DISTRIBUTION OF HAEMOLYTIC THIOBACILLI IN SEWAGE SAMPLES

Sulphur is one of the essential plant nutrients and it contributes to yield and quality of crops. Thiobacillus play an important role in sulphur oxidation in soil and domestic wastewater.The present study is sought to understand the frequency of Thiobacillus in sewage water samples collected from hospital and domestic area.Distribution of sulphur oxidizing bacteria were analysed by using modified Thiobacilli agar. Five isolates were selected and analysed their selected putative virulence properties phenotypically.Also they were showed biocompatible within the group of the Thiobacilli.The extracellular products (culture supernatant) of Thiobacilli showed a substantial level of haemolytic activity and antagonistic activity. Among the isolates from sewage sources, TB-D1 and TB-D2 produced higher level of bacteriocin against pathogenic bacteria due to the formation of maximum inhibition zone. It reveals that the bacteriocin from Thiobacillus showed the elevated antagonistic activity against most of the pathogenic bacteria, as we tested.The strains TB-D4 and TB-D5 showed beta and alpha haemolysis, it might have virulent properties and cause infection in human and animals.The strain TB-D5 showed the maximum inhibitions while compared with others. It is visibly showing presence of virulence factors in the extracellular metabolites of Thiobacilli.


INTRODUCTION
Sulphur is now considered the fourth major plant nutrient after N, P and K and is one of the sixteen elements which are essential for the growth and development of plants. The role of chemolithotrophic bacteria of the genus Thiobacillus in this process is essential.Organisms belonging to the group of colorlesssulfur bacteria oxidize sulfide to elemental sulfur under oxygen limiting conditions. Based on this feature many researchers worked for biological oxidation using various types of microorganisms (Gadre, 1989). The advantage of this biological sulfide oxidation system is that, no chemicals are required except oxygen (Buismanet al., 1990).
Thiobacillican use reduced inorganic sulphur compounds as an energy source (Kelly and Harrison, 1989) and are therefore used for removing sulphide from industrial outlets, domestic outlets and also from wastewater (Kim et al., 2002;Kleerebezem andMendez, 2002 andMartin et al., 2002;Cha et al 1999) have also studied the removal of organic sulphur compounds.Various workers have proposed the assignment of some species to other genera or the creation ofnew genera for most of the former species of Thiobacillus (Katayama et al., 1995;Moreira andAmils,1997;Hiraishiet al., 1998;Kelly and Wood, 2000).

Considerable interest has been shown in
Tferrooxidans because of its use in industrial mineral processing and its unusual physiology. The major contribution of T. ferrooxidans to metal extraction is its ability to attack sulphide containing minerals and convert the insoluble sulphides of metals such as copper, lead, zinc or nickel to their soluble metal sulphates.An alternative way to replace chemical methods in removing heavy metals is microbial leaching with Acidithiobacillussp. (Tyagi and Couillard, 1989;Tyagi et al., 1993;Blasis et al., 1993;Couillard and Mercier, 1993;Sreekrishnan et al., 1996). This method shows several advantages over chemical methods extraction, lower acids and alkali consumption and minimum reduction in sludge nutrients such as N and P (Tyagiet al., 1988;Couillard and Mercier, 1993).

Collection of samples
Sewage samples were collected from in and around Kongunadu College campus. Sewage discharge received from the hospitals, house, hotels, bakery and college hostel premises. Sterile bottles were taken to collect the samples from the respective places and transported to laboratory for further analysis.

Isolation and enumeration of total heterotrophic bacteria
All the sewage samples were subjected to enumerate the bacterial population in respective samples by total plate count method. The dilutions 10 -4 and 10 -5 of each sample were selected and 1 mL of suspension was transferred to Thiobacillus agar medium and incubated at 37°C for 24 h. After incubation colonies were counted and tabulated. Different colonies were selected and inoculated into Thiobacillusagar slants, and maintained it by frequent sub culturing and stored in refrigerator at 4˚C.

Presumptive and Biochemical Identification of Thiobacilli
Identification of Thiobacilluswas performed based on morphological, cultural and biochemical identification methods. The isolates were selected from Thiobacillus agar based on colony morphology with whitish blue colour.The presumptive colonies were subjected to Gram's stain;spore stain; motility test and biochemical test such as catalase test and oxidase test are followed.

Biocompatibility assay
Symbiotic relationship between the Thiobacilli were assessed by inhibiting within groups. Thiobacillus agar plates were prepared, the loop full of Thiobacillusisolates (n=5) were streaked on the agar plates. The plates were incubated at 37°C for 24 hrs. The growth and inhibitory activity of each organism were noticed.

