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Accumulation of heavy metals by living and dead bacteria as biosorbents: isolated from waste soil.

Introduction

In developing countries like Pakistan, the risk of heavy metal exposure to the environment is increasing day by day. Such heavy metals can cause impact on human health and are toxic to animals too (Tokar et al., 2011; Jomova and Valko, 2011). A considerable amount of heavy metals is present in waste water coming from different sources, i.e., electroplating, paint, leather, metal and tanning industries. Heavy metals are removed from the waste water through the process of biosorption and bioremediation using microorganisms and it has been proved as a very cost effective and environmental friendly process (Elekwachi et al., 2014; Joshi et al., 2011). Biosorption is a process, in which there maybe some chemical relationships between the metals and microbes used (Shumate and Stranberg, 1985). Significance of biosorption process over conventionally used method is that it is cost effective, very efficient and shows decreased chemical and biological waste products. Recovery of biomass used and possibility of metal recovery is also possible by biosorption (Kratochvil and Volesky, 1998). Biosorbents are a large subclass of low-cost adsorbents that can be subdivided into biomass (dead or living), agricultural wastes, and industrial solid wastes (Bhatnagar and Minocha, 2006). Dead biomass has been utilised by many researchers as a functional biosorbent to remove different pollutants (Li et al., 2010; Saraswat and Rai, 2010). Dead biomass is more readily desorbed than its living counterpart. Living biomasses including fungi (Kumar, 2014; Ismael et al., 2004; Fu and Viraghavan, 2002; 2000), algae (Navarro et al., 2012; Bishnoi and Pant, 2004), actinomycetes (Solecka et al., 2012; Fu and Virarghavan, 2002; 2000) and other microbial cultures with different strains (Kocberber and Donmez, 2007) were also used as low-cost biosorbents. Bacterial species like Bacillus has been identified as having a high potential of metal absorption and so it has been commercially used in biosorbent material preparation (Ghaima et al., 2013; Singh et al., 2012; Brierley, 1990).

Cadmium and chromium have highly deleterious effects on plants, animals as well as on human life. Cadmium is hazardous and highly toxic metal for environment and to human beings. This biologically non-essential element accumulates in body, especially in kidneys, liver, lung and brain. It can induce several toxic effects, depending on the concentration and exposure time. Cadmium has been linked to Alzheimer's disease (AD) as a probable risk factor (Meleleo et al., 2010). Cadmium is dangerous to virtually all vital systems in the animal body. On the other hand extreme usage of chromium supplements can cause stomach problems and hypoglycemia. Too much chromium uptake can also cause various injuries like liver, kidneys, and nerve problems, and it also causes irregular heart rhythm. Major sources of cadmium and chromium include refined foods, water containing foods, pipes of water, vegetables and fruits, coffee, tea, burning coal and cigarettes (Costa, 2003).

Understanding the problems occurring due to heavy metals exposure and their degradation by biological means is necessary. As according to the above discussion and literature review, it is an urgent need of time to explore different microbial strains as biosorbent to remove heavy metals from different sources. Therefore, this study was planned to isolate and identify the bacteria from waste sources which could be used to purify the contaminated water.

Materials and Methods

Sample collection. The contaminated soil samples viz., slaughter house waste, dairy waste, and household were collected in sterilised containers from different localities (Lower Chellah and Lower Plate: near workstation of automobiles) of Muzaffarabad, Azad Jammu and Kashmir, Pakistan. All samples were collected in triplicate.

Isolation and culturing of bacteria. Contaminated soil samples for isolation of bacterial strains were spread on nutrient agar and MacConkey agar media plates (NAM; Oxoid [CMOO.sub.3], MAM; Oxoid CM115), incubated at 37[degrees]C in incubator up to colony appearance. After 24 h incubation, single colony was picked up, streaked into nutrient broth medium (NBM; Oxoid CMI), placed in shaking incubator at 37[degrees]C for 24 h. Next day, the mixed cultures were again purified by quadrant streaking on nutrient agar and MacConkey agar plates. The pure culture was grown in nutrient broth medium for overnight at 37[degrees]C. Next day, bacteria were preserved in 60% glycerol and then placed at -20 [degrees]C for further processing.

