In vitro Assessment of Chromium, Lead, Cadmium and Nickel Tolerance of B. clausii, a Prospective Probiotic Microorganism for in vivo Bioremediation

Researches have demonstrated the ability of probiotic microorganisms to prevent and treat ailments especially those associated with the gastrointestinal tract. Probiotics like Lactobacillus spp. have also displayed the property of bioremediation of heavy metals under in vitro as well as in vivo conditions. The aim of this study was to assess the effects of chromium, lead, cadmium and nickel stress on the properties of B. clausii, a probiotic species of genus Bacillus. The minimum inhibitory concentration (MIC) of the organism under test was determined for Cr (VI), Pb (II), Cd (II) and Ni (II), followed by assessment of morphological and biochemical properties of the B. clausii, antibiotic sensitivity and probiotic efficacy by acid and bile tolerance assays. B. clausii exhibited exceptionally high MICs for the tested heavy metals. The organism did not exhibit any change in its morphological and biochemical characteristics after exposure to heavy metal stress. This stress also did not affect the test organism’s probiotic behaviour as assessed by acid and bile tolerance assays where it showed maximum growth at 3 hrs. for pH 3 and 0.3% bile concentration, respectively. However, after exposure to the four heavy metals, B. clausii showed a tremendous increase in its antibiotic sensitivity. The above study has indicated the capacity for B. clausii to survive in and tolerate high levels of Cr (VI), Pb (II), Cd (II) and Ni (II) while showing no change in its characteristics. Therefore, B. clausii appears to be an ideal candidate for potential bioremediation of Cr (VI), Pb (II) Cd (II) with Pb (II), Cd (II) and Ni (II), in vivo.

I n 2 0 0 1 , F o o d a n d A g r i c u l t u r e Organization of the United Nations/World Health Organization (FAO/WHO) defined probiotics as "live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host" 1 . However, probiotic food consumption has been a part of different cultures around the world for many centuries. Probiotic organisms generally belong to LAB (lactic acid producing bacteria) and include Lactobacillus, Lactococcus, Bifidobacterium and many other organisms 2 . Microorganisms of various other genera such as Bacillus, not belonging to LABs, have been identified and declared as probiotics as they exhibited probiotic characteristics 3 . In recent times, researchers have focussed on benefits of probiotics and many have established their health and nutritional benefits in humans. Beneficial effects of probiotics include improvement of gut health, reduction in symptoms of Irritable Bowel Syndrome (IBS) 4 , antibiotic associated diarrhoea 5,6 , infectious diarrhoea caused by rotavirus 7-9 , traveller's diarrhoea 10 and necrotizing enterocolitis 11 .
Many studies have been performed on bioremediation of heavy metals by microorganisms [12][13][14] and have reported an interesting observations that probiotics such as Lactobacillus spp. and Bacillus spp. also have the ability to bioremediate heavy metals [15][16][17][18][19] . This has been attributed to the structural components of their cell wall such as teichoic acid, lipoteichoic acid and peptidoglycan 20 .
Heavy metals such as lead, chromium, nickel, cadmium, mercury and arsenic reach human gut through food and water laden with them. These heavy metals enter the food chain as a result of anthropogenic activities and cause varying levels of toxicity in humans 21 . Hence, these microorganisms are the best and most effective agents to reduce toxicity caused by heavy metals since they are safe in terms of human usage, are ingested via diet, show gut remediation and can eliminate the heavy metals by defaecation 22 .
In this study, B. clausii was evaluated for tolerance and survival capacity in presence of Cr (VI), Pb (II), Cd (II) and Ni (II) and the effect of exposure of these heavy metals on it.

Methods Minimum inhibitory Concentration (MiC)
MIC of Cr (VI), Pb (II), Cd (II) and Ni (II) for B. clausii was assessed by method of Mistry et al 23 with some modifications. Spores were germinated in nutrient broth (NB) and inoculated on nutrient agar (NA) plates supplemented with two-fold concentration of respective heavy metals ranging from 1-512 ppm. These plates were incubated at 37°C for 24 hrs. The lowest concentration of the respective heavy metal which inhibited the growth of probiotic was considered as the MIC of that heavy metal for B. clausii.

