Short-term effect of aniline on soil microbial activity: a combined study by isothermal microcalorimetry, glucose analysis, and enzyme assay techniques.

The accidents of aniline spill and explosion happened almost every year in China, whereas the toxic effect of aniline on soil microbial activity remained largely unexplored. In this study, isothermal microcalorimetric technique, glucose analysis, and soil enzyme assay techniques were employed to investigate the toxic effect of aniline on microbial activity in Chinese soil for the first time. Soil samples were treated with aniline from 0 to 2.5 mg/g soil to tie in with the fact of aniline spill. Results from microcalorimetric analysis showed that the introduction of aniline had a significant adverse effect on soil microbial activity at the exposure concentrations ≥0.4 mg/g soil (p < 0.05) and ≥0.8 mg/g soil (p < 0.01), and the activity was totally inhibited when the concentration increased to 2.5 mg/g soil. The glucose analysis indicated that aniline significantly decreased the soil microbial respiratory activity at the concentrations ≥0.8 mg/g soil (p < 0.05) and ≥1.5 mg/g soil (p < 0.01). Soil enzyme activities for ß-glucosidase, urease, acid-phosphatase, and dehydrogenase revealed that aniline had a significant effect (p < 0.05) on the nutrient cycling of C, N, and P as well as the oxidative capacity of soil microorganisms, respectively. All of these results showed an intensively toxic effect of aniline on soil microbial activity. The proposed methods can provide toxicological information of aniline to soil microbes from the metabolic and biochemical point of views which are consistent with and correlated to each other.

Stability and toxicity of selected chlorinated benzophenone-type UV filters in waters.

In our study, the transformation of two most widely used UV filters, benzophenone-3 (BP3) and benzophenone-4 (BP4), in chlorinated water with disinfection reagents sodium hypochlorite (NaClO) and trichloroisocyanuric acid (TCCA) was studied. Based on the HPLC/MS and UV-Vis analysis the formation of two different chlorinated products (5-chloro-2-hydroxy-4-methoxybenzophenone and 3,5-dichloro-2-hydroxy-4-methoxybenzophenone) was established. Identity of chlorinated products was confirmed by means of comparison of retention times with independently synthesized standards. Photostability study showed that dichloro-derivative in water is less stable then parent compounds, which is not the case for monochloro-derivatives. Toxicity of chlorinated compounds tested by Vibrio fischeri was found to be in the same range as that of the starting compounds. Preliminary testing of real water samples from swimming pools and sea swimming areas confirmed the presence of BP3 and its 3,5-dichloro derivative.

A comparative study on the impact of phthalate esters on soil microbial activity.

In this study, an isothermal microcalorimetric technique was used to demonstrate the impact of dimethyl phthalate (DMP), di-n-octyl phthalate (DOP) and diethyl phthalate (DEP) on soil microbial activity. The effect of these phthalate esters (≤100 µg g⁻¹ soil) follows the order: DEP > DMP > DOP but changed to DMP > DEP > DOP when the concentrations of the phthalate esters are above 100 µg g⁻¹ soil. DMP, DEP and DOP significantly decreased (p < 0.05) the soil urease activity. The effect of phthalate esters on soil microbial activity is not the same with those on enzymatic activity. In addition, DEP, which has a lower bioavailability, is less toxic to soil microbes than that of DMP when its concentration is above 100 µg g⁻¹ soil.

Impact of beta-cypermethrin on soil microbial community associated with its bioavailability: a combined study by isothermal microcalorimetry and enzyme assay techniques.

In this study, an isothermal microcalorimetric technique has been used to show that beta-cypermethrin (CYP) had no significant effect (p > 0.05) on soil microbial activity at 80 µg g(-1) soil. Our soil enzyme data indicated that beta-CYP ranging 10-80 µg g(-1) soil had no significant effect (p > 0.05) on soil enzyme activities such as ß-glucosidase, urease, acid-phosphatase, and dehydrogenase. Therefore, our results infer that beta-CYP would not pose severe toxicity to soil microbial community, but its toxic level may vary greatly with environment that associates with its increase in bioavailability: the level in soil (at µg g(-1)) < the level in sediment (varying from µg g(-1) to µg L(-1)) 0.05). These results suggest that the heavy application of beta-CYP may not cause damage to soil microbial community which is very different from its high toxicity to the aquatic organism.