Identification of bacteria. Bacterial species were identified on the basis of colony morphology: Gram's staining and using biochemical tests (Cheesbrough, 2002; Collins et al., 1999; 1989). Further clarification and identification of all isolated bacteria were carried out at Department of Zoology, Government College University, Lahore, Pakistan.

Preparation of heavy metal concentrations. Chromium (Cr) and cadmium (Cd) heavy metals were used for biosorption assay. For cadmium Cd(II) and chromium Cr(IV) solutions Cd[Cl.sub.2] and [K.sub.2][Cr.sub.2][O.sub.7] salts were used. For each metal, a range of 50, 100, 150, 200, 250 and 300 [micro]g/mL were obtained. The metal solutions were prepared in sterilised distilled water so as to prevent any chances of contamination.

Resistogram analysis. Heavy metal resistance study for microbial isolates was performed using agar well diffusion method (Jagessar et al., 2008) with increasing concentrations of Cr and Cd ranging from 50 to 300. Nutrient agar (Oxide: [CMOO.sub.3]) and nutrient broth media (Oxide: CM1) were used for bacterial culture. The pH of media was adjusted to a final pH 7 by using sodium hydroxide. The microorganisms were activated by inoculating a loop full of strain in 25 mL of nutrient broth medium and incubated at 37[degrees]C on a rotatory shaker for 24 h. The overnight culture was mixed with freshly prepared nutrient agar medium (NAM) at 45[degrees]C and was poured into the sterilised petri dishes. All petri dishes were kept at room temperature in laminar flow for solidification. In each plate, 3 wells of 5 mm diameter were made using a 1 mL of sterilised micropipette tip and sterilised needle was used for the removal of agar plug. Approximately 30 [micro]L of each heavy metal concentration were placed in each prepared well and placed at 37[degrees]C for 24-48 h. Microbial growth was determined by measuring the diameter of zone of inhibition after 24 h (Seeley et al., 2001). Diameter of the clear zones (if greater than 1 mm) around each well was measured with the help of scale (Hammer et al., 1999). The results of the sensitivity test were expressed as (0) for no sensitivity, * (1-5 mm) for low sensitivity, ** (>5-10 mm) for moderate sensitivity and *** (>10-25 mm) for high sensitivity. The bacterial species having maximum resistance to these metals were selected for further study.

Antibiogram analysis. Sensitivity of antibiotics against test microbial strains was assessed through agar disc diffusion method (Prescott et al., 1999). This method is called antibiogram assay. Sensitivity was predicted with degree of clear zone of microbial growth inhibition surrounding the disc. The potency of standard antibiotics has been indicated as Ciprofloxacin (CIP; 5 [micro]g), Ampicillin (AMP; 10 [micro]g), Penicillin G (P; 10 [micro]g), Neomycin (N; 10 [micro]g), Vancomycin (VA; 30 [micro]g), Erythromycin (E; 15 [micro]g), Norfloxacin (NOR; 10 [micro]g), Tetracycline (TE; 30 [micro]g), Gentamicin (CN; 10 [micro]g), Oxytetracycline (OT; 30 [micro]g), Streptomycin (S; 10 [micro]g), Chloramphenicol (C; 30 [micro]g), Tobramycin (TOB; 10 [micro]g), Nalidixic acid (NA; 30 [micro]g), Sulfamethoxazole (SXT; 25 [micro]g) and Kanamycin (K; 30 [micro]g). All discs of antibiotics were purchased and made by Oxoid Company.

Bioaccumulation/biosorption study. In this study, each isolate was cultivated anaerobically in properly labelled test tubes containing nutrient broth medium by incubating the test tubes at 37[degrees]C in shaking incubator. Five mL of bacterial suspension (E. luteus, S. aureus and E. coli) having dried biomass (4, 11 and 27 mg) was mixed with 1 mL metal solution in sterilised test tubes which were then covered with aluminum foil and agitated at 150 rpm on a shaking incubator at 37[degrees]C. Biosorption was assayed by exposing the isolates to two different metals i.e., Cr(VI) and Cd(II) concentrations. The effects of different parameters like metal ion concentration, pH and incubation time on the adsorption capacity of each isolate were studied. After incubation, the samples were then centrifuged for 5 min at 13000 rpm and supernatant was used for the estimation of metal ion concentration using double beam spectrophotometer (Sshimadzu UV 1800). A control was also set containing nutrient broth medium along with metal solution keeping all other conditions same except bacterial culture. Each test was performed in triplicates and their average value was taken as result.

