Bioluminescence

environmental monitoring

1. Bioluminescence in Environmental monitoring Presented to :Ms Asifa Kiyani Presented by: Nayyab Nadeem & Sara Hassan

2. Abstract Bioluminescence is the production of light by living organisms such as bacteria, fungi and animals .The following review deals with detection and monitoring of pollutants using a biological method of rapid, cost effective and a sensitive technique for toxicology testing is the latest idea. The past decade has developed these novel tools which hold a far superior application compared to conventional methods. Previously most such assays were plant, animal or algae derived and as accurate as they were, they were expensive and required a large sample size in order to be standardized. One of the firsts assays based on bioluminescence was on the species V.fisheri and this has been reported to be versatile and test protocol is simple.Bioluminescene based assays / techniques are much sensitive when compared to wide range of chemicals or other bacterial based assays such as enzyme inhibition, respirometry etc.The assays have the added benefit of detecting toxicity across a wider range of toxic compounds.

3. Introduction Bioluminescence in its simplest definition is the light emission by a living organism. Their inherent beauty and eco friendliness has them marked for use in future environmental monitoring ventures . However they can be time consuming and often rather daunting (3).Due to these limitations the purpose is to find economic , quick and sturdy methods of environmental monitoring and ecosystem maintainace . The toxicity assays utilizing these microorganisms are based on the working principle that either light emission reduces or is inhibited in the presence of pollutants they interfere with the metabolism of these microorganisms.

4. What is Bioluminescence

5. Naturally occurring bioluminescent microorganisms in monitoring of environment The biolumiscenece emitting bacteria are found in freshwater, deep seas and marine habitats. They belong to gram negative cell wall group and are broadly divided into four types; vibrio, shewanella, photobacterium and photorhabdus . The light emission results from metabolic reactions and the process itself driven by co factors so when a particular toxic substance interferes the light emission is compromised giving an indication that a certain pollutant is present and extent to which it is affected confirms the concentration of the pollutant.

6. The bacterium photobacterium emits light which lies in the blue green region of the visible spectrum This method is sensitive, non invasive and provides real time monitoring of pollutants.

7. V.fisheri The first isolated microorganism whose bioluminesing ability was harnessed was vibrio fisheri The assay which was developed using a strain of V.fisheri was later given the name Microtox . The system contains freeze-dried bacteria which are activated prior to use and light emission is measured by a luminometer.

8. Hernando et al worked on v.fisheri strain and in 10 labs to observe its light inhibition capabilities and assays based on that. Their results were positive and confirmed that reliability and the reproducibility of the assay designed based on light inhibition of the V.fisheri. Scheerer et al carried out an experiment I n which they provided optimum conditions for V.fisheri and cultures in fermentors providing a long term continuous cultures for reproducible detection of pollutants and their measurement

9. The enzyme luciferase has two subunits which are alpha and beta and coded by lux A and lux B This operons also consists of other gene such as lux C , lux D, lux E and lux G along with lux A and lux B The greatest light emission occurs when cells are present in large concentrations and not in dilute conditions Different mechanisms exist for the regulation of the expression of the lux operons. The protein of lux R acts as a repressor. Lux I on the other hand codes for autoinducer which leaves the cells and also affects other surrounding cells. The repressor interacts with autoinducer and results in decrease in light emission .

10. Bioluminescent organism used in heavy metal monitoring and detection Heavy metal poisoning and its bioaccumulation is becoming an arising concern. bioluminescent organisms may be used for the rapid remediation of all such heavy metals polluted areas. These organisms usually have promoters which are also called as sensing elements which allow them to survive in such adverse environments. The luciferase genes are used as reported genes. The presence of the analyte triggers a reaction which then results in light emission

11. Angela et al constructed recombinant whole cell biosensors for the detection and assessment of heavy metals. Both gram positive and gram negative bacteria were used and were made to express the bioluminescence producing genes lux CDABE in response to heavy metals. Their results were supportive of the use of these microbial recombinant whole cells. Sulivan et al worked with a primary statistical model based on the crossings between the different detection ranges of five different bioluminescent strains for identification of four heavy metals cadmium, mercury, copper and arsenic. Their results showed that the statistical model could be used with confidence and that no sample pre treatment was required either.

12. The use of these biosensors which use bioluminescent microorganism as their biological agent have been used in rainwater, freshwater and estuarine areas containing high levels of mercury and they responded favorably. Another breakthrough which came was the online monitoring of heavy metals in waste water effluents Waste water treated effluents were found to contain heavy metals like lead , mercury etc and assays based on bioluminesing bacteria coupled to devices have been developed which allow online monitoring from remote far off areas of pollution.

