Isolation and Characterization of a Pseudomonas fl uorescens Strain Tolerant to Major Indian Water Pollutants

Heavy metals contamination of agriculture arable fields by frequent applications of deposited sewage sludge and industrial effluents can have long adverse effects on soil microorganisms [13]. Various developments in the fields of agriculture and industry have resulted in the introduction of new organic pollutants which find their use as fire retardants, paints, solvents, herbicides and fungicides. Phenolic compounds released from the petrochemical industry, chemical industry and coal gasification units are also among the pollutants found in industrial effluents [4,5]. It is also known that some of them are degraded by microorganisms [6,7]. Aligarh is a major lock-manufacturing city for more than 50 years. Large amounts of heavy metals are released from various lock manufacturing and electroplating industries and the emission rate has increased considerably during the last two decades [8-13]. These pollutants exert profound effects on the ecological status of the system and create problems related to public health [14-15]. Several studies conducted in India suggested the presence of various pollutants in surface waters with the major toxicants being heavy metals, pesticides and phenolics [12,13,16-19].


Introduction
Heavy metals contamination of agriculture arable fields by frequent applications of deposited sewage sludge and industrial effluents can have long adverse effects on soil microorganisms [1][2][3].Various developments in the fields of agriculture and industry have resulted in the introduction of new organic pollutants which find their use as fire retardants, paints, solvents, herbicides and fungicides.Phenolic compounds released from the petrochemical industry, chemical industry and coal gasification units are also among the pollutants found in industrial effluents [4,5].It is also known that some of them are degraded by microorganisms [6,7].Aligarh is a major lock-manufacturing city for more than 50 years.Large amounts of heavy metals are released from various lock manufacturing and electroplating industries and the emission rate has increased considerably during the last two decades [8][9][10][11][12][13].These pollutants exert profound effects on the ecological status of the system and create problems related to public health [14][15].Several studies conducted in India suggested the presence of various pollutants in surface waters with the major toxicants being heavy metals, pesticides and phenolics [12,13,[16][17][18][19].
Bioremediation has been used as a strategy to remove pollutants, not necessarily completely, but to compounds with decreased solubility, mobility and toxicity [20].Physiological and genetic features of Pseudomonas make it a promising agent for utilization in biotechnology, agriculture and environmental bioremediation applications.Some strains of Pseudomonas fluorescens were shown to have a significant role in the bioremediation of heavy metals, pesticides and phenolics [21][22][23][24][25].This paper deals with the isolation and characterization of a Pseudomonas fluorescens strain capable of degrading/detoxifying some selected members of the major water pollutants of India i.e. heavy metals, pesticides and phenolics.from the barren land receiving domestic and industrial wastes of Aligarh city for many years, and were kept moist with sterile distilled water [26].Aligarh wastewater samples were collected using APHA guidelines [27].

Analysis of soil and water samples
The soil and water samples were analyzed for the presence of various heavy metals and pesticides with the help of atomic absorption spectrophotometer GBC plus (Australia) and HPLC (Waters Milford, MA) containing a UV detector model 4490 coupled to Millenium 32  software respectively [28,29].

Isolation of Pseudomonas fluorescens strain from soil
The isolation of Pseudomonas fluorescens strains from soil was done according to the method described by Radjendirane et al. [30] with slight modification as follows: An enrichment technique was essentially employed for isolation of the test strain.A composite sample of 100 gm soil was suspended in 250 ml sterile distilled water and allowed to settle down after vigorous shaking.The aqueous layer containing the mixed bacterial population was filtered through ordinary filter paper.It was then centrifuged at 1400g for 5 min at 4°C using Remi (India) centrifuge model R8C.The supernatant was discarded from each tube and the several pellets obtained were pooled and further concentrated via centrifugation.The resulting pellet containing the microbial cells was resuspended in sterile normal saline and then spread on the Pseudomonas agar (Himeda, India) plates with different concentrations of toxicants.The seeded plates were incubated at 37°C overnight.The colonies obtained were checked for green fluorescence under ultraviolet light (250-350 nm) and the fluorescent clumps of colonies were separately streaked on a fresh toxicant supplemented plate for obtaining isolated colonies of resistant and most probably belonging to fluorescent group of Pseudomonas sp.Among several colonies, some well-separated single colonies were picked up to prepare the pure cultures.The concentrations of filter sterilized individual toxicants added to the molten agar medium were equal or simple multiples of those given in Table 1.