The following parameters on adsorption capacity of isolates were studied:

1. The effect of metal ion concentration was studied using metal concentration ranging from 50-300 [micro]g/mL for each metal ion.

2. The effect of incubation time of bacterial culture with metal solution was also studied. The incubation time given to the solution was 24 h, 48, 72 and 96 h.

3. The effect of pH 4, 6, 7, 8 and 10 was studied and pH was adjusted by using analytical grade sodium hydroxide and hydrochloric acid.

Cr(VI) was analysed by diphenylcarbazide method, and Cd(II) by dithizone method. The metal removal efficiency of each strain was calculated by equation 1.

R. % = ([C.sub.i]-[C.sub.e])/[C.sub.i] x 100. (1)

Where:

R = the removal efficiency (%), [C.sub.i] = the initial metal concentration before removal ([micro]g/mL) and [C.sub.e] = the final metal concentration after removal ([micro]g/mL) while, the amount of metal adsorption on the bacterial biomass can be calculated by equation 2.

[q.sub.eq] = ([C.sub.o]-[C.sub.eq])V/M. (2)

Where:

[q.sub.eq] (mg/g) = the metal adsorption capacity, [C.sub.o] (mg/L) = initial metal ion concentration, and [C.sub.eq] = final metal ion concentration, respectively. V = the solution volume and M (g) = the amount of biosorbent used.

Determination of metal concentration in the supernatant. Atomic absorption spectrophotometer, Perkin Elmer Analyst 300 was used to determine the heavy metal concentration (chromium and cadmium). The wavelength 544 nm and 545 nm were used for cadmium and chromium. It was done by using its specific lamp for each metal and at a specific wavelength.

Statistical analysis. Each experiment of resistogram and antibiogram analysis was repeated in triplicates and standard deviation from absolute data was calculated (http://easycalculation.com/statistics/standarddeviation.php).

Results and Discussion

Isolation and identification of bacteria. Three bacterial isolates such as Enterococcus luteus, Staphylococcus aureus and Escherichia coli were isolated from the contaminated soil samples. These strains were identified through gram's staining and biochemical tests (Table 1), while citrate, coagulase, oxidase, indole and nitrate tests were negative. S. aureus is gram positive cocci and motile. S. aureus showed catalase, coagulase, citrate, urease, methyl red, nitrate tests positive while oxidase, Voges proskeur tests were negative. Catalase, indole, methyl red, and nitrate tests were positive for E. coli while citrate, coagulase, oxidase and urease tests were recorded as negative results. Glucose and lactose fermentation tests were positive for E. coli and S. aureus whereas, E. luteus indicated non-fermenter of carbohydrates.

Resistogram analysis. For chromium metal concentrations. In resistogram analysis E. luteus showed resistance against Cr(IV) metal at 50 [micro]g/mL and 100 [micro]g/mL, but seemed to be sensitive against higher concentrations i.e., 150, 200, 250 and 300 [micro]g/mL. Zone of inhibition was measured as 14.0 [+ or -] 0.0 mm against 150 [micro]g/mL, 15.0 [+ or -] 0.0 mm against 200 [micro]g/mL, 15.0 [+ or -] 0.0 mm against 250 [micro]g/mL and 16.0 [+ or -] 0.0 mm against 300 [micro]g/mL. S. aureus showed resistance against all concentrations of Cr(IV) metal whereas, E. coli showed high sensitivity against all concentrations of Cr(IV) metal. Zone of inhibition measured against each concentration was recorded as; 12.0 [+ or -] 0.0 mm against 50 [micro]g/mL, 15.0 [+ or -] 0.0 mm against 100 [micro]g/mL, 19.0 [+ or -] 0.0 mm against 150 [micro]g/mL, 20.0 [+ or -] 0.0 mm against 200 [micro]g/mL, 20.0 [+ or -] 0.0 mm against 250 [micro]g/mL and 24.0 [+ or -] 0.0 mm against 300 [micro]g/mL. In each case it was observed that sensitivity increased with increase in metal concentrations (Table 2).