Investigation of the acute toxic effect of chlorpyrifos on Pseudomonas putida in a sterilized soil environment monitored by microcalorimetry.

Chlorpyrifos (CPF) is moderately persistent in soils. In our study, microcalorimetry was introduced for the first time to explore the acute toxic effect of CPF on a Pseudomonas strain in sterilized soil. Firstly, it was determined by microcalorimetry that P. putida failed to degrade CPF. Then the acute toxicity of increasing concentrations of CPF to P. putida was determined by its temporal effects on metabolism and counts of colony forming units. Results revealed that the increase of CPF concentration could induce a decrease of the growth rate constant (k) and the total thermal effect (Q (T)), representing an inhibiting action on P. putida. In addition, the colony forming units (CFU) for P. putida were counted. Results showed that the number of P. putida decreased with increasing CPF dose after 18 h of incubation in sterilized soil. Interestingly, the trend of the number of CFU was similar to the growth rate constant k, whereas the trend became irregular after 36 h of incubation. This indicated that P. putida resisted and also expresses high metabolic activity during the exponential growth phase of 18 h; thereafter the microorganisms showed a certain adaptation, even declining in number and activity.

Toxicity of three phenolic compounds and their mixtures on the gram-positive bacteria Bacillus subtilis in the aquatic environment.

Although phenolic compounds are intensively studied for their toxic effects on the environment, the toxicity of catechol, resorcinol and hydroquinone mixtures are still not well understood because most previous bioassays are conducted solely using single compound based on acute tests. In this work, the adverse effect of individual phenolic compounds (catechol, resorcinol and hydroquinone) and the interactive effect of the binary and tertiary mixtures on Bacillus subtilis (B. subtilis) using microcalorimetric method were examined. The toxicity of individual phenolic compounds follows the order catechol>resorcinol>hydroquinone with their respective half inhibitory concentration as 437, 728 and 934 microg mL(-)(1). The power-time curve of B. subtilis growth obtained by microcalorimetry is in complete agreement with the change in turbidity of B. subtilis against time, demonstrating that microcalorimetric method agrees well with the routine microbiological method. The toxicity data obtained from phenolic compound mixtures show that catechol and hydroquinone mixture possess synergistic effect while the other mixtures display additive joint actions. Furthermore, the concentration addition (CA) and independent action (IA) models were employed to predict the toxicities of the phenolic compounds. The experimental results of microcalorimetry show no significant difference on the toxicity of the phenolic compound mixtures from that predicted by CA. However, IA prediction underestimated the mixture effects in all the experiments.

Reduction in toxicity of arsenic(III) to Halobacillus sp. Y35 by kaolin and their related adsorption studies.

The growth of Halobacillus sp. Y35 has been investigated in HGM hypersaline medium with different doses of As(III) and kaolin. The metabolic heat flux decreases with the increase in As(III) concentration, indicating that strain Y35 lowers their metabolic activity in order to resist the As(III) toxicity. Carbon dioxide flux, cell growth and protein synthesis rates, and total thermal effect have been, for the first time, successfully employed simultaneously to assess the effect of As(III) on strain Y35 in the absence and presence of kaolin. The relative adsorption capacity and adsorption intensity of kaolin for As(III) are higher with strain Y35 than that without strain Y35, demonstrating that it is possible to reduce the toxicity of As(III) to our environment by both using mineral adsorption and biosorption technology. Our work shows the potential application of kaolin and strain Y35 for the removal of As(III) from contaminated groundwater.

A combination method to study microbial communities and activities in zinc contaminated soil.

