{"id":2221,"date":"2015-04-25T07:55:50","date_gmt":"2015-04-25T07:55:50","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=2221"},"modified":"2020-04-26T08:03:24","modified_gmt":"2020-04-26T08:03:24","slug":"effect-of-cu-on-pigmentation-and-survival-of-pseudomonas-stutzeri","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol5no1\/effect-of-cu-on-pigmentation-and-survival-of-pseudomonas-stutzeri\/","title":{"rendered":"Effect of Cu on Pigmentation and Survival of Pseudomonas stutzeri"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

The production under assured intensified environment of yellow-green, fluorescent, water-soluble stain is a feature property of some Pseudomonas <\/em>spp. (Stanier et al., <\/em>1966). They all belongs to the same intra-generic genetic homology group (Palleroni et al.,<\/em>1973) and \u00a0contain P. aeruginosa, P. putida, P. fluorescens <\/em>and phytopathogens of the P. syringae <\/em>type (Palleroni & Doudoroff, 1974).\u00a0 \u00a0<\/strong><\/p>\n

Teitzel et al<\/em> (2003) observed the effect of the heavy metals Cu, Pb, and Zn on bio-layer and planktonic Pseudomonas aeruginosa<\/em>. The bio- layer and live culture were tested for heavy metal tolerance through rotating-disk biofilm reactor and found that biofilm was more resistant to heavy metal as compared to live microbial cell.\u00a0 \u00a0A probable justification for this is that the extra cellular polymeric materials that encase a biofilm may be accountable for shielding \u00a0<\/sup>the cells from heavy metal pressure by combining the heavy metals and hold up their distribution within the biofilm.<\/p>\n

Meyer et al.,<\/em> (1978) reported that iron-deficiency was the major source in bioproduction of a yellow-green, fluorescent, water-soluble pigment by Pseudomonas fluorescens<\/em> which was not directly affected by the environment of the organic carbon basis. They also determined the spectral properties of the pure pigment, its molecular weight (1500 \u00b1 75) and its stability constant for Fe3+<\/sup> (of the order of 1032<\/sup>) and suggest that the biosynthesis and its chemical properties (formation of a stable Fe3+<\/sup> complex) showed that the fluorescent pigment was a desferrisiderophore.<\/p>\n

Detailed literature survey on the fluorescent pigment synthesized by a strain of Pseudomonas sp. <\/em>showed that it\u00a0 <\/em>\u00a0forms a stable complex with metals. But no report is available yet which describe the role of Pseudomonas stutzeri<\/em> as a bio monitoring tool to explain the soil contamination through its color of pigmentation. This article describes pigment synthesis in relation with heavy metal Cu, its effect on bacteria, Cu removal and role in complex formation.<\/p>\n

Material and methods<\/strong><\/p>\n

The strain of Pseudomonas stutzeri<\/em> employed was isolated from sample taken from heavy metal contaminated soil. The organism was held in nutrient agar as Ps 01B. The biochemical characteristics of oxidase positive organism were identified by QTS-NE Strips (Desto laboratories) (Table 1). Nutrient agar and nutrient broth were used, for the experiments, contains no source of Cu or Fe.<\/p>\n

Table 1: Identification Of Microorganism By Qts-Ne Kit<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
Test<\/strong><\/td>\nReaction<\/strong><\/td>\nIdentified Specie<\/strong><\/td>\n<\/tr>\n
IND<\/td>\nIndole<\/td>\nNegative<\/td>\n\u201cPseudomonas stutzeri\u201d<\/td>\n<\/tr>\n
GLU<\/p>\n