Isolation of heavy metals and xenobiotic tolerant Pseudomonas fluorescens strains
For the isolation of heavy metals and xenobiotic tolerant Pseudomonas fluorescens strain, a preliminary screening was done employing 1X concentrations of heavy metals and organic shown in table 1, taken in combination.From among the 100 moderately tolerant isolates, further secondary screening was conducted at a 4X rate of heavy metals, pesticides and phenolics as three separate assays.

Sub-screening of the toxicant tolerant strains in the liquid media
The 11 isolates displaying higher tolerance to either of the three groups of the toxicants were inoculated in 1X supplemented Pseudomonas broth with the overnight incubation at 37°C.Suitable volumes of the overnight grown culture were then 10 fold diluted in the Pseudomonas broth containing filter sterilized pollutants, viz.heavy metals, pesticides and phenolics at 4X concentration.Turbidity of each culture was recorded at 550 nm at different time points.Negative controls (medium with the respective pollutants without the microbes) were also run simultaneously.The two cultures exhibiting the maximum growth in the presence of the three types of toxicants separately and in combination of all toxicants were abbreviated as SM1 and SM6 strains.

Selection and taxonomic characterization of most tolerant strain
The microbial strains obtained as described above were further checked for growth in the presence of 4X toxicants.Thus the most tolerant isolate was finally characterized on the basis of morphological, cultural and biochemical properties [31,32].

Growth and viability of Pseudomonas fluorescens SM1 strain under stress
The test SM1 strain was incubated in Pseudomonas broth containing either sterile industrial sewage water as solvent instead of distilled water, or 4X toxicants (a mixture of test heavy metals, pesticides and phenolics serving as control).The efficiency of tolerance was also determined by suspending it in normal saline and in filtered polluted water.

Utilization of the test organic toxicants by the Pseudomonas fluorescens SM1 strain
Growth of the test Pseudomonas fluorescens SM1 strain was monitored in the liquid mineral medium containing either of the toxicants namely 2,4-D, BHC, mancozeb, catechol or m-cresol.The composition of mineral medium was (g/l): K  [33].For control experiments the test strain was inoculated in liquid mineral medium plus solvent.DMSO (Dimethyl sulphoxide) served as solvent in case of pesticides due to their poor solubility in water.The cell density in terms of optical density was recorded at different time points starting from 24 h up to 72 h at 37°C.

Enrichment and isolation of toxicant tolerant Pseudomonas fluorescens strain
Table 2 presents the results of preliminary screening for the toxicant tolerant Pseudomonas isolates.Out of the 100 suspected Pseudomonas clones exhibiting moderate tolerance (1X) to all toxicants, 11 isolates were found to tolerate higher doses (4X) of various groups of toxicants.In fact, 6 out of 11 were tolerant to the mixture of test heavy metals, 3 to the pesticides and 2 clones were obtained from the plates containing a mixture of test phenolics alone.In view of the better growth characteristics under stress, SM1 isolate was selected for further studies (Table 3).Routine identification tests [31] confirmed it to belong to the Pseudomonas fluorescens group of bacteria (Table 4).

Efficiency of tolerance of the test Pseudomonas fluorescens SM1 strain under stress
Tables 5 and 6 present the data of the growth and viability of the Pseudomonas fluorescens SM1 strain under various experimental whereas Bacillus megaterium was tolerant upto 0.3mM of total Pb (0.1mM soluble) in the same minimal medium.Interestingly, our Pseudomonas fluorescens SM1 strain tolerated Pb up to 2.3mM in the Pseudomonas medium at pH 6.8.Errasaquin and Vazquez [38] have isolated Trichoderma atroviride from a sludge sample polluted with heavy metals like Cu, Zn and Cd.They observed that in case of Cu, T. atroviride survived at concentrations up to 300 mg l -1 with almost constant levels of biomass.However, the growth dramatically decreased at 350 mg l -1 , while no growth was detected at 400 mg l -1 .The most toxic metal was Cd, with a 50% reduction in biomass at 125 mg l -1 and no detectable growth at 300 mg l -1 .Compared to above our isolate seems to be superior in terms of resistance or tolerance to Cu and Cd, since it could tolerate Cu and Cd up to the concentrations of 2942 and 496 mg l -1 respectively.

