The power-time curves of the growth metabolism of Bacillus subtilis and Candida humicola and the effect of Cd (II) on it were studied by using a TAM III microcalorimeter, ampoules method at 28 degrees C. For the evaluation of toxic effect on pure micro-organisms, the maximum peak-heat output power (P(max)) in the growth phase, the growth rate constants (k), the log phase heat effects (Q(log)), and the total heat effect (Q(T)) for Bacillus subtilis and Candida humicola were determined. Dissolved oxygen and biochemical oxygen demand (BOD) were evaluated by a biosensor. Cadmium has been regarded as the essential biological trace element. Cd (II) solutions of different concentration have different effects on Bacillus subtilis and Candida humicola growth metabolism. The higher concentrations of Cd (II) inhibit the growth of Candida humicola (1600-3200 microg.mL(-1)), Bacillus subtilis (240-480 microg.mL(-1)); the lower concentrations can promote the growth of both micro-organism. The values of cell dry weight is also showed in conformity in the cell dry weight changes to the micro-organisms' growth time. Comparison of growth curves of two micro-organisms showed that both the trends of biochemical oxygen demand were exhibiting regressive changes with the passage of time during their generation times (t(G)). Results from ultraviolet spectrophotometer and precision pH meter all showed that the control growth curves were visioning same trends with the thermodynamic curves of micro-organisms measured by microcalorimeter.

Toxic effect of inorganic arsenite [As(III)] on metabolic activity of Bacillus subtilis by combined methods.

In this study, microcalorimetry and measurement of culture turbidity were applied to evaluate the As(III) toxic effect on the metabolic growth of Bacillus subtilis. Using a multichannel thermal activity monitor, the power-time curves of the metabolic activity of B. subtilis during growth in the absence and presence of various concentrations of As(III) were obtained and studied. The turbidity changes during B. subtilis growth with As(III) were investigated by ultraviolet-visible spectrophotometry and the data agree with the results obtained by microcalorimetry. As(III) of various concentrations has different effects on the metabolic growth of B. subtilis with biphasic dose-response relationships called hormesis [i.e., low-concentration stimulation (10 microg/mL) and high-concentration inhibition (20-160 microg/mL). Typical J-shapes of the relationship between the growth rate constant (k) and c, and toxicity at the half-inhibitory concentration (IC(50)) of 98.82 +/- 7.32 microg/mL were obtained. The similarity between the two methods corroborates the validity and sensitivity of the microcalorimetric technique to investigate the toxic effect of As(III) on microorganisms.

Investigation of the toxic effect of cadmium on Candida humicola and Bacillus subtilis using a microcalorimetric method.

In this study, the technique of microcalorimetry based on heat-output by aerobic bacterial respiration was explored to evaluate the toxic effect of cadmium on Candida humicola, Bacillus subtilis, singularly or in a mixture of both. Power-time curves of the growth metabolism of C. humicola and B. subtilis and the effect of Cd(2+) were studied using the TAM III (the third generation thermal activity monitor) multi-channel microcalorimetric system, isothermal mode, at 28 degrees C. The differences in shape of the power-time curves and the thermodynamic and kinetic characteristics of microorganisms growth were compared. The effect of cadmium added into microorganism would significantly reduce the life cycle and change the thermal effect of microbial metabolic process with different concentrations of Cd(2+). The experimental results revealed that at the same concentration, the sequence of inhibitory ratio (I) and maximum thermal power (P(max)) of the Cd(2+) was: mixed microorganisms>C. humicola>B. subtilis. The sequence of total thermal effect (Q(total)) and growth rate constant (k) is mixed microorganisms>B. subtilis>C. humicola. These results are important to further studies of the physiology and pharmacology of C. humicola and B. subtilis and may support the theory of restoring contaminated soil.