(Acid)<\/td>\n

\u00a0\u00a0 a)Acid from Glucose<\/p>\n

b)Nitrate reduction<\/td>\n

Positive<\/td>\n<\/tr>\n
ADH<\/td>\nArginine dihydrolase<\/td>\nNegative<\/td>\n<\/tr>\n
URE<\/td>\nUrea hydrolysis<\/td>\nNegative<\/td>\n<\/tr>\n
ESC<\/td>\nEsculin hydrolysis<\/td>\nPositive<\/td>\n<\/tr>\n
GEL<\/td>\nGelatin hydrolysis<\/td>\nPositive<\/td>\n<\/tr>\n
ONPG<\/td>\nO, nitrophenyl \u03b2 D galactosidase<\/td>\nNegative<\/td>\n<\/tr>\n
GLU<\/td>\nGlucose assimilation<\/td>\nPositive<\/td>\n<\/tr>\n
MAL<\/td>\nMaltose assimilation<\/td>\nNegative<\/td>\n<\/tr>\n
MNE<\/td>\nMannose assimilation<\/td>\nPositive<\/td>\n<\/tr>\n
NAG<\/td>\nN-acetyl-glucose amine-assimilation<\/td>\nNegative<\/td>\n<\/tr>\n
CIT<\/td>\nSodium citrate assimilation<\/td>\nPositive<\/td>\n<\/tr>\n
CO<\/p>\n

Strip<\/td>\n

Spot test Cytochrome oxidase<\/td>\nPositive<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Culture was maintained in 5 ml nutrient broth. 200ug\/ml conc. of CuSO4<\/sub> was made in nutrient broth. Two fold serial dilution of CuSO4<\/sub> was made for the identification of survival and pigment production of Pseudomonas stutzeri<\/em>. 0.1 ml culture was inoculated in each tube separately and incubated at 370<\/sup> C.<\/p>\n

Bacterial intensification was estimated turbidometrically at 600 mm, to conclude the concentration of fluorescent pigment in culture media. Microorganisms were removed via centrifugation and the optical density of supernatant liquid was measured at 400nm. Survival of bacteria in different concentration of CuSO4<\/sub> was determined on nutrient agar by serial dilution method. Water and Hoagland solution in nutrient broth was taken as control.<\/p>\n

Results and Discussion<\/strong><\/p>\n

This study specifies some observations regarding the stress of Cu on the intensification and pigmentation of bacteria, Pseudomonas stutzeri<\/em> which were monitored on nutrient medium in presence of aqueous and Cu concentration. Activity was determined as a function of pigment secretion and survival of bacteria under metal stress (Meyer et al.,<\/em> 1978). Survival and optical density were represented in Figures (1-3). The change of color of pigmentation under the influence of copper on nutrient medium naturally suggested an affect of addition of the biologically important metal ions Cu2+<\/sup>, on the growth and the pigmentation of bacteria, as well as possible “detoxification” of the metals by bacteria. A probable mechanism of Cu detoxification or binding of Cu on dead biomass Pseudomonas stutzeri <\/em>was discussed at high concentration. Influence of Cu concentration in serial dilution viz 200 to .39 \u00b5gm\/ ml on growth and secretion of pigment gives some interesting results which were easily observe through the color of pigment of Pseudomonas stutzeri<\/em> presented in Fig. 1.<\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 1: Pigment production by Pseudomonas at different concentration of CuSO4<\/sub><\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Results suggest that growth of Pseudomonas stutzeri <\/em>in the typical medium (which included no Cu) was escorted by release of pigment whereas\u00a0\u00a0 excretion finished by adding different concentration of Cu.\u00a0 Highest concentration of Cu\u00a0\u00a0 to\u00a0 the\u00a0 culture medium\u00a0 almost\u00a0 inhibit\u00a0\u00a0 the growth yield\u00a0 with\u00a0 completely\u00a0 repressed\u00a0 formation of\u00a0 color of pigment when Cu was in the range \u00a0of 50 \u2013 12.5ug\/mL concentration. The last four dilution (3.13 \u2013 0.39ug\/mL) showed an increase in pigment production, indicates that Cu in its negligible amount had no effect on microbial growth and pigment production (Table 2&3).\u00a0 The quantity of stain produced per unit of cell mass was contrary to the initial Cu concentration of the medium.<\/p>\n

Table 2: \u00a0Effect of Variable concentration of Cu on pigment Color <\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
DILUTION<\/strong><\/td>\nCOPPER CONCENTRATION<\/strong><\/p>\n

