Characteristic of Hindon river water
The results obtained at the characterization of various parameters in the water samples collected from five different sites of Hindon River are summarized in Table 1. The mean values of pH in water samples from five sites, ranged from 7.25 to 7.69, were found within the permissible limits. The pH values of water samples were marginally alkaline at all five sites, showing that there is neither acid nor alkaline pollution in the water samples from Hindon river (Gupta et al., 2013). Electrical conductivity (EC) of water samples at all sites, where mean values varied from 1667.0 to 1916.75 μS/cm, were beyond the acceptable limit, where, what sample from Atali village site exhibited highest EC compared to that of other four sites. High EC was attributed to the existence of high number of dissolved ions (Verma et al., 2006). River water pollution by release of domestic and industrial waste rises the EC. High number of dissolved ions in water causes unpleasant taste and moreover distresses animals and plants (Kumar et al.,2018). Total dissolved solid (TDS) ranged from 773.25 to 1238.0 mg/L as mean value, was also beyond the permissible limit at all five sites of river. High level of TDS in river water is primarily due to the occurrence of carbonates (CO32−), bicarbonates (HCO3−), chlorides (Cl−), phosphates (PO43−) and nitrate compound such as Ca (NO3)2, Mg (NO3)2, KNO3 and Mn (NO3)2, organic salts, and other solid elements. Contagion of Hindon river by industrial effluent could be the responsible for increase in TDS as the dissolved ions are significantly existing in Industrial waste. The existence of high level of TDS in river water also results in unfit for drinking. The mean values of alkalinity varied from 261.75 to 368.75 mg/L was also significantly higher than the permissible limits at the all sites of Hindon river. High concentration of dissolved ions rises the alkalinity of river water, which possibly can also be one of the factors for enlarged alkalinity of water samples at all five sites. High alkalinity of river water results in unfit for drinking as well as for irrigation1. Hardness of water samples (mean value from 319.25 to 374.50 mg/L) exceeded the permissible limit at the all five sites. Well, hardness indicates the soap forming ability of a water sample where the two cations primarily Ca+ and Mg+ are accountable. Existence of other ions such as sulphate (SO²⁻₄), nitrate (NO⁻₃), and chloride (Cl⁻) in river water was found within the permissible limit. Sulphate is mostly natural in nature attributed to primarily by mineral sources like gypsum, etc. Verma et al.,2006 studied on high concentration sulphate in drinking water may cause several intestinal diseases. Chloride is extensively dispersed in nature in the form of salts of Na (NaCl), K (KCl) and Ca (CaCl2). COD and BOD values for Hindon river water were above the acceptable values at all five sites of river. A high BOD and COD values showed that the water has highly oxygen demanding waste attributed to the presence of organic pollutants which causes the depletion of dissolved oxygen (DO) which is a fundamental requirement for aquatic life. The high value of COD gives valuable information about the pollution potential of the Hindon river water (Gupta et al., 2017). The heavy metals Cd, Ni and Pb were found above permissible limits for drinking water as per BIS.
The physicochemical characterization of Hindon river water at five places revealed that the river had high levels of organic and inorganic pollution, which was most likely attributable to trash produced by companies located along the river's banks.
Table 1 Physicochemical analysis of the water samples collected along four sites of the Hindon river (March-June 2017).
