ISSN 00036838, Applied Biochemistry and Microbiology, 2011, Vol. 47, No. 3, pp. 293–297. © Pleiades Publishing, Inc., 2011. Original Russian Text © G.A. Vydryakova, A.A. Gusev, S.E. Medvedeva, 2011, published in Prikladnaya Biokhimiya i Mikrobiologiya, 2011, Vol. 47, No. 3, pp. 324–329.
Effect of Organic and Inorganic Toxic Compounds on Luminescence of Luminous Fungi G. A. Vydryakova, A. A. Gusev, and S. E. Medvedeva Institute of Biophysics, Siberian Branch, Russian Academy of Sciences, Krasnoyarsk, 660036 Russia email:
[email protected] Received November 17, 2009
Abstract—The possibility of the development of the solid phase bioluminescent biotest using aerial mycelium of luminous fungi was investigated. Effect of organic and inorganic toxic compounds (TC) at concentrations from 10–6 to 1 mg/ml on luminescence of aerial mycelia of four species of luminous fungi—Armillaria bore alis (Culture Collection of the Institute of Forest, Siberian Branch, Russian Academy of Sciences), A. mellea, A. gallica, and Lampteromyces japonicus (Fungi Collection of the Botanical Institute, Russian Academy of Sciences)—has been studied. Culture of A. mellea was shown to be most sensitive to solutions of the model TC. It was demonstrated that the sensitivity of the luminous fungi is comparable with the sensitivity of the bacteria that are used for environmental monitoring. Use of the aerial mycelium of luminous fungi on the solid support as a test object is a promising approach in biotesting for the development of continuous biosen sors for air monitoring. DOI: 10.1134/S0003683811010194
INTRODUCTION Bioluminescent analysis is one of the possible rapid methods for environmental monitoring for the detec tion of the toxic compounds (TC) of both organic and inorganic nature, including heavy metals. Luminous bacteria are the mostused in bioluminescent analysis. Inhibition of bacterial luminescence by TC is the basis of most bioluminescent tests with the most popular having lyophilized bacteria as test objects due to their high sensitivity to micro quantities of toxic substances and simplicity of their use [1–5]. Genetically modi fied bacteria carrying lux genes of luminous bacteria or other bioluminescence organisms are extensively employed in bioluminescent analysis [5–7]. However, biotests are not always able to estimate the effect of TC on eukaryotic organisms and to detect TC in the air. Development and application of biotests with higher organisms (yeast, worms, fungi) carrying genes of luminescent systems open new perspectives in the progress of bioluminescent analysis. Increase in the number of studies on luminous fungi has been observed in recent years due to the pos sibility of their use in bioluminescent analysis. The number of known luminous fungi [8–10] is advancing steadily in connection with the discovery of new spe cies [11, 12]. Eighty three species of luminous fungi are currently known, while only 17 are known among bacteria [14]. Luminous fungi of all known species emit bluegreen light with a maximum at approxi mately 530 nm [15]. Fungi are luminescent in a wide temperature range from 4 to 50°С depending on the species [16]. The luminescence of the mycelium on
agar medium and on wood can last from several days to several weeks depending on the fungus species. Longlasting luminescence of the fungi in a wide temperature range and availability of aerial mycelium hold a promise for development of new continuous bioluminescent biotests that could be used for toxicity detection in environments including air. The goal of this study is the comparison of the effect of toxic compounds (TC) of organic and inor ganic nature on the level of luminescence of the aerial mycelia of the Armillaria borealis, A. mellea, A. gallica, and Lampteromyces japonicus species. EXPERIMENTAL Fungi cultures with luminous mycelia Armillaria mellea, A. gallica, and Lampteromyces japonicus from the basidiomycete culture collection LE(BIN) of the Komarov Botanical Institute (Russian Academy of Sciences) and A. borealis from the working culture col lection of the Sukachev Institute of Forest (Siberian Branch, Russian Academy of Sciences) were the objects of this study (Table 1). Aqueous solutions of salts, including heavy metals Na2WO4, NiSO4, CoCl2, (NH4)2MoO4, CrSO4, ZnSO4, Li2SO4, AlCl3, MnCl2, CsCl, CdSO4, SnCl2, HgCl2, and lead acetate [Рb(ОСОСН3)2], as well as benzoquinone (BQ), phenanthrenequinone (PQ), 5amino2,3dihy dro1,4phthalazinedione (ADHPD), and 1,2naph thoquinone4sulfonic acid (NQSA) in concentra tions 10–5, 10–4, 10–3, 10–2, 10–1, and 1 mg/ml, were used as model TC. Toxicity of the investigated solu tions was estimated by the value of the Bioluminescent
293
294
VYDRYAKOVA et al.
