Keywords

Acinetobacter sp. Junii, Acinetobacter sp. Tandoii,, biosorption, Itogon, Benguet, heavy-metal resistant

Introduction

One of the pressing environmental problems facing our country today is the effluents from mining industries generating large volume of wastewater contaminated with heavy metals that pollute ground water, deplete aquatic life, which contaminate the food chain and deteriorate the ecosystem. Water discharge from mine tailings is usually acidic in nature and contains high concentration of heavy metals ions. The presence of heavy metals in aquatic environment is known to cause severe damage to aquatic life, and could eliminate the microorganisms needed for biological treatment of wastewater and consequently delay the process of water purification.

The study aims to apply microbial isolates for the biological treatment of acidic wastewater and reduction of heavy metals and to develop a cost-effective treatment system that would be able to remediate abandoned mine sites. In particular, a biological treatment system will be designed and operated employing microorganisms found on site. This will involve the screening, isolation and purification of best pollutant degrading bacteria found in Itogon, Benguet abandoned mine site. The preselected microorganisms will be tested for their removal efficiencies both on a batch and continuous basis. Physico-chemical methods such as: chemical precipitation, oxidation, and reduction, electrochemical treatment, evaporative recovery, filtration, ion exchange and membrane technologies have been widely used to remove heavy metal ions from industrial wastewater others (Das et al. 2008). These processes may be ineffective or expensive (Liu et al., 2003; Pamukoglu & Kargi, 2006). On the other hand, biological treatment is considered a promising technique for bioremediation of heavy metals wastewater, since it can degrade organic pollutant in the wastewater and simultaneously transform heavy metals (Wang et al. 2010). Biological treatments constitute an alternative method for the removal of heavy metals. Through his process we eliminate pollution in the area and restore aquatic life.

Materials and Methods

All reagents were analytical grade. Different concentrations of heavy metals such as: Copper Sulphate (CuSO4.5H2O) and Zinc Sulphate (ZnSO4.7H2O) were obtained from Univar. Potassium Dichromate (K2Cr2O7) were from Qualikems made in India, Lead Nitrate (Pb(NO3)2 and Nickel Sulphate (Ni2(SO4)) from Merck. Nutrient Agar (NA) and Nutrient Broth (NB) were from HiMedia made in India. Plastic cups were obtained from recycled bottles.

Wastewater Sampling

The water samples were collected from six (6) different locations in mine tailings at Itogon, Benguet, Mt. Province. The sampling sites are as follows:

Site Label Location (Barangay) in Itogon Benguet
1 Balatoc Virac
2 Ambalanga River, Phase 3, Pink Tunnel, Narba Tunnel Ucab
3 TSF 1 ( Gold Creek) Ucab
4 Convergence Ambalanga, Acupan River Poblacion
5 TSF 2 ( Active) Poblacion
6 Antamok 440 Loacan

Three (3) samples were collected from the center of the river and the 3 samples from the riverbanks. The collected water from each location was poured into a single sample bottle (1 Liter each) to a total of six bottles. Proper coding and identification of the sampling bottle with corresponding pH was labelled. The samples were stored in an ice cooler box and transported to the laboratory for isolation of heavy metal resistant microorganisms.

Isolation of Organisms from Contaminated Wastewater

Microbial samples were sourced from the contaminated wastewater from various locations at the mining sites in Benguet Province. One ml each from wastewater samples were diluted in 9 ml sterile water. Serial dilution up to 10-5 and a 0.1 ml diluted wastewater was then spread to the nutrient agar plates with different concentrations of heavy metals (5, 10, 15, 20 ppm each). The plates were then incubated at room temperature for 24 hours. Then picked and streaked the best selected isolates in NA plates with mixed heavy metals (60 and 100 ppm) concentrations, then incubated at room temperature for 24 hours. Select best isolates for growth at high concentration 100 ppm of heavy metal and streaked on NA test tubes slants, incubated at room temperature for 24 hours and stored in refrigerator for screening.