For Cd(II) metal concentrations. E. luteus showed resistance against four Cd(II) concentrations such as 50, 100, 250, and 300 [micro]g/mL. While against other two concentrations sensitivity was observed and the zone of inhibition was measured as 15.0 [+ or -] 0.0 mm against 150 [micro]g/mL and 12.0 [+ or -] 0.0 mm against 200 [micro]g/mL. S. aureus and E. coli seemed to be fully resistant against all concentrations of Cd(II) (Table 3).

Antibiogram analysis. In case of antibiogram analysis E. luteus showed sensitivity against all antibiotics. The maximum zones of inhibition were measured as 28.0 [+ or -] 0.0 mm against CIP, 13.0 [+ or -] 0.0 mm against N, 17.0 [+ or -] 0.0 mm against VA, 21.0 [+ or -] 0.0 mm against E, 19.0 [+ or -] 0.0 mm against NOR, 14.0 [+ or -] 0.0 mm against CN, 13.0 [+ or -] 0.0 mm against S, 17.0 [+ or -] 0.0 against NA, 18.0 [+ or -] 0.0 mm against C, and 12.0 [+ or -] 0.0 mm against OT (Table 4). S. aureus also seemed to be sensitive against most of the antibiotics. It showed resistance against P, AMP, and SXT. Sensitivity was recorded as 20.0 [+ or -] 0.0 mm against CIP, 11.0 [+ or -] 0.0 mm against VA, 17.0 [+ or -] 0.0 mm against E, 22.0 [+ or -] 0.0 mm against NOR, 20.0 [+ or -] 0.0 mm against CS, 14.0 [+ or -] 0.0 mm against S, 13.0 [+ or -] 0.0 mm against NA, and 11.0 [+ or -] 0.0 mm against C (Table 4). E. coli showed resistance against P, TET, AMP, OT and SXT while, sensitivity was observed as 19.0 [+ or -] 0.0 mm against CIP, 14.0 [+ or -] 0.0 mm against N, 23.0 [+ or -] 0.0 mm against E, 23.0 [+ or -] 0.0 mm against NOR, 17.0 [+ or -] 0.0 mm against CN, 19.0 [+ or -] 0.0 mm against S, and 11.0 [+ or -] 0.0 mm against NA (Table 4).

Biosorption of Cd(II) and Cr(IV) at various pH and incubation periods. Metal absorbance capacity of microbes is expressed in graphical way. The lines in graph show the amount of metal left in medium after microbial activity. Higher lines show much amount of metal left in medium and lower lines show less amount left while, no lines show zero % metal left behind, meaning that all the metals have been absorbed by the microbes. Different colours of lines are shown to express different concentrations of metals like dark blue colour used for 50 [micro]g/mL, red for 100 [micro]g/mL, green for 150 [micro]g/mL, purple for 200 [micro]g/mL, sky blue for 250 [micro]g/mL and for 300 [micro]g/mL orange.

Cd(II) bioaccumulation by E. luteus. With the decrease in pH up to acidic pH 4, E. luteus showed 100% absorbance of Cd(II) on all incubatory periods; 24, 48, 72, and 96 h (Fig. 1). Metal concentration seemed to had no effect on absorbance. At the pH 6, E. luteus showed decreased absorbance of Cd(II) after 24 h of incubation but after 48, 72, and 96 h of incubation, E. luteus showed 100% absorbance capacity. Even at each concentration, absorbance capacity remained constant (Fig. 1). At pH 7, Cd(II) absorbance activity was little decreased after 24 h of incubation at 37[degrees]C but with increase in incubatory periods i.e., after 48, 72 and 96 h the absorbance capacity of microbe decreased gradually (Fig. 1). Side by side the increase in metal concentration seemed to have negative impact on absorbance, as the absorbance decreased with increase in metal concentration. At pH 8 and 10, E. luteus showed absorbance which increased gradually with increase in incubation period . Maximum absorbance at pH 10 was observed after 96 h of incubation (Fig. 1).