Table 1. Fungi cultures used in experiments Culture
Strain, no. in the collection
Armillaria gallica (Vahl.) P. Kumm.
1043
Central Ural, Perm oblast, 1993 LE(BIN)
A. mellea (Vahl.) P. Kumm., s.l.
0356
Minsk oblast, Belarus, 1970
''
Lampteromyces japonicus (Kawam.) Singer
491
United Kingdom, 1947
''
A. borealis (Marxm. & Korhonen)
3k4
Krasnoyarsk krai, 2007
Working collection, Institute of Forest, Siberian Branch, Russian Academy of Sciences
Location and year of isolation
Fungi collection
Table 2. Level of remaining luminescence (%) 3 min after addition of toxic compounds Concentration, mg/ml Compound 1
10–1
10–2
10–3
10–4
10–5
10–6
Benzoquinone
27
35
42
63
65
74
79
CuSO4
33
52
58
88
67
81
85
Index (BI) that was calculated by the equation: BI = Io/Ic, where Io is luminescence intensity of the fungus mycelium normalized to the initial luminescence in the experiment and Ic is luminescence of the fungus mycelium normalized to the initial luminescence in the control. Cultures were grown on the agarized Saburo medium (40 g of glucose, 10 g of peptone, and 15 g of agar–agar per 1 L of distilled water) at room tempera ture. Experiments on the TC effect were performed when the visual luminescence of the mycelium was present. The luminescence intensity was measured on the plate luminometer Luminoscan Ascent (Thermo Electron Co, Finland). Blocks of the agar medium with surface approximately 3 × 3 mm2 were placed into the wells of a plate (where 50 µl of the sterile distilled water were placed prior to that to avoid the drying of the agar) and initial luminescence intensity was mea sured. This was followed by the addition of 200 µl of the test sample in the well and measuring the lumines cence after 5, 10, 15, 20, 25, and 30 min. Normalized values were calculated relative to the initial lumines cence. The solutions of the tested substances were considered toxic if the deviation of the bioluminescent index of the experiment from the value of 1 (biolumi nescent index of the control) was above 20%.
RESULTS AND DISCUSSION A preliminary experiment performed investigating shortterm (3 min) effect of the CuSO4 and benzo quinone solutions at concentrations 10–6, 10–5, 10–4, 10–3, 10–2, 10–1, and 1 mg/ml on the luminescence of the A. mellea mycelium (Table 2). Minimal benzo quinone concentration (10–6 mg/ml) caused a 21% decrease in luminescence in comparison with the ini tial level, while the 3 min exposure to benzoquinone at the concentration of 1 mg/ml resulted in 73% decline in luminescence. Exposure to the CuSO4 solution at concentration of 10–6 mg/ml caused a 15% decrease in luminescence, while the decline was 67% of the initial level at the concentration 1 mg/ml. Effect of a variety of organic and inorganic sub stances at concentrations from 10–5 to 1 mg/ml on the luminescence of the studied cultures of the luminous fungi was investigated in the following experiments (Figs. 1, 2). It was shown during the experiments that 5 min exposure was sufficient to determine the possi ble toxic effect of the model TC solutions on the lumi nescence of the fungus mycelium. Data on the estima tion of the bioluminescent index of the A. borealis culture upon action of the tested solutions during 5 (Fig. 1a) and 30 (Fig. 1b) min are presented in Figs. 1a and 1b. It follows from the data presented in the figures that the effect of the most potent toxicant is already evident after 5 min exposure; thus, hereafter we do not present
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
Vol. 47