Zinc (Zn) plays a special role in soil ecology and fertility because it can support the growth of soil organisms or inhibit their growth depending on its concentrations. In this work, the effects of different concentrations of Zn on soil microbial communities and activities were analyzed by loading five different doses of Zn (160-6000 microg g(-1)) into a wheat surface soil. The microbial metabolic process revealed a significant bimodal pattern at high concentrations of Zn (>1920 microg g(-1)). This phenomenon suggested that soil microorganisms were very sensitive to zincous poisoning. A variety of soil quality properties were also measured and assessed. The results showed slower bacterial growth in soil cultures polluted with high levels of Zn. In addition, two kinds of fungi were identified by morphology and glomalin-related soil protein content in the Zn-contaminated soil. The growth of the first kind was inhibited with increase in Zn concentration. By contrast, the second kind could survive and continue to grow with increasing doses of Zn at 160-1920 microg g(-1) and its growth began to decline with further increase in Zn concentration. Finally, the fungus could not survive at very high (6000 microg g(-1)) Zn concentration. In this work, we conclude that soil microbial communities and activities can adapt to Zn pollution to a certain extent.

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.

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.

Microcalorimetric investigation of the toxic action of ammonium ferric(III) sulfate on the metabolic activity of pure microbes.

A series of calorimetric experiments were performed to investigate toxic action of ammonium ferric sulfate (AFS) on Bacillus subtilis, Pseudomonas putida and Candida humicola. The power-time curves of micro-organism metabolism were obtained, and the action of them by addition of AFS was studied. C. humicola, B. subtilis and P. putida were inhibited completely when the concentrations were up to 320.0, 160.0 and 160.0µgmL(-1), respectively. The relationships between growth rate constant (k) and doses of AFS were approximately linear for three microbes, P. putida for 10.0-160.0µgmL(-1) (R=-0.9746), B. subtilis for 0-160.0µgmL(-1) (R=-0.9868) and C. humicola for 10.0-320.0µgmL(-1) (R=-0.9955). The total heat dissipated per milliliter (Q(T)) for three microbes remained balance approximately during the lower doses, P. putida and B. subtilis less than the dose of 20.0µgmL(-1), 0.56±0.01 and 0.26±0.01JmL(-1), respectively, C. humicola less than the dose of 40.0µgmL(-1), 0.58±0.03JmL(-1). The biomass and OD(600) of three micro-organisms growth in the absence of AFS also were obtained. The power-time curve of C. humicola growth coincided with its turbidity curve. It elucidates that microcalorimetric method agreed with the routine microbiology method.

Microcalorimetric study the toxic effect of hexavalent chromium on microbial activity of Wuhan brown sandy soil: an in vitro approach.

A multi-channel thermal activity monitor was applied to study soil microbial activity in Wuhan brown sandy soil in the presence of different concentrations of hexavalent chromium (K(2)Cr(2)O(7)). In order to stimulate the soil microbial activity, 5.0mg of glucose and 5.0mg of ammonium sulfate were added to a 1.20-g soil sample under a controlled humidity of 35%. The results show that the poisonous species of K(2)Cr(2)O(7) at an half inhibitory concentration (IC(50)) value of 4.27 microg mL(-1) against soil microbe, and an increase of the amount of hexavalent chromium is associated to a decrease in the microbial activity of the soil, probably due to an increase in the toxicity of hexavalent chromium, affecting strongly the life in this soil microbial environment. Our work also suggests that microcalorimetry is a fast, simple and more sensitive method that can be easily performed to study the toxicity of different species of heavy metals on microorganism compared to other biological methods.

Isolation and characterization of aniline-degrading Rhodococcus sp. strain AN5.

A pure culture using aniline as a sole source of carbon and energy was isolated by selective enrichment culturing on a minimum salt medium (MSM) from the Nanjing Chemical Plant, Nanjing, Jiangsu Province, China. An analysis of the 16S rRNA gene sequence and morphological and physiological characteristics showed that this strain was a member of the genus Rhodococcus, and it was designated as strain AN5. Its optimal conditions for aniline biodegradation were 30 degrees C (pH 7.0). Due to the water insolubility of aniline, growth of strain AN5 at various aniline concentrations was studied via spectrophotometry and microcalorimetry in different culture mediums. Strain AN5 not only utilized aniline as its sole carbon and energy resources, but also degraded phenol, benzoic acid and naphthalin. Glucose, peptone and ammonium sulfate, when utilized as its secondary carbon and nitrogen sources, had no active effect on the aniline biodegradation. Such findings would be valuable in the application of strain AN5 to depurate industrial waste water.