Parameters
|
Sites
|
|
|
|
|
BIS/
CPCB*
|
Unit
|
S1
Mean±SD
|
S2
Mean±SD
|
S3
Mean±SD
|
S4
Mean±SD
|
S5
Mean±SD
|
pH
|
7.25±0.49
|
7.25±0.47
|
7.33±0.33
|
7.48±0.57
|
7.69±0.44
|
6.5-8.5
|
|
EC
|
1916.75±327.90
|
1901.25±85.45
|
1667±178.41
|
1767.75±332.28
|
1843.75±423.86
|
-
|
μS/cm
|
TDS
|
1178.50±223.92
|
1238±34.12
|
1044±93.40
|
1117.25±136.27
|
773.25±84.70
|
500
|
mg/L
|
Alkalinity
|
344.75±21.23
|
368.75±33.24
|
327.75±56.14
|
348±103.19
|
261.75±44.97
|
200
|
mg/L
|
Hardness
|
319.25±35.94
|
362.50±35.42
|
374.50±71.92
|
359.25±55.4
|
339±121.57
|
200
|
mg/L
|
COD
|
356.50±66.52
|
365.25±92.88
|
307.25±42.51
|
327.25±34.95
|
339.5±67.13
|
-
|
mg/L
|
BOD
|
109.50±17.05
|
91.5±21.43
|
84.5±16.15
|
92.75±10.03
|
92.5±25.47
|
≤ 2*
|
mg/L
|
NO⁻₃
|
40.75±10.85
|
48.25±4.82
|
44±18.23
|
31±13.69
|
25±9.08
|
45
|
mg/L
|
SO²⁻₄
|
48.25±9.36
|
56.00±7.68
|
46.75±0.83
|
51±11
|
48±6.75
|
200
|
mg/L
|
Cl⁻
|
178.25±21.11
|
199.25±20.04
|
168±36.80
|
163.75±47.37
|
108±25.84
|
250
|
mg/L
|
Cd
|
0.022±0.02
|
0.037±0.02
|
0.027±0.02
|
0.04±0.06
|
0.002±0.00
|
0.003
|
mg/L
|
Ni
|
0.048±0.01
|
0.021±0.01
|
0.144±0.22
|
0.068±0.05
|
0.012±0.00
|
0.02
|
mg/L
|
Pb
|
0.052±0.04
|
0.046±0.04
|
0.051±0.04
|
0.054±0.04
|
0.016±0.01
|
0.01
|
mg/L
|
Selection of isolates
In the present investigation total number of 32 bacteria were isolated initially from the water samples following enrichment in nutrient media containing 200 mg/L heavy metals (Cd, Ni and Pb). When these strains were further studied for resistance and degradation of higher concentrations of heavy metals, two strains, viz., strain HIB2 and HIB11 were found to utilize and grow in nutrient containing up to 500 mg/L heavy metals and were selected for further study. The morphological and biochemical characteristics attentively suggested that these isolates were Bacillus sp. and Pseudomonas sp. (Tables 2).
Biochemical and Molecular characterization (16S rRNA)
As shown in Table 2, the bacterial isolate B2 was found to be gram-positive, small rod-shaped and motile. This isolate was able to ferment sucrose and glucose. It was MR negative and VP positive, and showed starch hydrolysis, but did not show H2S production. It showed positive results for the catalase test, nitrate reduction, oxidase test and citrate utilization tests. However, it showed negative results for urease test and indole test. The isolate B11 was found rod-shaped, gram negative and motile. It did not produce acid with sucrose and glucose. It showed negative results of MR test, indole test, urease test, and VP test, while positive results for catalase test, oxidase test, nitrate test, and citrate test. Further did not show starch hydrolysis and H2S production. Biochemical test results are also shown in Fig. 2.
Molecular identification was performed for the confirmation of selected isolates by amplification and sequencing of the 16S rRNA gene. A pure culture of selected isolates was grown on nutrient agar for 24h and the identification of isolates was further confirmed using 16S rRNA gene sequencing using the universal primers, the forward primer sequence 16SF (2f) 5'- AGAGTTTGATCMTGGCTCAG-3' and the reverse primer sequence was 16SR (1492r) 5'-TACGGYTACCTTGTTACGACTT-3'. The resulting sequences were compared with their closest relatives available in the GenBank database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the NCBI-BLAST tool. The nucleotide BLAST similarity search analysis showed that the two isolates matched with their closest relatives as follows: the closest sequence identity of Isolate B2 was found to be with Bacillus subtilis strain based on nucleotide homology and phylogenetic analysis (Fig. 3a). Similarly, isolate B11 had the closest sequence identity with Pseudomonas aeruginosa strain (Fig. 3b). Additionally, these bacterial sequences were deposited in the NCBI Gene bank and got assigned the accession numbers for further communication. Isolate B2 was named as Bacillus subtilis strain HIB2 with Accession Number: MK936323.1 and isolate B11 was named as Pseudomonas aeruginosa strain HIB11 with Accession Number: MK937645.1 (Table 3). The phylogenetic tree of each strain was constructed using the neighbor-joining algorithm from MEGA-V (Version 5.0) software and their phylogenetic relationship were inferred (Fig. 4). The approximate phylogenetic position of the strains B2 and B11 are depicted in (Fig 4).
