Bacterium Photobacterium Phosphoreum Research Articles

The investigation of different pollutants and operation processes on sludge toxicity in sequencing batch bioreactors

ABSTRACTThe influence of different target pollutants and operation modes in sequencing batch bioreactors (SBRs) on sludge toxicity was compared in this study. Sludge toxicity was characterized by the inhibiting luminosity through using luminescent bacterium Photobacterium phosphoreum (P. phosphoreum) during either gradual acclimation or impaction processes with synthetic wastewater containing high-strength bisphenol A (BPA) or N, N-dimethylformamide (DMF). When the activated sludge was first acclimated with either 120 mg/L DMF or 20 mg/L BPA, and then respectively increased to 200 mg/L DMF and 40 mg/L BPA it was defined as gradual acclimation process, whereas when the activated sludge was, respectively, injected with 200 mg/L DMF and 40 mg/L BPA directly it was defined as impaction process. Results showed that the toxicity of the impacted sludge was greater than that of the gradual acclimated sludge, especially in the initial stage before 10 d. Activated sludge treating BPA synthetic wastewater exhibited higher toxicity due to the more inhibition of BPA to sludge activity compared to that of DMF. The proteomics analysis indicated that the stress responses of activated sludge to DMF and BPA stimulation were both significant. In turn, the secretions from two kinds of sludge under stress conditions contributed to sludge toxicity.

Ozonation of the UV filter benzophenone-4 in aquatic environments: Intermediates and pathways

The occurrence of benzophenone-4 (BP-4) in water environments may pose a serious public health hazard due to its potential endocrine disrupting effects. In this work, the intermediates, probable degradation pathways and toxicity changes during ozonation of BP-4 in aqueous solution were systematically investigated. Results revealed that alkaline conditions favored the oxidation of BP-4. However, inorganic anions (Cl−, NO3−, SO42−), cations (K+, Ca2+, Mg2+) and humic acid had no remarkable effect on BP-4 removal within the tested concentrations. Ozonation was also effective for the fast removal of BP-4 in real waters. The TOC suggested a low mineralization rate, even after the complete BP-4 removal. Meanwhile, the treated mixtures exhibited an obvious inhibition to the bioluminescent bacteria Photobacterium phosphoreum, indicating the formation of transformation products with higher toxicities. Furthermore, fourteen products were identified by means of liquid chromatography-mass spectrometry. Notably, seven of them have not been reported previously. The quenching test indicated that the degradation processes probably were dominated by OH. Next, possible degradation pathways were proposed and further justified by theoretical calculations of frontier electron densities. This investigation will contribute to the systematic elucidation of the ozonation process of UV filters in aquatic environments.

Contrasting sludge toxicity under various starting modes (shocking or acclimating) and original organics (with or without N, N-dimethylformamide (DMF))

Sludge toxicity, as determined by the inhibition of luminescence in the bacterium Photobacterium phosphoreum (P. phosphoreum), was formed during either acclimating or shocking starting processes with high concentrations of N, N-dimethylformamide (DMF). In the acclimating starting process, the sludge was first acclimated with 120 mg L−1 DMF before 200 mg L−1 DMF was added, whereas in the shock starting process, the sludge was fed with 200 mg L−1 DMF directly. The results showed that the toxicity of the sludge shocked with 200 mg L−1 DMF was significantly higher than that of the acclimated sludge throughout the entire process. The maximum toxicity gap between the shocked and acclimated sludge appeared at the initial stage, and the gap decreased with operation time. In addition, the maximum toxicity existed for 6–8 h in a single SBR cycle, regardless of the starting process. Proteomics analysis indicated that the responses of the shocked and acclimated sludge to DMF stimulation were different. As a result, toxic substances accumulated in the sludge through various factors, including secretions from two types of sludge under DMF stress conditions.

Degradation of trichloroacetic acid by an efficient Fenton/UV/TiO2 hybrid process and investigation of synergetic effect

An efficient hybrid degradation process combined Fenton with TiO2 photocatalytic oxidation for trichloroacetic acid (TCAA) was first investigated, and then the synergetic degradation effect between Fenton reagent and TiO2 photocatalytic oxidation was discussed in this study. The dominant factors, such as dosages of TiO2 and Fe2+, TCAA concentration and pH were researched and optimized. The cooperation effect between Fenton and TiO2 photocatalytic oxidation probably occurred because (1) more OH radicals were produced in the Fe(OH)2+ and H2O2 decomposition reactions irradiated by UV light; and (2) photoinduced electrons voluntarily participated in the reduction from Fe3+ to Fe2+, improving the efficient separation of the photogenerated electrons and holes. Adsorption and degradation were both involved in the removal of TCAA in this hybrid process, and the removal of TCAA followed the Langmuir model and the degradation kinetics accorded with first order degradation kinetics. Toxicity test using a bioluminescent bacterium Photobacterium phosphoreum as the indicator proved that the hybrid process was a safe treatment because the toxicity decreased greatly with the degradation time.