No. 3
2011
EFFECT OF ORGANIC AND INORGANIC TOXIC COMPOUNDS BI 6
(а)
BI 32 28 24 20 16 12 8 4 0
1 2 3 4 5 6
5 4 3 2 1
(а) 1 2 3 4 5 6
I
I
II
III
IV
V
VI VII VIII IX
BI 7
X
XI XII XIII XIV XV XVI XVIIXVIII
(b) 1 2 3 4 5 6
6 5 4
II III IV
V
2
I
II
III
IV
I
II
III
IV
V
VI
VII VIII IX
X
XI XII XIII XIV XV XVI XVII XVIII
VI
VII VIII
IX
X
XI
1 2 3 4 5 6
1.5 1.0
Fig. 1. Effect of the model TC on the luminescence (BI) of the Armillaria borealis aerial mycelium after (a) 5 and (b) 30 min of exposure. (Normalized luminescence values are presented in Figs. 1 and 2: BI = 1 is bioluminescent index of the control; the area between the dash lines BI = 0.8– 1.2 designates the nontoxic zone for the compound). I is Na2WO4, II is NiSO4, III is CoCl2, IV is (NH4)2MoO4, V is CrSO4, VI is Lead acetate, VII is ZnSO4, VIII is Li2SO4, IX is AlCl3, X is MnCl2, XI is CsCl, XII is CdSO4 ⋅ 8H2O, XIII is SnCl2, XIV is HgCl2, XV is benzoquinone, XVI is phenanthrenequinone, XVII is amino23 dihydro1,4 phthalazinedione, and XVIII is 1,2naphthoquinone4 sulfonic acid. TC concentrations, mg/ml: 1 is 1, 2 is 10–1, 3 is 10–2, 4 is 10–3, 5 is 10–4, and 6 is 10–5.
0.5 0 I
II III IV
V
VI VII VIII IX X XI XII XIIIXIV XV XVIXVII XVIII
Fig. 2. Effect of the model TC (5 min) on the luminescence of the aerial mycelium (BI) of (a) Armillaria mellea, (b) Lampteromyces japonicus, and (c) A. gallica. TC desig nations and concentrations are the same as in Fig. 1.
the data obtained for 10, 15, 20, 25, and 30 min expo sure of the cultures of the tested fungi to the investi gated solutions. Effect of the model TC on the luminescence of the fungi mycelia was of various directions and depended on the concentration of the tested compound and the species of the fungus (Figs. 1, 2). The A. borealis culture was sensitive to the action of Mn, Cs, Pb, Cd, and Al metal ions. The majority of the investigated salt solutions of these metals at differ ent concentrations stimulated the mycelium lumines cence for this culture, while Co, Cr salts and low con centration of lead acetate solutions inhibited it. The effect of the organic substances solutions was not so pronounced (Figs. 1a, 1b). APPLIED BIOCHEMISTRY AND MICROBIOLOGY
V
(c)
2.0
0
XI XII XIII XIV XV XVIXVII XVIII
1 2 3 4 5 6
2.5
1
X
(b)
BI 3.0
3
VI VII VIII IX
BI 6 5 4 3 2 1 0
0
295
In the case of A. mellea culture, the highest effect (5 fold and higher increase in the level of lumines cence) was observed upon action of the Cd, Zn, Li, Al, Mn, and Hg salts. The effect of all the investigated organic substances on the luminescence of this culture after 5 min exposure was equally pronounced (Fig. 2a). Salt solutions of Zn, Li, Al, Mn, and lead acetate stimulated L. japonicus luminescence (Fig. 2b). Inhi bition of A. gallica luminescence was observed in the majority of cases for both organic and inorganic TC (Fig. 2c). Amongst the investigated cultures, the maximal level of the initial luminescence was observed for A. borealis. However, the A. mellea culture was found to be the most sensitive to the action of the model TC. The prevalent response of the A. mellea culture to the
Vol. 47
No. 3
2011
296
VYDRYAKOVA et al.
Table 3. Sensitivity of the luminous bacteria and fungi to the action of chemical compounds, mg/ml Sensitivity of bacteria, mg/ml [19]
Sensitivity of fungi, mg/ml 1
HgCl2
2.7 × 10–8
10–5
CuSO4
1.2 × 10–2
10–4–10–5
10–3
10–5
CoCl2
3 × 10–3
10–5
MnCl2
5 × 10–2
10–3–10–5
Benzoquinone
4 × 10–8
10–5
Compound
Lead acetate
1Results from the work of the authors.