Table 2 Morphological & Biochemical characteristic of the selected bacterial isolates.
Test
|
Bacillus Subtilis HIB2
|
Pseudomonas aeruginosa HIB11
|
Gram staining
|
+
|
-
|
Shape
|
Rods
|
Rods
|
Motility
|
+
|
+
|
Catalase
|
+
|
+
|
Oxidase
|
+
|
+
|
Indole production
|
-
|
-
|
Methyl red
|
-
|
-
|
VP (Voges Proskauer)
|
+
|
-
|
Citrate utilization
|
+
|
+
|
Urease test
|
-
|
-
|
Nitrate
|
+
|
+
|
H2S
|
-
|
-
|
Starch hydrolysis
|
+
|
-
|
Sucrose
|
+
|
-
|
Glucose
|
+
|
-
|
Table 3 Identification of effective bacterial isolates by 16S rRNA gene sequence analyses
Isolate
|
Organism
|
Accession no.
|
Identity (%)
|
HIB2
|
Bacillus subtilis
|
MK936323.1
|
99%
|
HIB11
|
Pseudomonas aeroginosa
|
MK937645.1
|
99%
|
Bioremediation of Heavy metals
In this study, metal removal was carried out using bacterial strains isolated from Hindon river water and after screening based on resistance against metal ions. Isolated bacteria in this study have unique metal accumulation characteristics and easy to cultivate. Tolerance and removal of Cd, Ni and Pb were determined in the aqueous medium. The amount of heavy metals removal was evaluated by comparing initial metal concentration and final concentration after treatment by bacterial isolates and their consortium (Table 4 to 9). The bacterial isolates in the medium were studied for:
(i). The reduction rate of heavy metals at different concentrations from 5 to 100 mg/L at 14 days incubation period (Fig. 5 B – 7B).
(ii). The reduction rate of heavy metal (10 mg/L concentration) at different time intervals of the incubation period for 14 days (Fig.5 A- 7A).
Heavy metal concentration was analyzed using the ICP-MS instrument. (Thermo fisher).
Removal of Lead (Pb2+)
The results displayed in Table 4 & 5 shows that both isolates Bacillus subtilis HIB2. and Pseudomonas aeruginosa HIB11 were well competent for removal of lead (Pb2+) from polluted water. There was significant influence on the removal efficacy of Pb2+ using Pseudomonas aeruginosa HIB11 when increased the initial concentration from 5 to 100 mg/L. The remaining values of Pb2+ in the media were ranged from 1.21±0.16 to 61.49±5.65 mg/L at rising the initial concentration from 5 to 100 mg/L respectively. In case of Bacillus subtilis HIB2, the removal efficacy of Pb2+ was also influenced at rising the initial concentration from 5 to 100 mg/L. The removal efficacy significantly varied from 78.8% to 41.9% at rising the initial concentration from 5 to 100 mg/L respectively. However, this showed that Bacillus subtilis HIB2 had slightly high removal efficiency of Pb2+ in comparison of Pseudomonas aeruginosa HIB11 (Fig 5). Similar findings were reported by Azzam et al., (2015) who found that Bacillus sp. and Pseudomonas sp. had capability of eliminating Pb2+ 99.7% and 99.6%. The key process for removal of Pb2+ by bacterial isolates includes primarily neutralization and adsorption. Dabir et al., (2019) reported Microbacterium oxydans CM3 and Rhodococcus sp. AM1 to reduce 39% and 58% of lead (Pb) at 400 mg/L after 72 h, respectively. Vimalnath et al., (2018) reported Pseudomonas aeruginosa cells to uptake 71.7% of Pb concentration ranging from 10 to 250 mg/L. Murthy et al., (2012) studied the biosorption of lead ions using Bacillus cereus at different concentrations of lead from 100 to 500 mg/L and found decrease in removal with increase in Pb concentration. Guo et al., (2010) studied the Bacillus sp. L14 which showed the 80.48% removal of Pb under the initial concentration of 10 mg/L concentration within 24 hrs. incubation period.