An analysis of the bioluminescence intensity dynamics of the luminous bacteria Photobacterium phosphoreum

The results of the analysis of the dynamics of the bioluminescence of the luminous bacteria Photobacterium phosphoreum IMB-7071 under optimal growth conditions are presented. A quasi-harmonic character of bacterial bioluminescence was revealed. The observed periods of these changes have similar values to the previously established periods of the changes in the physicochemical properties of water. Correlations between biorhythms and the quasi-harmonic character of the physicochemical properties of water are discussed.

Comparison of chronic low-dose effects of alpha- and beta-emitting radionuclides on marine bacteria

AbstractEffects of Americium-241 (241Am), alpha-emitting radionuclide of high specific radioactivity, and tritium (3H), beta-emitting radionuclide, on luminous bacteria Photobacterium phosphoreum were compared. Bioluminescence intensity served as a marker of bacterial physiological activity. Three successive stages in the bioluminescence response to 241Am and 3H were found under conditions of lowdose irradiation: (1) absence of effects, (2) activation, and (3) inhibition. They were interpreted in terms of bacterial response to stressfactor as stress recognition, adaptive response/syndrome, and suppression of physiological function (i.e. radiation toxicity). Times of bioluminescence activation (TBA) and inhibition (TBI) were suggested as parameters to characterize hormesis and toxic stages in a course of chronic low-dose irradiation of the microorganisms. Values of TBA and TBI of 241Am were shorter than those of 3H, revealing higher impact of alpha-irradiation (as compared to beta-irradiation) under comparable radiation doses. Increases of peroxide concentration and NADH oxidation rates in 241Am aquatic solutions were demonstrated; these were not found in tritiated water. The results reveal a biological role of reactive oxygen species generated in water solutions as secondary products of the radioactive decay. The study provides a scientific basis for elaboration of bioluminescence-based assay to monitor radiotoxicity of alpha- and beta-emitting radionuclides in aquatic solutions.

ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum

Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

Mechanism of toxicity formation and spatial distribution in activated sludge treating synthetic effluent containing bisphenol A (BPA)

Organic sludge toxicity is a critical issue for sludge land applications. To investigate sludge organic toxicity formation and spatial distribution properties, sludge was acclimated using synthetic effluent containing various bisphenol A (BPA) concentrations in sequential aerobic activated sludge reactors (SBRs). Acute sludge toxicity, which was characterized by the inhibition rate of the luminescent bacterium Photobacterium phosphoreum, was the focus of this study. The original influent COD of the SBR was approximately 300 mg/L; the sludge retention time (SRT) and hydraulic retention time (HRT) were controlled at 20 d and 12 h, respectively. The results indicated that a positive correlation existed between sludge toxicity and the influent BPA concentration. Furthermore, the toxicity was centralized in the intracellular and inner sections of extracellular polymeric substance (EPS) in sludge flocs. BPA concentrations were controlled at 2.5 mg/L, 5.0 mg/L, 7.5 mg/L and 20 mg/L; the sludge toxicities, measured by comparing the control SBR with the blank, were 2.99%, 4.05%, 6.99% and 10.64%, respectively, at the end of 28-day operation. In the 12-h SBR treatment process, aqueous and sludge phase BPA was completely removed within 2 h. However, the maximum sludge toxicity appeared in the 8th hour. The DGGE analysis suggested that the existence of BPA in wastewater increased bacterial diversity in activated sludge. However, the bacterial similarity between sludge in SBRs decreased with increasing BPA concentration. Lastly, the bacterial similarity between blank sludge and control sludge of 20 mg/L BPA was 0.47.

Biomarker discovery and biotherapeutics applications of photosynthetic light-harvesting and bioluminescence light-emitting chromophore-protein complexes in stem cell biology and regenerative medicine.