Utilization of BHC and certain phenolics as a source of carbon and energy
The test Pseudomonas fluorescens SM1 strain was allowed to grow for 72 h, in the synthetic medium containing 40x BHC as the sole source of carbon and energy.Positive and negative controls were also run simultaneously by using Pseudomonas broth and inorganic salts alone respectively for comparison [Table 7].Cell density was recorded to be enhanced in complete nutrient medium conditions.The cell density of SM1 strain for 48 h under different conditions of stress was roughly equal to that in Pseudomonas broth.Similarly viability of SM1 in the industrial waste did not reduce to a significant extent rather a little bit got enhanced from untreated control value (Table 6).Soil harbors many microbes which have evolved a number of mechanisms to survive under the hostile condition [34].Saxena et al. [35] isolated Pseudomonas putida S4 strain which was exhibiting resistance to several heavy metals like aluminium, zinc, nickel, cobalt, copper etc.This strain was able to grow in presence of 1mM Cu 2+ , 2mM Zn 2+ and 1mM each of Al 3+ , Co 2+ and Ni 2+ .Contrary to the above, our isolate Pseudomonas fluorescens SM1 could grow well in presence of 2.34 mM Pb 2+ , 5.07 mM Ni 2+ , 11.7 mM Cu 2+ , 0.13 mM Cr 6+ and 2.7 mM Cd 2+ taken in combination besides having the toxic levels of phenolics and pesticides in the culture medium (Table 5).Roane and Pepper [36] identified three isolates as Arthobacter, Bacillus and Pseudomonas sp. and further demonstrated that two of the isolates were highly resistant to soluble cadmium with maximum resistance at 275 mg l -1 while our strain tolerated cadmium up to a level of 496 mg l -1 .Moreover, two lead resistant strains namely Pseudomonas marginalis and Bacillus megaterium were isolated by Roane [37].Out of these two strains, Pseudomonas marginalis could withstand up to 2.5mM total (0.3 mM soluble) Pb in the defined minimal medium at pH 6.8 as well as in the known growth supporting salt medium containing phenolics.There was no enhancement in cell density in medium containing only salts.The same experiment was conducted using BHC (Benzene hexachloride) and it was found that our Pseudomonas fluorescens strain efficiently utilized BHC as the source of carbon and energy (Table 7).We could not succeed in obtaining appreciable growth in the presence of 40X mancozeb and 2,4-D probably due to the toxic effect of these pesticides and/or organic solvent (DMSO) which had also increased by 10 fold.Sahu et al. [39] isolated a Pseudomonas strain which degraded -HCH (Hexachlorocyclohexane) up to a level of 8120 ppb whilst our strain seems to have the ability to degrade as high concentration of BHC as 20,000 ppb (Table 7).It is also interesting to note that our isolate, Pseudomonas fluorescens SM1 was also capable of degrading comparable amount (16 mM) of phenols as reported by other investigators vis-à-vis utilizing them as sole source of carbon and energy [40][41][42] (Table 8).Some members of yeast belonging to genera Candida, Rhodotorula, Trichosporon and others metabolize phenolic compounds as a sole carbon and energy source [43][44][45].Moreover, we found that the utilization of catechol was faster than m-cresol by SM1 strain [46] (Table 8).The efficiency of utilization of the organic toxicants at the test concentration with respect to time by SM1 strain was as follows: Catechol > m-cresol > BHC The most attractive feature of the Pseudomonas fluorescens SM1 strain is its resistance to those substances which are the better representative of the major groups of pollutants of Indian water vis-à-vis a natural bacterium thriving under the unfavorable Indian environment conditions.Hence, the present study has demonstrated that the Pseudomonas fluorescens SM1 would play an important role in the remediation of some heavy metals, pesticides and phenolics in heavily polluted sites.

Table 1 :
Major toxicants in industrial sewage water and soil of Aligarh actually found and/or were taken for this study as 1X.The concentrations of phenolics and Cr (VI) were selected arbitrarily rather than actually found in the sewage water or soil of Aligarh sampling area *

Table 2 :
Screening and isolation of toxicant tolerant Pseudomonas fl uorescens strain.

Table 3 :
Growth characteristics of the two best isolates in the presence and absence of test toxicants.Optical density (O.D.) values are given as the statistical mean of the three independent experiments carried out using triplicate tubes

Table 4 :
Morphological and Biochemical Characteristics of Pseudomonas fl uorescens SM1 strain.
*O.D. values are given as the statistical mean of the three independent experiments carried out using experimental tubes in triplicate

Table 5 :
Growth characteristics of the most tolerant Pseudomonas fl uorescens strain SM1 in the presence and absence of major water toxicants.O.D. values are given as the statistical mean of the three independent experiments carried out using the tubes in triplicate *

Table 6 :
Tolerance of the test Pseudomonas fl uorescens SM1 strain exposed to the industrial wastewater for a period of 24 hours.

Table 7 :
BHC utilization effi ciency of the test Pseudomonas fl uorescens SM1 strain as a carbon source.O.D. values are given as the statistical mean of the three independent experiments carried out using triplicate tubes *