action of TC was the stimulation of the luminescence, while inhibition was observed for the A. gallica culture for the majority of TC, and both stimulation and inhi bition of luminescence were equally detected in the case of A. borealis. Luminescence stimulation by the salt solutions of some metals could be caused by the peculiarities of fungal metabolism. Thus, the enzymes—metallopro teins—are synthesized by the fungal cells that have metal ions in their composition, such as manganese dependent lignin peroxidases, as well as laccases and tyrosinases that contain mono and divalent copper ions and use phenol compounds as substrates [17]. It should be noted that fungus mycelium grown on solid nutrient medium was used in our experiments, while the experimental data known from literature were obtained using the globular mycelium grown in the liquid medium. The luminous aerial mycelium on the solid support seems to be a preferable test object for the development of the toxicity biotest in the air environment. However, the use of agar blocks was not without flaws, because, on the one hand, the damage of the mycelium structure occurs during block cutting, and, on the other hand, they could not be used for longterm monitoring. Hence, further search for pos sibilities to create a solid phase test object for the development of biosensors for toxicity detection not only in the aqueous but also in the air environment is necessary. Nevertheless, as follows from our experiments, the luminous aerial fungus mycelium on the solid phase support could be used as a test object in biosensors. Moreover, the mycelia cultures of the investigated fungi were shown to be close and, in some cases, even more sensitive to the action of the model TC in com parison with luminous bacteria (Table 3).
Lyophilized bacteria are used for the detection of TC in water sources [4, 18]. The bacterial biolumines cent test allows for the detection of small amounts of TC in the samples (Table 3). According to our data, the mycelium of the luminous fungus was more sensi tive to such TC as CuSO4, lead acetate, CoCl2, and MnCl2, and less sensitive to the action of benzo quinone and mercury chloride in comparison with the luminous bacteria. It is interesting to note the high sensitivity of the fungi luminescence to the presence of aluminum salts, and of some fungi to the solutions of tungsten and zinc salts. According to the data of foreign researchers, the comparison of the luminous fungi sensitivity to the variety of TC with luminescent biotests based on recombinant bacteria revealed that A. mellea fungus culture was more sensitive to copper than Pseudomonas fluorescence 8866 and Pseudomonas putida F1 bacterial cultures, while the culture of the Mycena citricolor fun gus was less sensitive to this TC [20, 21]. Currently several research groups around the world are attempting to create biosensors on the basis of luminous fungi. Brazilian researchers have used the novel species of luminous fungus Gerronema viridi lucens as a test object in the biosensor [22]. The lumi nous globular mycelium of A. mellea and M. citricolor was used as a test object by British and New Zealand researchers [20–23]. A 50% decline in luminescence (ЕС50) upon action of phenols and copper was of the same order of magnitude when the biotests based on the luminous fungi and bacterial biotests were used [23]. Successful attempts to construct recombinant lumi nous fungus of Neurospora sp. and Aspergillus sp. carrying genes of Gaussia copepod crustacean luciferase and the photoprotein obelin for biotest development are known [24]. Genetically modified bioluminescent fungus A. awamori carrying recombinant aequorin gene was used to detect changes in calcium ion concentra tion upon action of TC [25]. Hence, the performed experiments showed that aerial mycelium of luminous fungi could be used as a test object in a biosensor under appropriate adaptation to the measurement conditions. Five minute action of TC on the