\u00b5g\/ml<\/strong><\/td>\n

PIGMENTATION<\/strong><\/td>\n<\/tr>\n
Tube 1<\/td>\n200\u00b5g\/ml<\/td>\nNo<\/td>\n<\/tr>\n
Tube 2<\/td>\n100\u00b5g\/ml<\/td>\nNo<\/td>\n<\/tr>\n
Tube 3<\/td>\n50 \u00b5g\/ml<\/td>\n\u00a0Light Green color<\/td>\n<\/tr>\n
Tube 4<\/td>\n25\u00b5g\/ml<\/td>\nLight Green color<\/td>\n<\/tr>\n
Tube 5<\/td>\n12.5\u00b5g\/ml<\/td>\nLight Green color<\/td>\n<\/tr>\n
Tube 6<\/td>\n6.25\u00b5g\/ml<\/td>\nGreen color with blacking<\/td>\n<\/tr>\n
Tube 7<\/td>\n3.13\u00b5g\/ml<\/td>\nGreen color<\/td>\n<\/tr>\n
Tube 8<\/td>\n1.56\u00b5g\/ml<\/td>\nGreen color<\/td>\n<\/tr>\n
Tube 9<\/td>\n0.78\u00b5g\/ml<\/td>\nGreen color<\/td>\n<\/tr>\n
Tube 10<\/td>\n0.39\u00b5g\/ml<\/td>\nGreen color<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Table\u00a0 3: \u00a0Survival Of Pseudomonas In The Presence Of Different Concentration Of CuSo4<\/sub><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n
TUBES<\/strong><\/td>\nAVERAGE<\/strong><\/p>\n

NO. OF COLONIES<\/strong><\/td>\n

LOG10<\/sub><\/strong><\/td>\nO.D<\/strong><\/td>\n<\/tr>\n
01<\/strong><\/td>\nNo growth<\/td>\nNo growth<\/td>\n0.28<\/td>\n<\/tr>\n
02<\/strong><\/td>\nNo growth<\/td>\nNo growth<\/td>\n0.25<\/td>\n<\/tr>\n
03<\/strong><\/td>\n593<\/td>\n2.777<\/td>\n0.21<\/td>\n<\/tr>\n
04<\/strong><\/td>\n5370<\/td>\n3.729<\/td>\n0.24<\/td>\n<\/tr>\n
05<\/strong><\/td>\n20493<\/td>\n4.312<\/td>\n0.24<\/td>\n<\/tr>\n
06<\/strong><\/td>\n708<\/td>\n2.580<\/td>\n0.26<\/td>\n<\/tr>\n
07<\/strong><\/td>\n130<\/td>\n2.114<\/td>\n0.36<\/td>\n<\/tr>\n
08<\/strong><\/td>\n921<\/td>\n2.964<\/td>\n0.43<\/td>\n<\/tr>\n
09<\/strong><\/td>\n1946<\/td>\n3.299<\/td>\n0.40<\/td>\n<\/tr>\n
10<\/strong><\/td>\n108400<\/td>\n5.035<\/td>\n0.44<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Exposure of Pseudomonas stutzeri <\/em>cells to Cu2+<\/sup> at high concentration alone resulted in quick and marked cell loss, and binding of nearly all of the copper in solution (Finazzi-Agro et al.1970). The results illustrated that, at an effective concentration, feeble and reasonable copper-ligands can efficiently upset copper toxicity. Experiment showed number of interesting facts. In the Fig.1growth of bacteria in \u00a0\u00a0the tubes, indicates a distinct difference in tolerance of the diverse concentration of Cu. Thus, when both low and high copper concentration in dilutions of 200 to 100 \u00b5gm\/ ml, were added to agar, inhibit the growth of pseudomonas and pigmentation of interest (Muayad et al 2009). Low concentration of Cu added to the medias has no marked effect on pigmentation (Table 2 and Fig. 1). The addition of copper to the medium does not affect the growth at low concentration but effects on change of the color, (Fig. 1), although the shade was altered in some instances as reported in the Table (2). It may be related with growth of bacteria in Cu stress which showed varied results with the different concentration of Cu (Table 3). At higher concentration of Cu death of all bacteria were occurred which may be related with negative effects of Cu on bacteria growth or absorption of oxygen by Cu to form cupric oxide which act as a toxicant for bacteria but no color of Cu in the medium showed that Cu adsorbed on the dead mass of cell which was earlier investigated by Chang et al<\/em>.(2007 & 1997) who reported that the Pseudomonas aeruginosa<\/em> and Bacillus thuringiensis <\/em>\u00a0act as a\u00a0 good biosorbent for heavy metal removal\u00a0 in contaminated location specially for Hg and Cu. The burly interfaces of mercury and copper with organic stuff propose that these adverse elements might be eliminated from the surroundings by bacterial catching and appropriation (Hassen et al<\/em> 1998). It is interested that metal in polluted environments existed in different forms. The growth phase exhibited no effects on the adsorption of Cu (Chang et al 1997). The loss of Cu color at high concentration was not due to the reduction of Cu by the pigment but may be due to the adsorption of Cu on dead mass of Pseudomonas stutzeri<\/em> which indicated that the dead mass of Pseudomonas was effective to control the Cu toxicity as reported earlier that dead mass of the cells are effective in controlling the toxicity of heavy metal (Hassen et al<\/em> 1998).\u00a0 Chen et al (2007) reported that the results of laboratory equilibration studies which also indicated that biomass-adsorbed Cu (II) or Zn(II) portions may be included\u00a0 both reversibly and powerfully bounded or resistant components.<\/p>\n\n\n\n
\u00a0\"Figure<\/td>\n\n