Table 4. The efficiency of Bioremediation of Lead (Pb2+) at 10 mg/L at different time interval.
|
Residual values (mg/L)
|
Time (days)
|
Bacillus subtilis HIB2
|
%R
|
Pseudomonas aeruginosa HIB11
|
%R
|
1
|
7.19a±0.29
|
28.1
|
7.15a±0.72
|
28.5
|
3
|
5.56a±0.67
|
44.4
|
6.47b±0.55
|
35.3
|
5
|
4.60a±0.55
|
54.0
|
5.01b±0.14
|
49.9
|
7
|
3.39a±0.59
|
66.1
|
4.14b±0.51
|
58.6
|
14
|
2.83a±0.53
|
71.7
|
2.93a±0.08
|
70.7
|
Table 5. The efficiency of Bioremediation of Lead (Pb2+) at different time interval at different concentration after 14 incubation period.
|
Residual values (mg/L)
|
Conc. (mg/L)
|
Bacillus subtilis HIB2
|
%R
|
Pseudomonas aeruginosa HIB11
|
%R
|
5
|
1.06b±0.04
|
78.8
|
1.21a±0.16
|
75.8
|
10
|
2.83a±0.53
|
71.7
|
2.93a±0.08
|
70.7
|
50
|
25.64a±4.35
|
48.7
|
27.17b±7.86
|
45.6
|
100
|
58.04a±7.88
|
41.9
|
61.49a±5.65
|
38.5
|
Note: 1Mean ± SE; n=3, %R: Percent Removal
2Mean values with the similar superscripts in same raw are not significantly different while values with different superscripts are significantly different from one another at p<0.05 significance level (Duncan’s test).
Removal of Cadmium (Cd2+)
The results for Cadmium (Cd2+) exposed that Pseudomonas aeruginosa HIB11 attained better removal efficacy in comparison to Bacillus subtilis HIB2 at the same initial concentrations (Table 6 & 7). Furthermore, the removal efficacy of Cd2+ using Pseudomonas aeruginosa HIB11 was also somewhat influenced (72.4–37.9%) at mostly varied the initial concentration from 5 to 100 mg/L (Fig 6). Similarly, the removal efficacy of Cd2+ using Bacillus subtilis HIB2 was also affected at rising the initial concentration from 5 to 100 mg/L. This revealed that both Bacillus subtilis HIB2, and Pseudomonas aeruginosa HIB11 were significant competent for removal of Cd2+ from polluted water. However, the remaining concentration of Cd2+ after treatment were quite low using Pseudomonas aeruginosa HIB11 as compared to Bacillus subtilis HIB2 (Table 6 & 7). This metal biosorption efficiency of both isolates HIB2 and HIB11 could possibly be involvement of bio-flocculant formed by these bacteria. The bacterial constituent like cell wall and extracellular polysaccharide attributed to Ion uptake, has significant roles in curbing heavy metal contamination in the treatment procedures4. Similar studies were carried. Dabir et al., (2019) informed lead reduction of 200 mg/L concentration up to 20% by Bacillus sp. CM4. Li et al., (2019) reported average 54.7%, 43.2% and 7.34% removal of Cd at concentrations of 0.05mg/L, 0.5mg/L and 5mg/L by B. subtilis respectively after 24 days incubation period. Guo et al., (2010) studied the Bacillus sp. L14 which showed 75.78%, removal of 10 mg/L concentration of Cd within 24 hrs. incubation period. Khan et al., (2015) reported E. coli P4 to reduce 18.8%, 37%, and 56% Cd2+ after 48h, 96h, and 144h, respectively. Zeng et al., (2009) studied the removal efficiency of Pseudomonas aeruginosa for Cd and found 43.3% removal at initial concentration of 110.2 mg/L with 24 hours incubation period. Ziagova et al., (2007) reported Pseudomonas sp. Removed the 75% Cd at 200 mg/L initial concentration after 5 hours of incubation.
Table 6. Bioremediation efficiency of Cadmium (Cd2+) at 10 mg/L at different time interval.
|
Residual values (mg/L)
|
Time (days)
|
Bacillus subtilis HIB2
|
%R
|
Pseudomonas aeruginosa HIB11
|
%R
|
1
|
8.75a±0.48
|
12.5
|
8.57a±0.73
|
14.3
|
3
|
8.02a±0.58
|
19.8
|
8.26a±0.75
|
17.4
|
5
|
6.36a±0.49
|
36.4
|
6.42a±0.43
|
35.8
|
7
|
5.56a±0.81
|
44.4
|
5.80a±0.22
|
42.0
|
14
|
4.61a±0.85
|
53.9
|
4.24a±1.26
|
57.6
|
Table 7. The efficiency of Bioremediation of Cadmium (Cd2+) at different time interval at different concentration after 14 incubation period.