Characterization of heavy Metal resistant B. clausii
Various characterization parameters of B. clausii were evaluated before and after 24 hrs. exposure to Cr (VI), Pb (II), Cd (II) and Ni (II). This was done to assess whether the respective heavy metal exposure affects characteristics of the test organism.

Morphological Characterization
Observations on cell size, shape, colony characteristics and staining properties were performed on B. clausii before and after exposure to test heavy metals.

Biochemical Characterization Catalase test
Unexposed and exposed cultures of B. clausii for Cr (VI), Pb (II), Cd (II) and Ni (II) were cultured on NA slants. Few drops of H 2 O 2 were added to the slant after 24 hrs. and observed for immediate bubbling 24 .

Methyl red and Voges Proskauer (MrVP) test
MRVP test was performed to determine glucose fermentation products. MR test was done according to methodology of Clark and Lub 25 while for VP test method of Voges and Proskauer 26 modified by Barritt 27 was followed. This was done on unexposed and exposed B. clausii to Cr (VI), Pb (II), Cd (II) and Ni (II) respectively, in sets of glucose phosphate broth tubes. The tubes were inoculated and incubated for 72-96 hrs. Methyl red was added to one tube of the set to check development of acid. To the other tube, 12 drops of reagent VP-I was added followed by 2-3 drops of reagent VP-II. The tubes were exposed to air and were shaken intermittently. One tube remained uninoculated and acted as control.

sugar Fermentation test
Sugar fermentation test was performed according to procedure of Cappuccino and Sherman 28 . A set of sugar fermentation tubes were prepared with different sugars (lactose, sucrose, glucose, sorbitol and mannitol). Phenol red was added to the broth as pH indicator to check for acid production and Durham's tube were placed in the broth to check for gas production. The broth was inoculated with unexposed and exposed B. clausii to each heavy metal respectively. An uninoculated tube was kept as control. antibiotic sensitivity test To analyse whether Cr (VI), Pb (II), Cd (II) and Ni (II) exposure caused any change in antibiotic sensitivity of B. clausii, Kirby-Bauer disc diffusion method was performed using antibiotic discs 29 . Zone of inhibition was measured after 24 hrs. for the unexposed and exposed cultures of B. clausii.

Probiotic Characterization
Efficacy of B. clausii as a probiotic was studied by acid and bile tolerance assays after exposure to heavy metals under study. For both assays, methodology of Hassanzadazar et al 30 was followed with some modifications.
For acid tolerance assay, B. clausii exposed to Cr (VI), Pb (II), Cd (II) and Ni (II) respectively, was added to different broths of pH 2, 3 and 4. Growth of exposed B. clausii was monitored by viable cell count that was performed by pour plate technique for every hour for 3 hrs. Prior to plating, ten-fold serial dilution of the inoculum was prepared in 0.1% peptone water. All the experiments were done in triplicates. Cell counting was done after 24 hrs. of incubation at 37°C. Glucose fermentation Acid formation and gas production Acid formation and gas production 6.
Sucrose fermentation Acid formation and gas production Acid formation and gas production 7.
Lactose fermentation No acid and gas production No acid and gas production 8.
Mannitol fermentation Acid formation and gas production Acid formation and gas production 9.
Sorbitol fermentation Acid formation and gas production Acid formation and gas production 10. Methyl red test Positive Positive 11.
Voges-Proskauer test Negative Negative For bile tolerance assay, NB with bile salt concentration of 0.2, 0.3 and 0.4% were prepared. B. clausii after 24 hrs. exposure to Cr (VI), Pb (II), Cd (II) and Ni (II) was added to the respective broths. Viable cell count was performed by pour plate technique. The inoculum was prepared hourly for 3 hrs. by ten-fold serial dilution in 0.1% peptone water. Colony count was done after 24 hrs. incubation at 37°C.