Cd(II) bioaccumulation by E. coli. At acidic pH 4 and 6, microbe E. coli showed very good absorbance activity. The metal was absorbed 100% at each concentration level and incubatory period. Metal concentration even did not affect the absorbance capacity of E. coli (Fig. 1). At pH 7 after 24 and 48 h, E. coli showed maximum absorbance, and even 100% at 300 [micro]g/mL. While this activity decreased after 72 h and same results were observed after 96 h as well (Fig. 1). At pH 8, E. coli showed good absorbance activity after 72 and 96 h of incubation except 300 [micro]g/mL and with increase in incubation this activity also increased. E. coli showed absorbance of Cd(II) at pH 10, which increased with increase in incubatory periods (Fig. 1).

Cd(II) bioaccumulation by S. aureus. Staphylococcus aureus showed good and complete absorbance activity after 24 h, at pH 6, 7 and 8. While increase in concentration had no effect on absorbance capacity of microbes (Fig. 1). S. aureus showed average results for absorbance of Cd(II) at pH 4, but the absorbance capacity increased with increase in incubatory period. Basic pH 10 and acidic pH 4 seemed to be very ineffective for the S. aureus to absorb Cd(II) metal (Fig. 1).

Cr(IV) bioaccumulation by E. luteus. At pH 4 E. luteus showed almost 100% absorbance capacity for Cr(IV) for all metal concentrations after 24 h of incubation but the absorbance ability was decreased gradually along with increase in incubation after each 24 h and finally the capacity decreased much after 96 h (Fig. 2). At pH 6, E. luteus showed gradual decrease in absorbance capacity for Cr(IV) metal concentrations with increase in incubatory period i.e., after each 24 h of incubation. At pH 7, metal concentration in media showed a little decrease after 48 h but after 72 h E. luteus showed high absorbance capacity showing decrease in metal concentration but after 96 h capacity again decreased. At basic pH 8, E. luteus showed good absorbance capacity after each 24 h of incubation. At high basic pH 10 microbe showed decrease in absorbance capacity after each 24 h of incubation. After 24 h E. luteus absorbed 100% of each metal concentration except 50 and 200 [micro]g/mL.

Cr(IV) bioaccumulation by E coli. Escherichia coli absorbance activity for Cr(IV) metal concentration showed good activity after 24 h of incubation at pH 4. At acidic pH 6, E. coli showed near to 100% absorbance at 24 h incubation but after that with increase in incubation the absorbance capacity of microbe decreased gradually. At pH 7, E. coli showed good absorbance even after 24 h of incubation and this capacity increased with increase in incubatory period. After 96 h of incubation the capacity remained 100% while at others like 48 and 72 h the capacity remained close to 100%. Another effect seen was that the absorbance capacity was decreased with increase in concentration of metal i.e., from 50 to 250 [micro]g/mL. At highest concentration i.e., 300 [micro]g/mL the microbe showed maximum absorbance that remained close to 100% at last two incubatory periods (Fig. 2). At pH 8, microbe showed good absorbance capacity after 24 h of incubation but this capacity was decreased gradually with increase in incubation. At high basic pH 10, E. coli showed 100% absorbance after 24 h of incubation. But after further incubation, the capacity of metal absorbance decreased gradually.

Cr(IV) bioaccumulation by S. aureus. Staphylococcus aureus at acidic pH 4 showed best absorbance capacity after 48 h of incubation (Fig. 2). After some incubations metal concentration showed negative impact on absorbance e.g., after 24, 72 and 96 h of incubation. At neutral pH S. aureus showed maximum capacity to absorb Cr(IV) metal up to 72 of incubations but decline absorbance was recorded after 96 h. Metal concentration had no clear effect on absorbance. At pH 6, S. aureus showed good absorbance activity after 24 h of incubation. S. aureus showed near to 100% absorbance capacity for the concentrations from 50 to 200 [micro]g/mL but for 250 [micro]g/mL and 300 [micro]g/mL the capacity was decreased. After further incubation of 48, 72 and 96 h, the capacity of metal absorbance of S. aureus decreased gradually (Fig. 2). At basic pH 8 after 24 h incubation the estimated capacity of absorbance was good but maximum absorbance capacity was seen after 48 h incubation. The maximum capacity of absorbance of S. aureus at basic pH 10 was seen after 24 h of incubation. After that the capacity decreased with increase in incubation i.e., 48, 72 and 96 h of incubation (Fig. 2). Increase in metal concentration gradually decreased the absorbance of metal.