We have since the 1970's embarked on the development of biologically derived fluorophore-protein complexes that will find applications in the communicable and non-communicable disease etiology processes and their cures. We have since then become largely successful in these endeavors along with interspersed contributions also from investigators who have generally restricted to working in confined disciplines. Their encompassment with our works as this investigator has traversed his definitely chosen and not merely a circumstantial, coincidental, or accidental step-wise multi-disciplinary scientific path from biophysics to regenerative medicine spanning these lines of investigations for last four decades have finally yielded the much necessitated disease related applied biological interventions for human benefits. Taking a cue from our early investigations and findings on which we call attention to the identification and characterization of the use of the primary light-emitting lumazine precursor of riboflavin which is 6,7-dimethyl-8-ribityl lumazine-protein complex which we had derived from the bioluminescence bacterium (Photobacterium phosphoreum) wherein it functions as a naturally occurring fluorescence light emitter (LumP). These in vivo phenomena have been a precursor to the subsequent developments in vitro. This in vivo to in vitro investigation path of ours has been also comprised among others of binding of photosynthetic light-harvesting marine dinoflagellate algae (Glenodinium sp.) derived peridinin-chlorophyll a-protein (PerCP) complex-labeled monoclonal antibodies useful in the development of flow cytometry. These fluorescence labeled antibodies bound antigens which include those of communicable infectious diseases (HIV/AIDS - env-gp160, gag-p24), non-communicable but also potential hereditary and malignant disorders (Cancer/Tumor Markers - Melan-A/Mart-1 of melanoma), normal immune response cells (Human/Mouse/species cellular MHC/TCR/CD45/CD33/CD56/CD19/CD41), and of types of stem cells (CD34/CD38/c-Mpl/Oct4/Neuropilin-1/SOX17). Such antigens have been analyzed by us and other investigators by fluorescence-activated cell sorting (FACS - cell surface and intracellular binding), confocal fluorescence microscopy, or/and immunohistochemistry, to determine qualitative and quantitative antigen expression levels and their mechanistic implications. We have followed stem cell differentiation patterns and signaling mechanisms through marker antigen-antibody binding wherein the antibodies are labeled with covalently linked fluorophore-protein complexes or fluorescence emitting chromophores. These complexes among others also have included PerCP. We are also now in the process of developing flow cytometry applications of additional visible light emitting chromophore-protein complexes through industrial collaborations. The United States Navy has long been known for interest in the estimation of changes in illumination intensity in and under the oceans to track movements of enemy ships and other naval vessels.

Effects of Hydrostatic Pressure on Growth and Luminescence of a Moderately-Piezophilic Luminous Bacteria Photobacterium phosphoreum ANT-2200

Bacterial bioluminescence is commonly found in the deep sea and depends on environmental conditions. Photobacterium phosphoreum ANT-2200 has been isolated from the NW Mediterranean Sea at 2200-m depth (in situ temperature of 13°C) close to the ANTARES neutrino telescope. The effects of hydrostatic pressure on its growth and luminescence have been investigated under controlled laboratory conditions, using a specifically developed high-pressure bioluminescence system. The growth rate and the maximum population density of the strain were determined at different temperatures (from 4 to 37°C) and pressures (from 0.1 to 40 MPa), using the logistic model to define these two growth parameters. Indeed, using the growth rate only, no optimal temperature and pressure could be determined. However, when both growth rate and maximum population density were jointly taken into account, a cross coefficient was calculated. By this way, the optimum growth conditions for P. phosphoreum ANT-2200 were found to be 30°C and, 10 MPa defining this strain as mesophile and moderately piezophile. Moreover, the ratio of unsaturated vs. saturated cellular fatty acids was found higher at 22 MPa, in agreement with previously described piezophile strains. P. phosphoreum ANT-2200 also appeared to respond to high pressure by forming cell aggregates. Its maximum population density was 1.2 times higher, with a similar growth rate, than at 0.1 MPa. Strain ANT-2200 grown at 22 MPa produced 3 times more bioluminescence. The proposed approach, mimicking, as close as possible, the in situ conditions, could help studying deep-sea bacterial bioluminescence and validating hypotheses concerning its role into the carbon cycle in the deep ocean.

Ozonation of trimethoprim in aqueous solution: Identification of reaction products and their toxicity

This work aimed to better understand the ozonation process of a typical antibiotic pharmaceutical, trimethoprim in aqueous solution. The parent compound was almost completely degraded with ozone dose up to 3.5 mg/L with no mineralization. Twenty one degradation products were identified using an electrospray quadrupole time-of-flight mass spectrometer. Several ozonation pathways were proposed including hydroxylation, demethylation, carbonylation, deamination and methylene group cleavage. Two species of luminescent bacteria Photobacterium phosphoreum and Vibrio qinghaiensis were selected to assess the toxicity of ozonation products. For P. phosphoreum, higher level of toxicity was observed compared to the parent compound, but a negligible toxicity change was observed for V. qinghaiensis, indicating different modes of action for the same water sample. This was further confirmed by quantitative structure–active relationship analysis. This work proves the dominant role of ozone rather than hydroxyl radicals in the reaction and the potential risk after ozonation.