Figure 2: Survival of Pseudomonas sp. in different CuSO4<\/sub> concentration<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n\n
\"Figure<\/td>\n\n

Figure 3: Optical Density of Pseudomonas sp. Pigment in different CuSO4<\/sub> concentration.<\/strong><\/p>\n

Click here to View figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Some authors (Meyer et al \u00a0\u00a01978) have recognized an important role of nature of the organic carbon and energy source in pigment synthesis; showed influence on pigment production\u00a0 \u00a0\u00a0which are classified as \u2018chromogenic\u2019 or \u2018anti-chromogenic\u2019. It was observed that when Cu concentration decreases, the growth of Pseudomonas stutzeri<\/em> increases and significant quantity of fluorescent pigment were produced\u00a0 (Table 1)when\u00a0 standard\u00a0 nutrient medium was\u00a0 pretreated with Hoagland nutrient medium\u00a0 to\u00a0 check the effect on production of pigment,\u00a0 an increase yield of Pseudomonas stutzeri <\/em>was reduced with low pigment production or may be due to an \u2018antichromogenic\u2019 substrate can thus be converted into a \u2018chromogenic\u2019 one by a specific reduction of the Cu concentration of\u00a0 the medium (Jo\u00e3o P.S. Cabral 1994 and 1991). However, the precise Cu supplies of Pseudomonas stutzeri <\/em>may vary as a function of the organic carbon and energy source.<\/p>\n

Conclusion<\/strong><\/p>\n

The general conclusion of above report based on justification of the effect of different doses of Cu metal in general on pigmentation and survival. Naturally, there would be an optimum effective metal concentration on each organism and in this study it was above 50 \u00b5gm\/ ml,<\/p>\n

The addition of Cu, on the \u00a0medium containing low concentration of copper produced a good intensification of pigment with change in color from light green to blue which clearly indicate the binding of Cu with the pigment but however at\u00a0 a concentration higher than 100 \u00b5gm\/ ml, it turns colorless which indicate two possibilities 1) reduced state of pigment and 2) Cu binding with dead mass of strain or may be the fact that in the presence of metals some ordinarily pigmented bacteria grow without producing a pigment as in the case of Staphylococcus aureus<\/em>, Pseudomonas pyocyanea<\/em>, or to a different color as in the case of Chlorella<\/em> (Kharasch et al<\/em> 1936). This study also suggest that pigment secretion in presence of metal can also be used as a bio tool for detection of heavy metal concentration in soil through inoculation of bacteria in soil where survival of bacteria determine the toxicity level \u00a0however more researches are required to take under consideration with different metal and different bacterial strain<\/p>\n

