Application of bacterial biomass as a potential heavy metal bio-removal agent

Water has been the most important element for saving life; the major global health problem has been water pollution that may be due to the wastewater discharge into the water bodies. Several techniques have been used for water treatment that is, physical, chemical and biological methods. Recently, the third method was the most effective one for the wastewater treatment. In this work twenty bacterial isolates were isolated from River Nile, Egypt to study their capability to remove some heavy metals from its solution. Agar plates amended with different concentrations of some heavy metals were used for screening the bacterial capability for removing the tested heavy metals. According to the identification procedures based on the BIOLOG system the bacterial isolate MSNIOF11 showed a similarity of 97% to Bacillus subtilis var. globigii, so it was given the name as Bacillus subtilis var. globigii MSNIOF11. The heavy metal removal process was pH and temperature dependent, where the maximum growth and heavy metal removal was recorded at 30°C with neutral pH (7.0). In the first 24 h there was an increase of the metal removal and there was no significant change after 30 h.


INTRODUCTION
Water pollution is an acute problem in the River Nile.In the rise of the increasing urbanization and industrializetion, the pollution potential of the River is gaining momentum day by day.Dumping wastewater and toxic wastes into the main channel of the River has caused serve pollution in the River to the extent that its water is posing a threat to the survival of aquatic flora and fauna (Dalman et al., 2006).
Water quality has been decreased during this century due to discharge of wastewater into water channels as well as environmental pollutants.This is considered as one of the major global health problems, and cross adaptation of microbial population to structurally related chemicals may play an important role in the practical development and application of bioremediation techniques (Liu and Jones, 1995;Monachese et al., 2012).Pollution of the natural environment by heavy metals is a worldwide problem because these metals are indestructible and most of them have toxic effects on living organisms when they exceed a certain concentration (MacFarlane and Burchett, 2000;Ekeanyanwu et al., 2010;Raphael et al., 2011;Aikpokpodion et al., 2012;Ahmed et al., 2013).Heavy metals are of high ecological significance since they are not removed from water as a result of self purification, but accumulate and enter the food chain which inevitably affects the human health resulting in extremely disrupted biological processes (Loska and Wiechula, 2003;Igwe and Abia, 2006;Akpor and Muchie, 2010;Young et al., 2012).
The bio-removal/ processes could offer the possibility to destroy or render various contaminants using natural biological activity.As such, it uses relatively low-cost, low technology techniques, generally have a high public acceptance and can often be carried out on site using a microbial source which has received much attention recently due to the awareness of environmental problems (Saithong and Poonsuk, 2002;Rani et al., 2010;Abioye, 2011).Microorganisms such as bacteria (Daboor and Sabae, 2007;Nanda et al., 2011;Lin and Harichund, 2011;Samarth et al., 2012), algae (El-Sherif et al., 2008;Tamilselvan et al., 2011;Mane and Bhosle, 2012;Kumar and Oommen, 2012), fungi (Selvam et al., 2002;Joshi et al., 2011;Simonescu and Ferdes, 2012;Hemambika et al., 2011) andyeast (Abdul Rehman et al., 2008;Machado et al., 2009Machado et al., , 2010) ) persist a variety of mechanisms exist for the removal of heavy metals from aqueous solution.Regarding the same process Zhou et al. (2007) used Bacillus cells as a factor to remove the chromium ions from the aqueous solution.Jarosławiecka and Piotrowska-Seget (2014) reported that the bacterial extracellular polysaccharides are involved with the lead adsorption.
The main objective of the present study was to isolate and screen heavy metal tolerance of bacterial isolates and evaluate their competence to remove heavy metals from its solution and detect the suitable conditions for the maximum activities under laboratory conditions.

Sampling and strain isolation
Water samples were collected from five stations at Demitta Branch of RiverNile.These stations are highly polluted with high concentrations of heavy metals due to discharge of industrial effluent.Twenty bacterial isolates (12 as Gram positive and eight as Gram negative) were isolated after seeding the samples on Glu-cose Mineral Salt (GMS) agar plates, (Daboor and Sabae, 2007).

Heavy metal
Heavy metals solutions of zinc sulfate (ZnSO 4 ), lead chloride (PbCl 2 ) and cadmium chloride (CdCl 2 ) were prepared with a final concentration 100 mg/L and kept sterilized for further use.