Effect of tritium on luminous marine bacteria and enzyme reactions

The paper studies chronic effect of tritiated water, HTO, (0.0002–200 MBq/L) on bioluminescent assay systems: marine bacteria Photobacterium phosphoreum (intact and lyophilized) and coupled enzyme reactions. Bioluminescence intensity serves as a marker of physiological activity. Linear dependencies of bioluminescent intensity on exposure time or radioactivity were not revealed. Three successive stages in bacterial bioluminescence response to HTO were found: (1) absence of the effect, (2) activation, and (3) inhibition. They were interpreted in terms of reaction of organisms to stress-factor i.e. stress recognition, adaptive response/syndrome, and suppression of physiological function. In enzyme system, in contrast, the kinetic stages mentioned above were not revealed, but the dependence of bioluminescence intensity on HTO specific radioactivity was found. Damage of bacteria cells in HTO (100 MBq/L) was visualized by electron microscopy. Time of bioluminescence inhibition is suggested as a parameter to evaluate the bacterial sensitivity to ionizing radiation.

Bioluminescence as a tool for studying detoxification processes in metal salt solutions involving humic substances

The paper considers effects of humic substances (HS), as natural attenuators of toxicity, on solutions of model inorganic pollutants, metal salts – Pb(NO3)2, СоСl2, CuSO4, Eu(NO3)3, СrСl3, and K3[Fe(СN)6]. Luminous bacteria Photobacterium phosphoreum and bioluminescent system of coupled enzymatic reactions were used as bioassays to monitor toxicity of salt solutions. The ability of HS to decrease or increase toxicity was demonstrated. Detoxifying concentrations of HS were determined; detoxification coefficients were calculated at different times of exposure of salt solutions to HS. To study the combined effects of HS and salts on bioluminescent assay systems, the rates of biochemical reactions and bacterial ultrastructure were analyzed. The detoxifying effects were explained by: (1) decrease of free metal content in water solutions under metal–HS binding; (2) increase of biochemical reaction rates in a bioluminescent assay system under HS effect; (3) enhancement of mucous layers on cell surface as a response to unfavorable impact of toxicants. Detoxifying mechanisms (2) and (3) reveal the active role of bioassay systems in detoxification processes.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

Acute Toxicity of Alkylpolyglucosides to Vibrio fischeri, Daphnia magna and Microalgae: A Comparative Study

In this paper, toxicity values of alkylpolyglucosides have been determined by applying the 24-h immobilization test with Daphnia magna, the LumiStox(®) 300 test which employs the luminescent bacteria Photobacterium phosphoreum and the test with Selenastrum capricornutum. Three alkylpolyglucosides with different alkyl chain and degree of polymerisation have been tested. For all tests, the results indicated that Vibrio fischeri was more sensitive to toxic effects from alkylpolyglucosides than was D. magna or S. capricornutum. The results demonstrate considerable variation in toxicity responses within structurally related glucose-based surfactants regardless of the species tested. The toxicity increased as the critical micelle concentration decreased, and as the alkyl chain length and resultant hydrophobicity increased.

Effect of americium-241 on luminous bacteria. Role of peroxides

The effect of americium-241 ( 241Am), an alpha-emitting radionuclide of high specific activity, on luminous bacteria Photobacterium phosphoreum was studied. Traces of 241Am in nutrient media (0.16–6.67 kBq/L) suppressed the growth of bacteria, but enhanced luminescence intensity and quantum yield at room temperature. Lower temperature (4 °C) increased the time of bacterial luminescence and revealed a stage of bioluminescence inhibition after 150 h of bioluminescence registration start. The role of conditions of exposure the bacterial cells to the 241Am is discussed. The effect of 241Am on luminous bacteria was attributed to peroxide compounds generated in water solutions as secondary products of radioactive decay. Increase of peroxide concentration in 241Am solutions was demonstrated; and the similarity of 241Am and hydrogen peroxide effects on bacterial luminescence was revealed. The study provides a scientific basis for elaboration of bioluminescence-based assay to monitor radiotoxicity of alpha-emitting radionuclides in aquatic solutions.