When metals are dissolved in huge volumes at relatively low concentrations, metal removing technologies such as chemical precipitation, ion exchange, evaporation floatation and filtration become generally ineffective (e.g., less than 100 mg/L) (Patterson, 1985). The research is for efficient and particularly cost-effective remedies (Blocher et al., 2003; Volesky, 2001). Results of this study demonstrated that the biomass concentration strongly affected the amount of metal removed from aqueous solution. Moreover, as the biomass concentration rises, the maximum biosorption capacity drops, indicating poorer biomass utilisation. It shows that the sorption of metals is more correctly described by more than one model. In recent research, the same effect was observed in most of the results of metal absorbance, that the increase in metal concentration caused decrease in absorbance of metal, such as Cd(II) absorbance by E. luteus at pH 7, 8, 10, by E. coli at pH 8, and by S. aureus at pH 4, 8, 10 and for Cr(IV), S. aureus at pH 4, 8, 10, E. coli at pH 4, 6, 10, and E. luteus at pH 4, 6, and 10. In general, E. luteus seemed to be effective biosorbent at acidic pH 6 for Cr(IV) metal, while at basic pH 10, showed minimum absorbance. E. coli also absorbed significant quantity of Cr(IV) metal in acidic pH, while at basic pH, the absorbance capacity decreased, at pH 7, the microbe remained active absorbent. In case of S. aureus pH 6, 7 and 8 was favourable to absorb Cr(IV).

From bioaacumulation assay it was revealed that maximum biosorption was recorded after 24 h. It means that exponential phase of microbial growth is very crucial for bioaccumulation of heavy metals. At this phase maximum absorbance could be possible due to less biomass of microbes. Our findings are consistent wih Abdel Aty et al. (2013), who reported that biosorption of metals was rapid in the first 20 min then was gradually increased till the equilibrium attained at 60 and 90 min for Cd and Pb, respectively and the biosorption became almost constant thereafter. It was observed that when heavy metal concentration was more used, E. luteus showed sensitivity whereas resistant at 50 [micro]g/mL. E. luteus showed biosorption of Cr(IV) when concentration is increased upto 200-300 [micro]g/mL at pH 10 but greater absoption was measured at pH 6, 7 and 8. It shows that both dead and live biomass was used for the Cr(IV) biosorption. Similar results were recorded for Cd(II) and Pb(II) onto A. sphaerica biomass. It may be carried out chemically via involving valence forces through sharing or exchange of electrons between sorbent and sorbate (Smith, 1981). Kefala et al. (1999) examined two specific strains of gram-positive Actinomycetes, living, non-living bacterial biomass for Cd(II) removal. They revealed that non-living biomass exhibited higher metal uptake. Our results are consistent with them that S. aureus and E. coli showed maximum absorption of Cd(II) as dead biomass. It is concluded that bacteria have the ability to absorb the metals within the cell body or on their cell wall and cell membrane as well.

Various types of biomasses such as bacteria, fungi, mushrooms, plants as well as chemically modified biosorbents were used for the removal of heavy metals (Kumar, 2014; Ghaima et al., 2013; Singh et al., 2012; Volesky, 1986). The attraction or affiliation of removal is dependent upon the chemical composition or structure of the organisms. The results show that for Cd(II) and Cr(IV) removal both dead and live biomass could be used. Chemical composition play vital role in heavy metal affiliation for removal from waste materials e.g., the overexpression of metallothioneins result in enhanced metal accumulation which provide an excellent strategy for the development of microbial based biosorbents for the remediation of metal contamination (Pazirandeh et al., 1995). Similarly, the expression of proteins on the surface of bacterial cell provides an inexpensive and affinity adsorbents.

Conclusion

It was concluded that optimisation of exponential phase of microbial growth, pH of media, incubation period, attraction between sorbent and sorbate and biomass (dead or alive) are important for biosorption process. More research is needed to clarify the impact of heavy metals on cellular structure and which part or organelle is efficient absorber for heavy metals.