13. Environmental monitoring by recombinant bioluminescent microorganisms One of the downside of the assays designed from V.fisheri is the need for appropriate salt concentration due to their marine habitats They also function within a narrow pH and temperature range and may not be suitable for terrestrial systems. E.coli is an example of transgenic bioluminescent microorganism which has been used in evaluating pollutants in air, land and water due to the vast genetic knowledge regarding these and they are easy to use.

14. One such example is a strain of E.coli called E.coli HB101 which consists of the plasmid Pucd607 which consists of luxCDABE from V.fisheri and the tet (tetracycline) promoter The other bacterial strains which have been transformed using this Puc plasmid include pseudomonas fluroscens 10586 , P.putida F1 which is a bacterium able to breakdown toluene. The P.fluroscens is especially sensitive towards copper as well as cadmium and has been used in monitoring of industrial effluents such as malt whiskey distilleries. MacGrath et al reported that soils which had a low pH had high levels of zinc and showed greater sensitivity towards the p.fluroscens transgenic organism compared to E.coli HB101.

15. Why do we need Biomonitoring ? Water quality may be affected by: Spills of oil and industrial products from tanks, pipelines Pesticides from agricultural area, leaching pathogens Endocrine disrupting chemicals Neurotoxins, hepatotoxins from algae blooms Contamination from terrorist attack (toxins, microbes, viruses, radioactive compounds) Accidents, sabotage etc.

16. MICROTOX ASSAY Microtox assay uses marine bioluminescent bacterium strain Vibrio fischeri (24) V. Fischeri is exposed to a range of concentrations of toxic agents like heavy metals Zn, Cd , Ni, Hg, Cu etc . As the concentration of toxins in the sample increases the consequential reduction in the light intensity emitted from the bacterium is measured and compared to standard. This shift in the light intensity output measured by a luminometer . The concentration of the toxic substances fabricate a dose / light response relationship.

17. Normal bioluminescence Contaminated sample is added!! Bioluminescence inhibition Photobacterium Vibrio fischeri ( Microtox test) Photobacterium Vibrio fischeri Bioluminescence measurement at exposure time 5, 15 and 30 min Microtox M500 analyzer

18. Lights off lights on assay

19. Toxicity tests have been used for : Marine and fresh waters biomonitoring Toxicity testing of wastewaters and soils Toxicity testing of fly ash leachates Toxicity assessments of pure compounds, heavy metals and pesticides Wastewater treatment plant applications Applications of ecotoxicity testing

20. Dinoflagellates and Bioluminescence

21. Dinoflagellates for environmental risk detection Bioluminescent dinoflagellates are also used in assays for ecotoxicological testing. The dinoflagellates Bioluminescence is dependant on circadian rhythms. In various studies Lingulodinium polyedrum , Pyrocystis noctiluca , Pyrocystis fusiformis , Ceratocorys horrida , Pyrophacus steinii and Pyrocystis lunula have been employed to find the effects of xenobiotic contaminants on intensity of bioluminescence.

22. Use of dinoflagellates as a metal toxicity assessment tool in aquatic system Dinoflagellates selected Gonvaulax polvedra and Pyrocystis lunula for the assessment of biological toxicity of the selected contaminants present in the aquatic water samples. This study used the bioluminescence of dinoflagellates with QwikLite assay developed by the US Navy for the heavy metals Hg, Cu, Cd , As, Pb , Cr, etc. The results obtained in the form of single metal toxicity in the order Hg+ > Cu > Cd > As > Pb > Cr Comparison was made to standard solutions. Dinoflagellates demonstrated great sensitivity to metal concentrations hence showing great prospect for testing heavy metal toxicity in aquatic systems (28).

23. ROLE OF BIOLUMINESENCE IN MARINE WATER AND WASTER WATER TREATMENTS.

24. Assessment of heavy metals by bacterial bioluminescence in bench-scale wastewater treatment system According to one study the bioluminescent strain Shk1 was used to determine the toxicity during the activated sludge wastewater treatment in batch experiments using bench-scale activated sludge system to determine presence of heavy metals Ni, Cd , Cu and Zn using both sources influent wastewater and activated sludge from the municipal wastewater treatment plant in batch experiments. According to the findings of this study the Shk1 strain bioluminescence demonstrated the highest sensitivity to Cd and Zn, then towards Cu, and least sensitivity to Ni.