Detoxification of AM-241 solutions by humic substances: bioluminescent monitoring

The study addresses the effect of humic substances on marine luminous bacteria Photobacterium phosphoreum exposed to Am-241 (3,000BqL(-1), water solution). Luminescent intensity of the bacteria was applied as a marker of their physiological activity. Humic substances have been found to reduce the effect of Am-241 on luminescence, decrease damage to cells, and change distribution of Am-241 between bacterial cells and intercellular media. It was shown that water-soluble humic substances, being products of natural transformation of organic substances in soil and bottom sediments, can serve as protecting agents for water microorganisms exposed to alpha radionuclides.

Acute toxicity evaluation of explosive wastewater by bacterial bioluminescence assays using a freshwater luminescent bacterium, Vibrio qinghaiensis sp. Nov.

The compositions of explosive wastewater generated from TNT (2,4,6-trinitrotoluene) purification stage were characterized by using UV–vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), high performance liquid chromatograph (HPLC) and gas chromatograph/mass spectroscopy (GC/MS). The acute toxicity was evaluated by bacterium bioluminescence assay using a freshwater luminescent bacterium (Vibrio qinghaiensis sp. Nov.) and a marine luminescent bacterium (Photobacterium phosphoreum). The results showed that the wastewater's biodegradability was poor due to the high amount of chemical oxygen demand (COD). The main organic components were dinitrotoluene sulfonates (DNTS) with small amount of TNT, dinitrotoluene (DNT), mononitrotoluene (MNT) and other derivatives of nitrobenzene. It was highly toxic to luminescent bacteria P. phosphoreum and V. qinghaiensis sp. Nov. After reaction time of 15min, the relative concentration of toxic pollutants (expressed as reciprocal of dilution ratio of wastewater) at 50% of luminescence inhibition ratio was 5.32×10−4 for P. phosphoreu, while that was 4.34×10−4 for V. qinghaiensis. V. qinghaiensis is more sensitive and suitable for evaluating the wastewater's acute toxicity than P. phosphoreum. After adsorption by resin, the acute toxicity can be greatly reduced, which is helpful for further treatment by biological methods.

Enhanced nitrogen removal from pharmaceutical wastewater using SBA-ANAMMOX process

Efficient biological nitrogen removal from pharmaceutical wastewater has been focused recently. The present study dealt with the treatment of colistin sulfate and kitasamycin manufacturing wastewater through anaerobic ammonium oxidation (ANAMMOX). The biotoxicity assay on luminescent bacterium Photobacterium phosphoreum (T3 mutation) showed that the pharmaceutical wastewater imparted severe toxicity with a relative luminosity of 3.46% ± 0.45%. During long-term operation, the cumulative toxicity from toxic pollutants in wastewater resulted in the performance collapse of conventional ANAMMOX process. A novel ANAMMOX process with sequential biocatalyst (ANAMMOX granules) addition (SBA-ANAMMOX process) was developed by combining high-rate ANAMMOX reactor with sequential biocatalyst addition (SBA). At biocatalyst addition rate of 0.025 g VSS (L wastewater) −1 day −1, the nitrogen removal rate of the process reached up to 9.4 kg N m −3 day −1 in pharmaceutical wastewater treatment. The effluent ammonium concentration was lower than 50 mg N L −1, which met the Discharge Standard of Water Pollutants for Pharmaceutical Industry in China (GB 21903-2008). The application of SBA-ANAMMOX process in refractory ammonium-rich wastewater is promising.

Improved detection of toxic chemicals by Photobacterium phosphoreum using modified Boss medium

A bioluminescent bacterium Photobacterium phosphoreum has been used widely as an indicator of pollutants where the presence of toxic chemicals decreases light output. The optimum conditions for the growth and bioluminescence of P. phosphoreum KCTC 2852 were investigated using modified Boss medium and two environmental conditions (pH and temperature). Optimized conditions that supported growth of P. phosphoreum with high bioluminescence intensities were: NaCl (20 g/L), glycerol (2.5 g/L), peptone (1.0 g/L), and yeast extract (1.0 g/L). Growth and bioluminescence gradually increased as pH increased reaching a maximum at pH 7.0. The maximum temperature for growth and bioluminescence was 20 °C. Based on these optimum conditions for bioluminescence, a continuous culture reactor of P. phosphoreum was operated. During the continuous operation, the optimized medium maintained bioluminescence with a high relative light unit (RLU) around 13,600 for more than 120 h. When zinc (0.5 mg/L) was added to the reactor, an EC 50 was observed in 15 min. The detection limit was improved by using the modified Boss medium since the modified Boss medium contained less organic matter which minimizes the complexation and precipitation of heavy metals compared to other enrichment media with high levels of organic matter.