Screening of Microorganisms and Sample Digestion with HCl

From 150 initial isolates, fifty (50) selected strains were screened in NA plates with different concentrations of heavy metals (i.e., 5, 10, 15, 20, 60 and 100 ppm). Then 4 isolates were grown in 100 ppm concentration of heavy metals Cr, Zn, Ni, and Pb. The resulting 4 strains were cultured in flasks using various concentrations of heavy metals 25, 50, 75 and 100 ppm and nutrient broth. Instead of distilled water, synthetic mines water (SMW) containing (CuSO4.5H2O, ZnSO4.5H2O, K2Cr2O7, Pb(NO3)2, Ni2(SO4) was used as diluent. Then a 100 ml of synthetic mines water and nutrient broth was cultured with 10% inoculant of a selected isolate. After 5 days in a shaker at room temperature the samples were filtered and digested with concentrated HCl and read at the atomic absorption spectrophotometer (PinAAcle 500 AAS, Perkin Elmer, USA).

Characterization of Microorganisms

The 4 best selected isolates were identified at the Philippine Genome Center (UP Diliman, Quezon City, Philippines) The sequences were compared in GenBank database via nucleotide BLAST search (Altschul et al.1990) as isolates A) Acinetobacter sp. junii, B) Acinetobacter sp. tandoii, C) Bacillus cereus, and D) Bacillus toyonensis. The Acinetobacter sp. junii was resistant to zinc, Acinetobacter sp. tandoii to lead, Bacillus cereus to chromium and Bacillus toyonensis to lead. Growth kinetics of the 4 isolates was determined. The growth of strains were optimized for pH and the dry cell weight were analyzed.

Semi Continuous Biosorption Experiment

Inoculum propagation for both screening and for biosorption of heavy metals was done in nutrient broth media. Nutrient agar slants were streaked with stock culture of each chosen organism (Acinetobacter sp. junii) and incubated for 24 hours at room temperature. The organism was further grown in nutrient broth for another 24 hours. The inoculum equivalent to 10 % (vol) was used for immobilization of the isolates in recycled plastic bottle cups. Biosorption was performed in an aerobic filter bed baffled bioreactor containing the immobilized isolate. This was acclimatized for 1 week, then loading of simulated wastewater containing 1000 ppm zinc. Collections of samples were every four hours for 1 month and analyzed using Atomic Absorption Spectrophotometer.

Results and Discussion

Isolates A), Acinetobacter sp. junii, B), Acinetobacter sp. tandoii, C), Bacillus cereus, and D), Bacillus toyonensis

Figure 1: Isolates A), Acinetobacter sp. junii, B), Acinetobacter sp. tandoii, C), Bacillus cereus, and D), Bacillus toyonensis

Sample collection and characterization

Table 1: Characteristics of mine tailings (water samples) from Itogon, Benguet

Parameters
Site pH Color Turbidity, FAU**
1 6.26 Light brown 621
2 6.33 Light brown 50
3 6.65 Dark brown 145
4 6.78 Light brown 176
5 6.96 Clear 10
6 6.84 Clear 29
Standard values* 6.0-9.0

*Based from DENR Administrative Order 2016-08 for Class C waters (Inland waters), **Formazine attenuation unit

Isolation and Screening of Microorganisms

A total of 150 potential microbial strains were isolated from the six (6) different locations in mine tailings at Itogon, Benguet, Mt Province, Philippines. From 150 initial isolates, fifty (50) selected strains were screened in nutrient agar plate with different concentrations of heavy metals (i.e., 5, 10,15, 20, 60 and 100 ppm). Then 4 isolates were grown in 100 ppm concentrations of heavy metals Cr, Zn, Ni, and Pb.

Number of bacterial isolates that survived at different mixed metal (Cu, Ni, Pb, Cr and Zn) concentrations

Figure 2: Number of bacterial isolates that survived at different mixed metal (Cu, Ni, Pb, Cr and Zn) concentrations

Characterization of Microorganisms

Based on the 16S RNA gene sequence the isolates were identified as shown in Table 2.

Table 2: Identification of selected bacterial isolates

Bacterial isolate Identification*
A Acenitobacter junii (82%)
B Acenitobacter tandoii (72%)
C Bacillus cereus (95%)
D Bacillus toyonensis (98%)

*Based from the comparison of sequences in GenBank via nucleotide BLAST search (http://www.ncbi.nlm.nih.gov/BLA)

Batch biosorption experiments

Copper biosorption using bacterial isolates

Results of sample digestion (Table 3.1) showed that at 75 ppm, the removal rate of Cu was highest with Bacillus toyonensis (Figure 3). Undigested samples results were generally lower compared with the digested samples for the 4 identified bacterial isolates.