|
Residual values (mg/L)
|
|
Conc. (mg/L)
|
Bacillus subtilis HIB2
|
%R
|
Pseudomonas aeruginosa HIB11
|
%R
|
5
|
1.40a±0.46
|
72.0
|
1.38a±0.45
|
72.4
|
10
|
4.61a±0.85
|
53.
|
4.24a±1.26
|
57.6
|
50
|
26.81a±1.64
|
46.3
|
25.04a±2.72
|
49.9
|
100
|
58.86a±13.67
|
41.1
|
62.02a±7.80
|
37.9
|
Note: 1Mean ± SE; n=3, %R: Percent Removal
2Mean values with the similar superscripts in same raw are not significantly different while values with different superscripts are significantly different from one another at p<0.05 significance level (Duncan’s test).
Removal of Nickel (Ni2+)
Similar patterns were observed for the reduction of Nickel (Ni2+) ions using Bacillus subtilis HIB2 and Pseudomonas aeruginosa HIB11 as presented in Table 8 and 9. The removal in Ni2+ value was considerably higher using Bacillus subtilis HIB2 in comparison to Pseudomonas aeruginosa HIB11 Nonetheless, the removal efficacy of Ni2+ was highly declined at rising the initial concentration from 5 to 100 mg/L. Bacillus subtilis HIB2 had the highest nickel removal proportion (69.4%) than Pseudomonas aeruginosa HIB11 (68.8%) at initial concentration of 5 mg/L. The removal efficacy of Ni2+ was significantly declined from 68.8% to 33.8% at rising the initial concentration from 5 to 100 mg/L using Pseudomonas aeruginosa HIB11. Similarly, in case of Bacillus subtilis HIB2, the removal efficacy of Ni2+ was significantly declined from 69.40% to 37.36% (Fig.7). This showed that the efficacy of both isolates is initial concentration dependent. Similar studies were reported. Das et al., (2014) observed the removal of Ni by Bacillus thuringiensis where observed a substantial percentage (82%) removal of Ni from the medium during in vitro culture. Similarly, there are other studies on the removal of Ni such as 95% Ni removal by Microbacterium sp. (Sathyavathi et al., 2014), biosorption of Ni & Cd, by E. coli sp. (Ansari & Malik, 2007) and removal of Ni by Proteus vulgaris strain, Stenotrophomonas sp. and Bacillus thuringiensis respectively (Kumar et al., 2016).
Table 8. Bioremediation efficiency of Nickel (Ni2+) at 10 mg/L at different time interval.
|
Residual values (mg/L)
|
|
Time (days)
|
Bacillus subtilis HIB2
|
%R
|
Pseudomonas aeruginosa HIB11
|
%R
|
1
|
8.95a±0.75
|
10.5
|
8.10b±0.24
|
19.0
|
3
|
7.77a±0.66
|
22.3
|
5.56b±0.10
|
44.4
|
5
|
7.01a±0.14
|
29.9
|
4.96c±0.10
|
50.4
|
7
|
6.39a±0.11
|
36.1
|
4.47c±0.12
|
55.3
|
14
|
3.14a±0.08
|
68.6
|
3.36a±0.34
|
66.4
|
Table 9. The efficiency of Bioremediation of Nickel (Ni2+) at different time interval at different concentration after 14 incubation period.
|
Residual values (mg/L)
|
Conc. (mg/L)
|
Bacillus subtilis HIB2
|
%R
|
Pseudomonas aeruginosa HIB11
|
%R
|
5
|
1.53a±0.39
|
69.4
|
1.56a±0.36
|
68.8
|
10
|
3.14a±0.08
|
68.6
|
3.36a±0.34
|
66.4
|
50
|
23.89a±7.43
|
52.2
|
30.38c±6.49
|
39.2
|
100
|
62.64b±10.14
|
37.3
|
66.14a±7.10
|
33.8
|
Note: 1Mean ± SE; n=3, %R: Percent Removal
2Mean values with the similar superscripts in same raw are not significantly different while values with different superscripts are significantly different from one another at p<0.05 significance level (Duncan’s test).