Minimum inhibitory Concentration (MiC)
In presence of nickel and chromium, B. clausii showed growth till 128 ppm and hence MIC was obtained as 256 ppm. In case of cadmium exposure, MIC was obtained at 64 ppm. However, after lead exposure, B. clausii showed good growth till 512 ppm and thus no MIC was recorded.
Earlier, environmental species of genus Bacillus have shown bioremediation of Cr (VI) 31 , Pb (II) 32 , Cd (II) 33 and Ni (II) 34 . The genetic determinants for heavy metal tolerance might be borne either on plasmids, transposons or genomic DNA 35,36 . In fact, these determinants are responsible for increased tolerance of microorganisms for heavy metals and are reported more in heavy metal polluted sites rather than in unpolluted sites 37 .
The results of the assay for MIC indicate combined resistance of B. clausii and high MIC values for the heavy metals tested. The earlier studies conducted by Silver 38 and Alam and Imran 39  According to WHO, the guideline values for Cr (VI), Pb (II), Cd (II) and Ni (II) in drinking water are 0.05 mg/L, 0.01 mg/L, 0.003mg/L and 0.07mg/L, respectively 42 . B. clausii has shown MIC values much above the permissible values of these heavy metals in drinking water, indicating that it has the potential for bioremediation of the above mentioned heavy metals.

M o r p h o l o g i c a l a n d B i o c h e m i c a l Characterization
Vegetative cells of B. clausii exposed to heavy metal stress did not exhibit any change in their various morphological characteristics like cell shape, cell size, colony characters etc. Similar observations were made when heavy metal exposure did not affect the biochemical properties test organism. These results indicate that 24 hrs. exposure of B. clausii to Cr (VI), Pb (II), Cd (II) and Ni (II) could not modify or transform the organism's basic characteristics as depicted in Table 1.

antibiotic sensitivity test
An organism can be either tolerant or resistant to antibiotics. For an organism to be resistant, it must grow in constant presence of low concentration of antibiotic. If an organism is categorized as tolerant, it means that it can survive for short duration of high concentration of antibiotic 43 . This helps to determine the successful clinical use of antibiotics. In this study, upon exposure to heavy metal stress, a marked increase was observed in antibiotic sensitivity of B. clausii for the antibiotics used (Fig 1). Against amoxicillin, Pb (II) exposed B. clausii showed maximum change (from 9 mm to 33 mm) (Fig 2,  Fig 4) while minimum change was recorded for Ni (II) (Fig 2, Fig 5) exposed and Cd (II) exposed culture (from 9 mm to 17 mm) (Fig 2, Fig 6b). Lead exposed culture recorded maximum (from 15 mm to 34 mm) (Fig 2, Fig 4) and cadmium exposed culture recorded minimum (from 15 mm to 21 mm) change against ciprofloxacin (Fig 2, Fig 6a). After exposure to lead, B. clausii showed doubling of antibiotic sensitivity (from 11 mm to 22 mm) against norfloxacin (Fig 2, Fig 4) while minimum was for chromium exposed culture (from 11 mm to 17 mm) (Fig 2, Fig 3). The zone of inhibition increased from 8 mm to 28 mm after lead exposure which was maximum for ampicillin (Fig 2, Fig  4) while minimum increase was for Cr (VI) (Fig  2, Fig 3) and Cd (II) (Fig 2, Fig 6c) exposed B. clausii (from 8 mm to 17 mm respectively). Pb (II) exposed test organism recorded maximum change against kanamycin (13 mm to 31 mm) (Fig 2, Fig 4) while Cd (II) exposed B. clausii showed negligible change (13 mm to 15 mm) (Fig 2, Fig 6b). For chloramphenicol, after exposure to nickel, B. clausii showed a tremendous increase in the zone of inhibition from 7 mm to 32 mm (Fig 2, Fig 5) while negligible change was found in Cd (II) exposed test probiotic (7 mm to 10 mm) (Fig 2, Fig 6a). This behaviour may be related to presence of proteins involved in chloramphenicol and kanamycin resistance have also been identified in proteome of B. clausii 44 . Thus, lead exposure caused maximum change in antibiotic sensitivity while cadmium exposure led to minimum change. According to above definition, B. clausii can be considered as resistant to the antibiotics tested against and can therefore be used to replenish the microflora of the gut after antibiotic treatment, even after heavy metal exposure. The results of above assay indicate that the antibiotic resistance of B. clausii decreases upon exposure to Cr (VI), Pb (II), Cd (II) and Ni (II). Similar results obtained by Tuckfield  From the above results, it can also been concluded that B. clausii has potential to resist and tolerate both heavy metals and antibiotics. In bacteria, antibiotic resistance and heavy metal tolerance are genetically linked through R-plasmid [49][50][51] . According to the study of Alam and Imran 39 selective pressure of the heavy metals causes co-selection of antibiotic resistance, albeit indirectly.