25. USING OSTEOCOCCUS TAURI TO DETECT ANTIFOULING BIOCIDES IN MARINE WATER A recombinant was constructed using Ostreococcus tauri , known to be the smallest eukaryotic cell luminescent biosensor with the aim to detect antifouling biocides. Diuron and Irgarol (antifouling biocides) and their degradation products prevalent in coastal waters were selected to investigate the novel biosensor for ecotoxicological testing. Cyclin -dependent kinase ( a cell cycle protein) fused to luciferase (CDKA-Luc) proved to be a highly sensitive biosensors showing precise determination of diuron and Irgarol . Luminometer was employed to measure luminescence compared to inhibition of growth. Luminescence is a more sensitive indicator of toxicity than growth inhibition in marine phytoplankton.

26. Detection of drugs in surface water and wastewater samples preliminary testing of toxicity studies using  vibrio fischeri Effluent from various industries including municipal waste water find their final resting place in the large water bodies like seas and oceans. To detect these pharmaceutical compounds in the water sample a preliminary test is performed using the bioluminescent strain Vibrio Fischeri for executing Microtox ® . detect the presence of analgesic drugs, primarily acidic and polar in nature. The drugs investigated according to a particular study were ibuprofen, gemfibrozil , diclofenac , naproxen, with their decomposition products like salicylic acid. This preliminary step was followed by combined analytical method using (LC–ESI-MS) toxicity and liquid chromatography– electrospray ionization-mass spectrometry for determination of pharmaceutical compounds in the water samples

27. GENETICALLY ENGINEERED MICROORGANISMS FOR POLLUTION MONITORING

28. Immobilization and integration into biosensors No matter the type of modification used the bacteria reporter strain has to be sophisticated and for its specific use it must be immobilized to a bioactive membrane to achieve and attain its final purpose of detection of whatever particular type of substance it has been tailored for .So for this purpose it is take away from its boundaries of the lab and taken to the be adhered or incorporated into a biosensor divide that ensures protection and maintenance along with ease of storage ease of sample access and transduction of signals. For this it may be impregnated into strontium alginate. onto optic fiber tips (48) immobilization in agar (49)and encapsulation in gel matrices

29. bioavailability of nutrients. After observing the phenomenon of eutrophication of algal blooms which occur in the presence of nitrogen and phosphorus in excess a novel branch of bioreporters was constructed which worked on the principle of nutrient bioavailability. Nitrogen and phosphorus clearly don’t make it t the top of the most deleterious pollutants list but the principle of eutrophication was used to tailor   Synechococcus sp. with a glnA :: lux fusion and a Synechocystis sp containing a nblA :: lux fusion each serves as a sensitive reporters nitrogen bioavailability. The former is specific for ammonia, nitrite and organic nitrogen on the contrary the latter is specific for nitrate alone .

30. Detection of specific pollutants The first constructs to detect specific inorganic and organic pollutants for determining the bioavailability and catabolic activity and potential in water bodies like streams and rivers a genetically modified bioluminescent bacteria Psuedomonas flouresens HK44 was employed. It bears a fused transcriptional fragment nahG -" uxCDABE responsible for naphthalene and salicyclate catabolism . exposure of either compounds induced bioluminescence. The cells of P. flouresens were immobilized in strontium alginate to make an optical based whole-cell biosensor for monitoring of bioavailability of naphthalene and salicylate in streams of waste water. On exposure to additional compounds for instance glucose,toluene , complex nutrient medium either showed nill or weak bioluminescence increases produced after delayed response times compared to naphthalene.

31. References Sagi, E., Hever, N., Rosen, R., Bartolome, A. J., Premkumar, J. R., Ulber, R., Lev, O., T., Belkin , S., 2003, Fluorescence and bioluminescence reporter functions in genetically modified bacterial sensor strains, Sens. Actuators B-Chem. 90:2–8. Sayler , G. S., Simpson, M. L., Cox, C. D., 2004, Emerging foundations: nano -engineering and bio-microelectronics for environmental biotechnology, Curr . Opin . Microbiol . 7:267– 273. Selifonova , O., Burlage , R., Barkay , T., 1993, Bioluminescent sensors for detection of bioavailable Hg(II) in the environment, Appl. Environ. Microbiol . 59:3083–3090. Shao , C. Y., Howe, C. J., Porter, A. J. R., Glover, L. A., 2002, Novel cyanobacterial biosensor for detection of herbicides, Appl. Environ. Microbiol . 68:5026–5033. Stiner , L., Halverson, L. J., 2002, Development and characterization of a green fluorescent protein-based bacterial biosensor for bioavailable toluene and related compounds, Appl. Environ. Microbiol . 68:1962–1971. Southward, C. M., Surette , M. G., 2002, The dynamic microbe: green fluorescent protein brings bacteria to light, Mol. Microbiol . 45:1191–1196.