Antagonistic activity
Thiobacilli isolates (n=5) were qualitatively tested for the production of antimicrobial compounds like bacteriocin at 37C for 24hrs. Overnight culture of indicator bacterium Aeromonashydrophila, Pseudomonas aeruginosa, Acinetobactorbaumanii, Serratiamarcescens and Bacillus subtilis) approximately 5mm10 -7 cells were swabbed over nutrient agar plate and 0.5mm0.5 mm sized wells were made using well cutter and kept it for incubation for 30 min. About 500L filter sterilized (Sartorius) bacterial supernatant (culture colonies of producer strains is 0.5 mm or larger. Inhibition zone could be around the wells, thus demonstrating bacteriocins-mediated inhibition of the sensitive microorganism.

Hemolytic activity
Haemolytic activity was determined as a zone of haemolysis around the colonies on blood agar plates containing 5 % (v/v) human blood, after 24 hrs incubation at 37 o C (Brenden and Janda, 1987).Blood agar plates were prepared and the loop full of Thiobacillusisolates [n=5] were streaked on the blood agar plates and the growth and inhibition zone was noticed in all the plates tested.

Determination of haemolysin production
Extracellular products: Brain heart infusion broth (BHIB) (15mL) was prepared and loop full of Thiobacillusisolates [n=5] were inoculated. The tubes were incubated at 37℃at 24 hrs. After 24 hrs, broth was taken and centrifuged at 12,000 rpm for 15 min. After centrifugation, collect the supernatant in fresh tubes and ammonium sulphate (5mL) was added to it and kept the content for overnight in cold room. The content was then centrifuged at 15,000rpm for 30 minutes and discards the supernatant immediately.
Phosphate buffer saline [1X concentration] (1mL) was addedto the pellet and stored at cold room.
Haemolysin assay: Thiobacilluswas subjected to haemolytic efficiency by well diffusion method. About 10 of each extracellular protein was loaded on plates and incubated at 37℃ for 24 hrs. After 24 hours incubation the zone was observed.

Total heterotrophic population in sewage samples
Collected samples were serially diluted and the total heterotrophic bacterial population were noticed on the respective dilutions. Sewage samples and Hospital sewage/discharges were labelled as TB-D1 to D5 and TB-H1 to TB-H2 respectively. The population count was maximum (68X10 -7 cfu mL -1 ) in sewage samples collected from Saibaba colony, Coimbatore. Hospital samples showed too numerous countable numbered colonies which indicates the existence of bacteria is more in hospital sewage.  biochemical tests such as catalase, hydrogen suphide production ferrous ion oxidation and nitrate respiration were studied and noticed that results were favour for Thiobacillus. The strains TB-D4 and TB-D5 were showed beta and alpha haemolysis; it might have virulent properties and cause infection in human and animals.
In a study, Wong et al. (1996) reported bacterial haemolysin -positive genotype was Nitrate respiration Negative

Biocompatibility of the thiobacillus
The results of biocompatible assay showed the significant level of symbiotic relationship with in the group of sewage isolates by showing the noninhibitory growth on Thiobacillus agar after 24 and 48 hrs growth. It exposes the biocompatibility of the isolates and provides an evidence for supporting the growth of each other.

Antagonistic Activity
Influence of environmental factor on the production of extracellular products that could account for the differences found in virulence for trout and mile of the strains studied, a comparative study was made of the enzymatic and toxic activities contained in culture supernatant fluids growth at 28 o C and 37 o C after incubation for 24 to 48 hrs (Mateoset al., 1993).
Bacteriocin as extracellular metabolites, which has a potential inhibitory activity against pathogenic microorganisms. In the present study, we qualitatively measured the efficiency of bacteriolytic activity of sulphur oxidizing bacteria, Thiobacillus, which produces bacteriocin, were used to analyse bacteriolytic activity by the method of antagonistic activity with pathogenic microorganisms (Aeramonashydrophila, Pseudomonas aeruginosa, virulent in the suckling mouse model assay. They also observed that after 24 hours at 37 C, the production of hemolysin was found high, whereas Wretlindet al. (1973) and Riddle et al., (1981) observed the haemolytic activity during the exponential growth phase, reaching a maximum before maximal growth, and then falling on prolonged incubation.

Hemolytic activity in extracellular protein
Allen and Stevenson (1981) reported that haemolytic activity appeared extracellularly during the early stages of growth, reading a peak just before an increase in the haemolytic activity. The cell free culture supernatant was showed significant level of haemolytic activity on blood agar. The extracts were showed inhibition zone around the well with clear inhibition zone and it was revealed the RBC lytic performing Gram's staining; motility assay and