Probiotic Characterization
As B. clausii is a probiotic microorganism, it can survive low pH conditions of stomach as well as in presence of bile salts in the intestinal tract.
To determine the effect of heavy metal stress on its probiotic efficacy of B. clausii, acid and bile tolerance assays were performed. acid tolerance assay When Cr (VI) exposed B. clausii was inoculated in pH 2, 3 and 4 respectively and growth was monitored for 3 consecutive hours, it was observed that maximum CFU (colony forming units) were obtained at pH 3 after 3 hrs. (Fig. 7). Similar result was obtained for Pb (II), Cd (II) and Ni (II) exposed B. clausii (Fig. 8-10). This is in corroboration with a study conducted by Sahadeva et al 52 which says that for an organism to be acid tolerant it must show maximum growth at pH 3 after 3 hrs.
Mechanisms of acid tolerance are more prominent in Gram positive bacteria like Bacillus spp. as they attain higher cell densities which enhance biofilm formation and communication amongst cells 53 . These mechanisms are (i) change in types of fatty acids of cell membrane 54 (ii) exchange of proton for an amino acid in presence of amino acid carboxylase 55,56 (iii) conserving structures of macromolecules like proteins with the help of chaperones 54,57,58 and (iv) counteracting the low pH with production of alkaline molecules like ammonia 59 . Since the test organism belongs to genus Bacillus, it probably suggests use one or more of these mechanisms. Interestingly, despite high heavy metal stress, acid tolerance of B. clausii was not affected as concluded by the results of this assay.

Bile tolerance assay
For a probiotic to be categorized as bile tolerant, it must have maximal growth at 0.3% bile salt concentration after 3 hrs. 60,61 When B. clausii exposed to heavy metals under study was inoculated in bile salt concentrations of 0.2%, 0.3% and 0.4%, highest CFUs were observed at 0.3% bile after 3 hrs. Hence, subjection of B. clausii to heavy metal stress did not change its bile tolerance property ( Fig. 11-14).
In contrast to defined mechanisms of acid tolerance, the methods by which bacterial cell counters bile stress is not well defined. One of the mechanism is with the use of enzyme bile salt hydrolase 62 . Apart from this, complex gene expression is also known to be responsible for bile tolerant property of bacteria [63][64][65][66] .
It may be noted that even under heavy metal stress, B. clausii is able to retain its probiotic efficacy by probable use of different mechanisms and can be applicable for bioremediation of heavy metal, in vivo.

ConClusion
Anthropogenic and natural activities are the causes of Cr (VI), Pb (II), Cd (II) and Ni (II) contamination of soil and water and air. These are toxic heavy metals which reach humans through the food chain and cause detrimental effects on their health. Chromium and lead are systemic toxicants while nickel and cadmium are carcinogens. When the effects of these heavy metals were tested on a probiotic microorganism, B. clausii, no change was observed in its morphological and biochemical properties and its probiotic efficacy. Hence, it can be concluded that B. clausii, a commercial probiotic has the potential for in vivo bioremediation of Cr (VI), Pb (II), Cd (II) and Ni (II) and can therefore benefit the society at large by reducing the toxicity of these heavy metals in the human gut.

aCknowledgeMents
The authors wish to thank Ar. Achal Choudhary, President and Dr. Sanjay Nagar, Head, Department of Biotechnology, IPS Academy, Indore for their valuable assistance regarding infrastructural and laboratory facilities.