Screening for bacterial isolates resistant to metal ions
The bacterial isolates were separately streaked on GMS having 100 mg/L of each of the metal solution.After two days of incubation at 30°C the plates were checked for bacterial growth (Daboor and Sabae, 2007).

Heavy metal removal efficiency detection
The bacterial isolates that showed a positive growth on the agar plates (having heavy metals solutions) were sub cultured again onto modified T-medium (Duxbury, 1981) amended with mixture of heavy metal ions Cd 2+ , Pb 2+ and Zn 2+ (50 mg/L of each one).The plates were swabbed and the growth was measured by the periodic determination of culture density absorbance at wave length of 600 nm using a spectrophotometer (Ultrospec 1100-pro, Amersham Pharmacia Biotech) based on McFarland's scale (Sutton, 2011).The most resistant isolates were inoculated (0.2 g) into 250 mL conical flask containing 50 mL T-medium which has metal ions with 50 mg/L final concentration of each metal ion and incubated in shake condition (50 rpm) for 24 h at 30°C.

Identification and characterization of the metal resistant isolates
Bacterial colonies from the plates showed highly intensiveness growth were picked and streaked on Tryptic Soy Agar (TSA) medium, after overnight incubation at 30°C pure colonies were stained with Gram stain, hence figure out which Biolog Micro Plate could be used.
According to the manufacturer's directions of MicroLog System (Biolog, Hayward, CA, USA) pure bacterial isolates were transferred into Biolog Universal Growth (BUG) medium (Biolog, Hayward, CA, USA) and incubated overnight at 30°C.Bacterial growth were collected and suspended in 0.15 M NaCl using cotton swabs.(Tanase et al., 2011).

Biolog MicroPlates preparation and identification
The isolate was tested to utilize 95 different carbon sources in Gram-positive (GP) MicroPlate (Biolog, Hayward, CA, USA) as recommended by the manufacturer's manual.A plastic disposable loop was used to collect colonies carefully so that there would be minimum carryover of nutrients from the agar when the growth was suspended in 0.15 M NaCl.The turbidimeter (which measured the turbidity at wave length at 590 nm) was blanked with a tube of uninoculated saline.The suspension was then adjusted to fall within the low-limit and high-limit GP MicroPlate turbidity standards supplied by manufacturer.The inoculum, which was always used within 10 min. of preparation, was poured into a disposable plastic reservoir just prior to use.MicroPlates (GP) were inoculated with an eight-channel multi-pipette, with 150 μl of the inoculum being dispensed per well; plates were then generally incubated at 30°C.The carbon source utilization patterns were read with a MicroPlate reader and analyzed for the differentiation of bacterial strains by a cluster analysis program using Biolog database and software (Biolog, Hayward, Calif), with the MicroLog GP data base colour formation in the individual cells of the microtitre plates was measured at 590 nm (Miller and Rhoden, 1991;Holmes et al., 1994).
Biolog system provides identifications if the similarity index of the genus or species was 0.750 or greater after four hours incubation.When a lower similarity value is obtained, the user is prompted to continue the incubation for 24 h.In this study, all MicroPlates were read at both four hours and confirmed after 24 h even when identification was reported at four hours, a similarity index of less than 0.50 results in an instrument report of not identified (NI).Similarity indices of 0.50 result in a computer report of identification to either the genus or the species level (Miller and Rhoden, 1991;Holmes et al., 1994).

Effect of temperatures on bacterial cells and heavy metals removal
The effect of temperature degree was investigated by using the isolate B. subtilis var.globigii MSNIOF11.Heavy metal removal was conducted in 250 mL conical flasks containing 50 mL of GMS broth having heavy metals with final concentration 150 mg/L (50 mg/L of each metal).The flasks (three replicates) were inoculated with 0.2 g bacterial cells and incubated for 48 h at 20, 25, 30, 35 and 40°C.Samples were taken and centrifuged at 10.000 rpm for half hour.The supernatant was analyzed to state the heavy metal remaining in the solution (Daboor and Sabae, 2007) and heavy metal removal was calculated based on its initial concentration according to the equation of Kuycak and Volesky (1988).

Effect of pH on bacterial cells and heavy metals removal
The effect of different pH values (5.0, 6.0, 7.0, 8.0 and 9.0) was investigated.Adjusting the pH of the medium using 0.1 N HCl and 0.1 N NaOH, and incubating for 24 h at 30°C, remaining heavy metals in the solution were calculated as described previously based on the equation of Kuycak and Volesky (1988).