References<\/strong><\/p>\n

    \n
  1. Chen X, Wu W, Shi J, Xu X, Wang H, Chen Y.2007 Adsorption of copper and zinc on Pseudomonas putida CZ1: particle concentration effect and adsorption reversibility. Colloids Surf B Biointerfaces.\u00a0 15;54(1):46-52.<\/li>\n
  2. Finazzi-Agro, A., Rotilio, G, Avigliano,L., Guerrieri,P\u00a0 Boffi, V., Mondovi, B.1970 Environment of copper in Pseudomonas fluorescens Azurin: fluorometric approach\u00a0 Biochemistry, 9 (9): 2009\u20132014<\/li>\n
  3. Hassen A., Saidi, N., Cherif and Boudabous ,A. , 1998 Effects of heavy metals on Pseudomonas aeruginosa<\/em> and Bacillus thuringiensis\u00a0\u00a0\u00a0\u00a0 <\/em>Bioresource Technology\u00a0 65(1-2), 73-82<\/li>\n
  4. Jo-Shu Chang, Robin Law and Chung-Cheng Chang (1997) Biosorption of lead, copper and cadmium by biomass of Pseudomonas aeruginosa<\/em> PU21 .Water Research\u00a0 31(7):1651-1658<\/li>\n
  5. Jo\u00e3o P.S. Cabral 1991The antibacterial action of cupric ions in Pseudomonas syringae\u00a0 <\/em>FEMS\u00a0 Microbiology Letters 79(2-3):303-308<\/li>\n
  6. Jo\u00e3o P.S. Cabral 1994 Influence of organic ligands on the toxicity of copper to Pseudomonas syringae\u00a0 <\/em>FEMS Microbiology Letters.\u00a0\u00a0\u00a0 117,(3): 341-344<\/li>\n
  7. Kharasch \u00a0M. S., Conway E. A. and Bloom W. 1936 Some chemical factors influencing growth and pigmentation of certain microorganisms’ Journal of bacteriology, 32( 5)533-540.<\/li>\n
  8. Gail M. Teitzel and Matthew R. Parsek., 2003 Heavy Metal Resistance of Biofilm and Planktonic Pseudomonas aeruginosa<\/em> Applied and Environmental Microbiology,69(4):2313-2320<\/li>\n
  9. Muayad M. Abboud, Humodi A. Saeed, Khaled A. Tarawneh, Khaled M. Khleifat and Amjad Al Tarawneh 2009 Copper Uptake by Pseudomonas aeruginosa<\/em> Isolated from Infected Burn Patients\u00a0 Current Microbiology 59(3)282-287<\/li>\n
  10. Meyer J. M. and Abdallah M. A. (1978), The Fluorescent Pigment of Pseudomonas fluorescens: Biosynthesis, Purification and Physicochemical Properties Journal of General Microbiology 107 319-328; DOI\u00a0\u00a010.1099\/00221287-107-2-319<\/li>\n
  11. Palleroni, N. J., Kunisawa, R., Contopoulou, R. & Doudoroff, M. (1973). Nucleic acid homologies in the genus International Journal of Systematic Bacteriology <\/em>23, 333-339.<\/li>\n
  12. Palleroni, N. J. & Doudoroff, M. (1974). The genus In Bergey\u2019s Manual of Determinative Bacteriology, <\/em>8th edn, pp. 217-243. Edited by R.E. Buchanan & N.E. Gibbons. Baltimore: Williams & Wilkins.<\/li>\n
  13. Stanier, R. , <\/em>Palleroni, N. J. & Doudoroff, M. (1966). The aerobic pseudomonas : a taxonomic study. Journal of General Microbiology <\/em>43, 159- 271.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

    Introduction The production under assured intensified environment of yellow-green, fluorescent, […]<\/p>\n","protected":false},"author":2,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[14],"tags":[],"class_list":["post-2221","post","type-post","status-publish","format-standard","hentry","category-vol5no1"],"_links":{"self":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/2221","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/comments?post=2221"}],"version-history":[{"count":5,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/2221\/revisions"}],"predecessor-version":[{"id":33277,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/posts\/2221\/revisions\/33277"}],"wp:attachment":[{"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/media?parent=2221"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/categories?post=2221"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/biomedpharmajournal.org\/staging\/wp-json\/wp\/v2\/tags?post=2221"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}