luminescent mycelium is sufficient for rapid testing. The results of our experiments confirmed and supplemented the data available in the literature that luminous fungi compare well to luminescent bacteria cells in sensitivity to the action of the model toxic compounds. Application of luminous fungus myce lium on solid support is a promising approach in biotesting as it allows us to use a test object with pro longed luminescence and to perform continuous monitoring of the air environment. ACKNOWLEDGEMENTS We are grateful to N.V. Psurtseva and N.V. Belova (Komarov Botanical Institute, Russian Academy of Sciences), and to N.V. Pashenova (Institute of Forest,
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
Vol. 47
No. 3
2011
EFFECT OF ORGANIC AND INORGANIC TOXIC COMPOUNDS
Siberian Branch, Russian Academy of Sciences) for providing the cultures of the luminous fungi. This work was supported in part by the Federal Tar get Program (State contract no. 02.512.11.2008). REFERENCES 1. Kuznetsov, A.M., Rodicheva, E.K., and Medvedeva, S.E., Luminescence, 1999, vol. 14, no. 5, pp. 263–265. 2. Gellert, G., Ecotoxicol. Environ. Saf., 2000, vol. 45, no. 1, pp. 87–91. 3. Ulitzur, S., Lahav, T., and Ulitzur, N., Environ. Toxi col., 2002, vol. 17, no. 3, pp. 291–296. 4. Rodicheva, E.K., Kuznetsov, A.M., and Medvedeva, S.E., Vestn. OGU, 2004, vol. 5, pp. 96–100. 5. Girotti, S., Ferri, E.N., Fumo, M.G., and Maiolin, E., Anal. Chim. Acta, 2008, vol. 608, no. 1, pp. 2–29. 6. Bechor, O., Smulski, D.R., Van Dyk, T.K., LaRossa, R.A., and Belkin, S., J. Biotechnol., 2002, vol. 94, no. 1, pp. 125–132. 7. Roda, A., Pasini, P., Mirasoli, M., Michelini, E., and Guardigli, M., Trends Biotechnol., 2004, vol. 22, no. 6, pp. 295–303. 8. Wassink, E.C., in Bioluminescence in Action, Herring, P.J., Ed., London: Academic, 1978, pp. 171–197. 9. Herring, P.J., Mycologist, 1994, vol. 8, no. 4, pp. 181–183. 10. Shimomura, O., Bioluminescence: Chemical Principles and Methods, New York: World Sci. Publ., 2006. 11. Desjardin, D.E., Capelari, M., and Stevani, C.V., Mycologia, 2007, vol. 99, no. 2, pp. 317–331. 12. Desjardin, D.E., Oliveira, A.G., and Stevani, C.V., Photochem. Photobiol. Sci., 2008, vol. 7, no. 2, pp. 170– 182.
APPLIED BIOCHEMISTRY AND MICROBIOLOGY
297
13. Vydryakova, G.A., Psurtseva, N.V., Belova, N.V., Pashenova, N.V., and Gitel’zon, I.I., Mikol. Fitopatol., 2009, vol. 43, no. 5, pp. 369–376. 14. Dunlap, P.V. and KitaTsukamoto, K., Luminous Bac teria, in The Prokaryotes, Dworkin, M., Falkow, S., Rosenberg, E., Schleifer, K.H., and Stackebrandt, E., New York: Academic Press, 2006, vol. 2, pp. 863–892. 15. O’Kane, D.J., Lingle, W.L., Porter, D., and Wampler, J.E., Mycologia, 1990, vol. 82, no. 5, pp. 607–616. 16. Yachevskii, A.A., Osnovy mikologii (Fundamentals of Mycology), Naumov, N.A., Ed., Leningrad: 1933. 17. Decker, H. and Terwilliger, N., J. Exp. Biol., 2000, vol. 203, no. 12, pp. 1777–1782. 18. Stom, D.I., Geel, T.A., Balayan, A.E., Shachova, G.I., Kuznetsov, A.M., and Medvedeva, S.E., Arch. Environ. Contam. Toxicol., 1992, vol. 2, pp. 203–208. 19. Rodicheva, E.K., Vydryakova, G.A., and Medvedeva, S.E., Katalog kul’tur svetyashchikhsya bakterii (Catalogue of Cultures of Luminescent Bacteria), Novosibirsk: Nauka, 1997. 20. Horswell, J., Weitz, H.J., Percival, H.J., and Speir, T.W., Biol. Fertil. Soils, 2006, vol. 42, pp. 569–576. 21. Weitz, H.J., Colin, D., Campbell, C.D., and Killham, K., Environ. Microbiol., 2002, vol. 4, no. 7, pp. 422–429. 22. Mendes, L., Prada, S., and Stevani, C., Proceedings of the SETAC 26th Annual Meeting in North America (USA), 2005, http://abstracts.co.allenpress.com/pweb/ setac2005/document/57331. 23. Paton, G.I., Viventsova, E., Kumpene, J., Wilson, M.J., Weitz, H.J., and Dawson, J.J., Sci. Total. Environ., 2006, vol. 355, nos. 1–3, pp. 106–17. 24. GB Patent No. WO/2004/076685, 2004. 25. Kozlova, O., Zwinderman, M., and Christofi, N., BMC Microbiol., 2005, vol. 5, no. 40, doi: 10.1186/1471 2180540.
Vol. 47
No. 3
2011