Table 3.1: Effect of sample digestion for the removal rate of Copper for 4 isolates

A) Acinetobacter sp. junii B) Acinetobacter sp. tandoii
Cu ppm Undigested (Cu) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 6.872 29.483 10.303 9.69 4.674 1.53
6.951 7.28 15.288 24.47 9.078 1.529
8.014 `9.458 -61.249 -25.039
50 ppm 6.059 32.018 11.278 12.93 7.95 13.85
11.986 11.44 16.009 21.01 13.769 17.626
16.275 15.01 13.734 15.975
75 ppm 9.879 2.856 9.713 8.52 12.254 25.32
11.516 10.53 7.979 6.55 7.989 60.701
10.202 8.819 7.855 3.013
100 ppm 4.812 20.392 5.851 11.02 29.79 14.27
12.712 9.77 6.684 20.39 11.779 2.79
11.79 5.503 15.435 10.137

C) Bacillus cereus D) Bacillus toyonensis
Cu ppm Undigested (Cu) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 21.215 -6.3614 17.541 15.76 22.9958 14.58
18.393 20.15 -2.7723 12.98 15.619 -5.9274
20.849 12.9802 14.113 6.173
50 ppm 16.03 21.0884 15.98 15.43 21.4207 20.04
16.918 16.43 28.2978 19.41 15.306 20.6219
16.338 21.0299 15.017 18.0738
75 ppm 2.389 16.1161 4.662 4.78 18.1571 38.26
3.261 3.3 8.181 20.21 5.275 16.1052
1.772 -0.1623 4.402 80.5075
100 ppm 5.729 -3.1611 `10.627 9.55 20.6909 20.51
4.467 5.8 53.3306 27.2 7.91 10.9206
7.216 1.0494 10.109 29.9096

Table 3.2: Selected parameters for highest copper removal

Parameter Result
Bacteria D (Bacillus toyonensis)
Initial concentration (ppm) 75 ppm
Removal 38.26%

Bacteria vs percent removal plot at different initial copper concentrations (ppm)

Figure 3: Bacteria vs percent removal plot at different initial copper concentrations (ppm)

Chromium biosorption using bacterial isolates

At 25 ppm of Cr, results of digested samples (Table 4.1) showed that three isolates, Acinetobacter sp. junii, Bacillus cereus, and Bacillus toyonesis produced the highest removal rates for chromium (Fig. 4), compared with the lower results with the undigested.

Table 4.1: Effect of sample digestion for the removal rate of Chromium for 4 isolates

A) Acinetobacter sp. junii B) Acinetobacter sp. tandoii
Cr ppm Undigested (Cr) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 2.505 66.114 30.538 29.88 34.727 32.4
7.989 7.45 62.981 75.63 28.869 32.873
11.186 97.784 30.232 29.607
50 ppm 11.852 62.358 37.569 31.11 36.43 30.95
7.548 9.21 39.377 55.33 37.114 17.661
8.238 64.241 37.95 38.747
75 ppm -2.347 -7543 18.265 20.11 33.446 43.81
-5.125 -8.195 24.719 49.137
-14.028 -58.23 17.336 48.856
100 ppm 6.678 -7.539 25.594 24.27 28.547 27.73
8.904 7.38 50.755 28.8 22.863 27.812
6.548 6.829 24.347 26.836

C) Bacillus cereus D) Bacillus toyonensis
Cr ppm Undigested (Cr) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 10.563 83.123 86.045 9.421 12.43 100 82.34
16.27 13.91 87.001 11.812 76.399
14.894 88.012 16.055 70.629
50 ppm 10.549 75.129 58.22 13.972 13.35 80.976 68.66
8.251 8.95 69.065 15.026 61.546
8.035 30.464 11.065 63.45
75 ppm -12.775 -62.809 -23.536 -368
-12.986 -195.65 -14.073 -109.104
-16.082 -216.185 -26.184 -192.77
100 ppm 5.795 31.628 33.32 7.674 7.8 7.232 9.63
5.982 6.7 36.011 10.014 -14.16
8.299 32.319 5.717 12.032

Table 4.2: Selected parameters for highest chromium removal.