Acknowledgement

Authors are thankful to Department of Zoology, University of Azad Jammu and Kashmir, Pakistan for financial support to carry out this research work.

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Iqra Batool (a), Saiqa Andleeb (a)*, Shaukat Ali (b), Kalsoom Akhtar (c) and Nazish Mazhar Ali (d)

(a) Microbial Biotechnology Laboratory, Department of Zoology, University of Azad Jammu and Kashmir, Muzaffarabad-3100, Pakistan

(b) Toxicology Laboratory, Department of Zoology, University of Azad Jammu and Kashmir, Muzaffarabad-3100, Pakistan

(c) Department of Chemistry, University of Azad Jammu and Kashmir, Muzaffarabad-13100, Pakistan

(d) Microbiology Laboratory, Department of Zoology, Government College University, Lahore, Pakistan

(received February 3, 2016; revised June 22, 2016; accepted July 12, 2016)

* Author for correspondence; E-mail:drsaiqa@gmail.com

Caption: Fig. 1. Bioaccumulation of cadmium concentrations by bacterial isolates at various pH and incubation periods. A) E. luteus, B) S. aureus, C) E. coli.

Caption: Fig. 2. Bioaccumulation of chromium concentrations by bacterial isolates at various pH and incubation periods. A) E. luteus, A) S. aureus, C) E. coli.
Table 1. Identification of bacterial isolates through
gram's staining and biochemical tests

Biochemical               Enterococcus    Escherichia   Staphylococcus
tests for bacterial       luteus          coli          aureus
identification

Gram staining             +ve             -ve           +ve
Colony morphology         Coccus          Rod           Coccus
Citrate test              -ve             -ve           +ve
Catalase test             +ve             +ve           +ve
Coagulase test            -ve             -ve           +ve
Indole test               -ve             +ve           -ve
Methyl red test           -ve             +ve           +ve
Oxidase test              -ve             -ve           -ve
Urease test               +ve             -ve           +ve
Motility test             -ve             +ve           +ve
Carbohydrate test
  (glucose and lactose)   -ve             +ve           +ve
Nitrate test              -ve             +ve           +ve
Voges Proskeur test       -ve             -ve           -ve

+ve and -ve indicate positive and negative results.

Table 2. Resistogram analysis of chromium
concentrations against bacterial isolates

                    Chromium metal concentrations

Microbes        50                   100
used                  (micro]g/mL)

                Zone of inhibition in mm (M [+ or -] SD)

E. luteus       R                    R
S. aureus       R                    R
E. coli         12.0 [+ or -] 0.0    15.0 [+ or -] 0.0

                    Chromium metal concentrations

Microbes        150                  200
used                  (micro]g/mL)

                Zone of inhibition in mm (M [+ or -] SD)

E. luteus       14.0 [+ or -] 0.0    15.0 [+ or -] 0.0
S. aureus       R                    R
E. coli         19.0 [+ or -] 0.0    20.0 [+ or -] 0.0

                    Chromium metal concentrations

Microbes        250                  300                  pH
used                  (micro]g/mL)

                Zone of inhibition in mm (M [+ or -] SD)

E. luteus       15.0 [+ or -] 0.0    16.0 [+ or -] 0.0    7
S. aureus       R                    R                    7
E. coli         20.0 [+ or -] 0.0    24.0 [+ or -] 0.0    7

Growth inhibition were expressed as (0) for no sensitivity,
(1-5 mm) for low sensitivity, (>5-10 mm) for moderate
sensitivity and (>10-25 mm) for high sensitivity. R for
resistance; (M [+ or -] SD) Mean [+ or -] Standard deviation.