Effect of incubation time on bacterial cells and heavy metals removal
To study the effect of different incubation periods on both bacterial growth and heavy metals removal, the pH was adjusted to pH 7.0.After several intervals of time 12, 18, 24, 30 and 36 h of incubation at 30°C, heavy metals residue in the solution were calculated following the method reported by Kuycak and Volesky (1988).

Statistical analysis
The arithmetic means of the three replicates estimations were tabulated and the least significant difference (L.S.D.) at 0.05% confidence limit was calculated according to Pielou (1966).

Biolog MicroLog identification
Only one of the twenty bacterial isolates showed a very good growth in the presence of heavy metals high con-centration (data not shown).This isolate was rood shaped positive to Gram staining.After four hours and 24 h the color change within each well in the MicroPlate was red by the automated reader.The tested strain showed 97% similarity with Bacillus subtilis var.globigii and the data also revealed a very low similarity (2.8%) with B. pumilus.The relationship between Bacillus strains by carbon sources utilization pattern was shown in Figure 1.The analyzed data by Biolog software illustrated the arrangement and distances of the Bacillus species, clarified that both B3 (the selected strain) and B2 have the same unit of taxonomic distances, hence B3 is Bacillus subtilis var.globigii, for differentiation between the selected strain and others it given the name Bacillus subtilis var.globigii MSNIOF11.

Effect of different temperatures
Effect of incubation temperatures on the growth of B.. subtilis var.globigii MSNIOF11 and heavy metal removal were represented in Figure 2. The temperature effect represented significant differences (P>0.05) between the percentage of metal ion removal, where maximum activity of the heavy metal removal was recorded at 30 o C with the values of 72.30, 68.30 and 70.00% of Cd, Pb and Zn ions.

Effect of different hydrogen ion concentrations
pH values play an important role on heavy metal removal by B. subtilis var.globigii MSNIOF11,as shown in Figure 3. Hydrogen ion concentrations -pH values-affected both the growth and heavy metal removal by the bacterial isolate.At pH levels of 5.0 and 9.0 no obvious growth was seen and metal removal was indemonstrable, nevertheless, the maximum growth and the maximum metal removal were obtained at pH of 7.0, the logarithmic numbers of viable cells /ml were 7.70, 8.39 and 9.20 for Cd 2+ , Pb 3+ and Zn 2+ at 30 o C, respectively and at the same time metal removal percentage was in the order of Cd 2+ >Pb 2+ > Fe 2+ with 69.30, 68.70 and 67.00%,, respec-tively.It was also clear that, there was a significant drop in both growth and metal uptake when the pH was shifted towards both acidic and alkaline media (pH of 6.0 and 8.0).
The percentages of metal uptake were 36.30,56.70 and 48.70% at pH 6.0, while it was 53.30, 43.30 and 46.6% at pH 8.00 for Cd +2 , Pb +2 and Zn +2 , respectively.The maximum values for Cd, Pb and Zn ions were recorded at pH 7.0, where the removal percentage was in the order Cd +2 > Zn +2 > Pb +2 .
Regarding to the effect of the incubation periods the data represented in Figure 4 showed that after 12 h incubation the microbial growth gradually increased.The metal removal was increased by time until 24 h at which the highest values of Cd 2+ , Pb 2+ and Zn 2+ removal were detected, but there was no significant effect with time increasing up to 30 h.