Table 3. Resistogram analysis of cadmium
concentrations against bacterial isolates

Microbes used       Cadmium metal concentrations

                 50        100       150

                           ([micro]g/mL)

                 Zone of inhibition in mm (M [+ or -] SD)

E. luteus        R         R         15.0 [+ or -] 0.0
S. aureus        R         R         R
E. coli          R         R         R

Microbes used       Cadmium metal concentrations         pH

                 200                 250       300

                        ([micro]g/mL)

                       Zone of inhibition in mm
                             (M [+ or -] SD)

E. luteus        12.0 [+ or -] 0.0   R         R         7
S. aureus        R                   R         R         7
E. coli          R                   R         R         7

Growth inhibition were expressed as (0) for no sensitivity,
(1-5 mm) for low sensitivity, (>5-10 mm) for moderate
sensitivity and (>10-5 mm) for high sensitivity. R for resistance;
(M [+ or -] SD) Mean [+ or -] Standard deviation.

Table 4. Sensitivity test of selected standard antibiotics against
bacterial strains

                                  Antibiotics
bacterial
pathogens       CIP              P                TET

                   Zone of inhibition in mm (Mean [+ or -] SD)

E. luteus       28.0 [+ or -]    3.0 [+ or -]     3.0 [+ or -]
                0.0              0.0              0.0

S. aureus       20.0 [+ or -]    R                4.0 [+ or -]
                0.0              0.0              0.0

E. coli         19.0 [+ or -]    R                R
                0.0

                                  Antibiotics
bacterial
pathogens       N                VA               E

                   Zone of inhibition in mm (Mean [+ or -] SD)

E. luteus       13.0 [+ or -]    17.0 [+ or -]    21.0 [+ or -]
                0.0              0.0              0.0

S. aureus       10.0 [+ or -]    11.0 [+ or -]    17.0 [+ or -]
                0.0              0.0              0.0

E. coli         14.0 [+ or -]    6.0 [+ or -]     23.0 [+ or -]
                0.0              0.0              0.0

                                  Antibiotics
bacterial
pathogens       NOR              AMP              CN

                   Zone of inhibition in mm (Mean [+ or -] SD)

E. luteus       19.0 [+ or -]    5.0 [+ or -]     14.0 [+ or -]
                0.0              0.0              0.0

S. aureus       22.0 [+ or -]    R                20.0 [+ or -]
                0.0                               0.0

E. coli         23.0 [+ or -]    R                17.0 [+ or -]
                0.0

                                  Antibiotics
bacterial
pathogens       S                NA               TOB

                   Zone of inhibition in mm (Mean [+ or -] SD)

E. luteus       13.0 [+ or -]    17.0 [+ or -]    2.0 [+ or -]
                0.0              0.0              0.0

S. aureus       14.0 [+ or -]    13.0 [+ or -]    6.0 [+ or -]
                0.0              0.0              0.0

E. coli         19.0 [+ or -]    11.0 [+ or -]    2.0 [+ or -]
                0.0              0.0              0.0

                          Antibiotics
bacterial
pathogens       K                C

                  Zone of inhibition in mm
                     (Mean [+ or -] SD)

E. luteus       8.0 [+ or -]     18.0 [+ or -]
                0.0              0.0

S. aureus       10.0 [+ or -]    11.0 [+ or -]
                0.0              0.0

E. coli         7.0 [+ or -]     7.0 [+ or -]
                0.0              0.0

                          Antibiotics
bacterial
pathogens       OT               SXT

                  Zone of inhibition in mm
                     (Mean [+ or -] SD)

E. luteus       12.0 [+ or -]    10.0 [+ or -]
                0.0              0.0

S. aureus       5.0 [+ or -]     R
                0.0

E. coli         R                R

The results of the sensitivity tests were expressed as (0) for no
sensitivity, (1-10 mm) for low sensitivity, (11-19 mm) for moderate
sensitivity and (20-35 mm) for high sensitivity. R indicates
resistance. CIP (ciproflaxin); P = pencillin G; TE tetracycline; N
= neomycin; VA = vancomycin; E = erytheromycin; NOR = norflaxin; AMP
= ampicillin; CN getamycin; S = streptomycin; NA = nalidixic acid;
TOB  = tobramycin, K = kanamycin; C chloramphenicol; OT =
oxytetracyclin, SXT = sulfmethoxyzole.
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Author:Batool, Iqra; Andleeb, Saiqa; Ali, Shaukat; Akhtar, Kalsoom; Ali, Nazish Mazhar
Publication:Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences
Article Type:Report
Geographic Code:9PAKI
Date:Jul 1, 2017
Words:6196
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