DISCUSSION
The results here illustrated the ability of some bacterial isolates to resist the heavy metals toxicity.Only one bacterial isolate showed resistant to the toxicity of all the tested heavy metals, this may be due to biochemical and structural properties, physiological and/or genetic adaptation of microorganisms and environmental modification of metal specification control the surviving of microorganisms in solutions having toxic metals (Cooksey, 1993;Blackwell et al., 1995).Hence this bacterial isolate was selected for the heavy metal bioremoval study and identification.It is not possible to isolate and culture microorganisms from their natural habitat and stay behind identification.This will lead to shortage of microbial community composition and function information (Wagner et al., 1993).
In this study, different carbon source profiles were generated by inoculating Biolog GP microtitre plates.The colour development in each well of the GP Biolog microtitre plates reflected the ability of the bacterial community to utilize that specific carbon source.The data indicated that metabolic diversity (substrate utilization), as determined with the Biolog system, could be used to learn more about functional diversity (number of different substrates utilized) and evenness (distribution of species abundance within the community) in natural habitats (Miller andRhoden, 1991, Heerden et al., 2002;De Paolis and Lippi, 2008).Some of the B. subtilis could survive in the presence of Cd +2 , Pb +2 and Zn +2 at concentration 100 mg/L, while others cannot.Bioaccumulation of heavy metals such as copper, zinc, cadmium, and nickel were reported by several Bacillus species ( Mayer andBeveridge, 1989, Samarth et al., 2012;Odokuma and Akponah, 2012 ).Heavy metal bioremoval includes the formation of stable complexes between heavy metals and nuclides of microbial biomass and these complexes are generally the result of electrostatic interactions between the metal ligands and negatively charged cellular biopolymers which were produced in both Gram negative and Gram positive bacteria (Ledin and Pedersen, 1996).
The metal bioremoval capacity of living cells from aqueous solutions was influenced by environmental growth conditions, such as temperature, pH value, biomass concentration, and incubation time.It was clear from the present data that, the temperature degree had an important effect on the bioremoval percentage of heavy metal by B. subtilis var.globigii MSNIOF11 where the uptake of the tested metals increased gradually by increasing temperature from 20 to 30°C and decreased at the higher temperature 35 and 40 °C.The maximum metals (Cd +2 , Pb +2 and Zn +2 ) bioremoval were recorded at 30°C, which may depend on cell metabolism that are most likely to be inhibited by low temperature, meanwhile the higher temperature also affect the integrity of the cell membrane, these present results are in line with those reported by Brady and Duncan (1994 a,b).
The metal binding to bacterial cell was influenced by many factors including pH, buffer type, ionic strength and incubation time.The present results showed the maximum removal percentage of Cd 2+ , Pb 2+ and Zn 2+ ions by B. subtilis var.globigii MSNIOF11 at pH7.0.The change of pH values from five to seven may result in an increase in the bacterial cell wall negative charge which favored electrochemical attraction and adsorption of metal (Gourdon et al., 1990).The results here were in accordance with those of Lo et al. (2001), they reported that, the biosorption increased by increasing pH from two to seven, and as the pH value was increased the solubility of the metal decreased which enhanced metal sorption (Tsezos and Volesky, 1982).On the other hand at low pH most of nitrogen containing groups at the bacterial cell wall would be neutral and so preclude the metal sorption (Elliot et al., 1986;Korenevskii et al., 1999) and the high concentrate of hydrogen ion compete with the cations of sorption sites (Mclean and Beveridge, 1990).The results also revealed that, both growth and metal bioremoval were not observed at pH five.In this respect Mera et al. (1992) suggested a delicate competition between H + and metal ions for binding into the cells in the presence of competitive cations (Cd 2+ , Pb 2+ and Zn 2+ ) would alter energy states of these cells.Also, metal removal was completely inhibited at pH nine that may be due to the formation of insoluble oxides, hydroxides and carbonates at pH above neutrality which reduced the free metal ions (Brady and Duncan, 1994 a, b).
The present results showed that the metal removal also varied with incubation time.When cell suspension of B. subtilis var.globigii MSNIOF11was exposed to heavy metal in their solutions, the utmost removal was recorded after 24 h the increase of incubation time had a little effect on the metal removal, the stage of the life cycle and cultural conditions affected metal accumulation, the structural features of the cell wall as affected by cell age provide a mechanism to immobilization metals and prevent their entry into the cell (Remacle, 1990;Delgado et al., 1996).
In conclusion the selected bacterial isolate B. subtilis var.globigii MSNIOF11 had the capability of accumulation of the test metals.The bioremoval process of the tested heavy metals from its solutions was pH and temperature dependant.B. subtilis var.globigii MSNIOF11 recorded maximum growth and heavy metal removal at 30°C with neutral pH (7.0) during the first 24 h.By pas-sing time that is, after 30 h there was no significant change.For that reason the major directions in the bioremediation technology research contains, studying the microbial communities from contaminated sites with special emphasis on those strains that play major functional roles in pollutant removal.In this respect, characterization of the catabolic potential, as well as accurate taxonomical identification of such types of bacteria is very important.In this study, we identified phenotypic and metabolic traits.