Parameter Result
Bacteria C (Bacillus cereus)
Initial concentration (ppm) 25 ppm
Removal 86.05%

Bacteria vs percent removal plot at different initial chromium concentrations (ppm)

Figure 4: Bacteria vs percent removal plot at different initial chromium concentrations (ppm)

Lead biosorption using bacterial isolates

In contrast, the elimination of Pb revealed far better efficiencies for the four bacteria. Four strains attained removal efficiencies in the range of 42 -92% at all concentrations investigated (Figure 5). Bacterial isolate A (Acenitobacter junii) was not effective in removing Pb at higher concentrations of 75 mgL-1 and 100 mg L-1. The highest performance efficiency obtained was 92.43% at 75 mg L-1 that was ascribed to isolate D (Bacillus toyonensis).

Table 5.1: Effect of sample digestion for the removal rate of Lead for 4 isolates.

A) Acinetobacter sp. junii B) Acinetobacter sp. tandoii
Pb ppm Undigested (Pb) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 34.445 31.738 50.611 49.84 56.459 55.3
80.417 53.91 58.762 40.31 58.66 58.597
46.868 30.428 40.257 50.829
50 ppm -3.279 21.882 54.903 34.38 49.819 42.2
1.36 9.06 2.428 14.03 43.342 34.022
16.751 17.779 48.229 42.716
75 ppm -2.243 9.629 55.268 67.24 65.589 79.54
-0.021 21.957 60.5 72.6 83.402
-5.322 28.906 73.851 89.616
100 ppm -5.16 83.745 81.79
-6.903 77.773
-10.312 83.863

C) Bacillus cereus D) Bacillus toyonensis
Pb ppm Undigested (Pb) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 94.319 81.343 54.633 41.67 48.367 40.84
88.518 91.12 82.116 80.34 26.461 34.202
90.521 77.548 43.906 39.941
50 ppm 61.65 60.608 56.583 51.12 62.412 63.34
65.165 59.72 68.899 66.192 46.272 68.284
52.352 69.069 50.494 59.337
75 ppm 82.129 92.859 83.887 83.04 94.314 92.43
82.04 77.58 90.611 83.08 80.536 89.003
68.584 65.78 84.706 93.963
100 ppm 89.626 88.596 88.08
88.5 88.68 88.397
87.911 87.261

Table 5.2: Selected parameters for highest lead removal

Parameter Result
Bacteria D (Bacillus toyonensis)
Initial concentration (ppm) 75 ppm
Removal 92.43%

Bacteria vs percent removal plot at different initial lead concentrations (ppm)

Figure 5: Bacteria vs percent removal plot at different initial lead concentrations (ppm)

Nickel biosorption using bacterial isolates

With the case of nickel at 25 ppm the undigested samples (Table 6.1) resulted in the highest removal rate for the three isolates, Acinetobacter sp. tandoii, Bacillus cereus, and Bacillus toyonesis compared with the digested samples, which showed lower removal rate for Ni.

Table 6.1: Effect of sample digestion for the removal rate of Nickel for 4 isolates

A) Acinetobacter sp. junii B) Acinetobacter sp. tandoii
Ni ppm Udigested (Ni) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 22.285 -20.678 20.206 34.55 -24.579
23.687 27.88 -3.304 40.76 -9.759
37.654 -19.721 42.684 -12.21
50 ppm 30.499 -6.275 -10.945 38.87 8.954 8.95
35.352 28.6 -0.34 36.42 49.042 -5.975
19.952 36.423 28.69 -4.874
75 ppm -0.929 0.047 -25.412 14.49 4.649 4.56
-3.322 6.38 7.18 3.64 23.316 5.109
6.382 -5.044 5.668 3.932
100 ppm -16.379 -15.574 -13.706 12.94 -14.39
51.23 47.72 -12.009 11.032 -12.021
44.215 -13.145 14.838 -20.254

C) Bacillus cereus D) Bacillus toyonensis
Ni ppm Undigested (Ni) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 38.864 -2.448 27.104 55.35 -13.124
8.111 38.41 -103.335 1.63 64.936 -8.878
68.267 1.633 73.996 -9.417
50 ppm -21.847 4.076 5.194 10.41 9.997 10.58
-17.463 8.978 5.7 22.059 15.468
-8.312 4.05 3.974 6.277
75 ppm 41.967 7.96 42.973 35.63 -91.878 9.74
32.323 27.61 17.032 11.49 24.933 9.119
8.543 9.472 38.971 10.354
100 ppm -8.637 -9.555 17.064 12.68 0.737 0.737
-25.645 23.75 -10.349 10.558 -2.281
23.746 -10.049 10.407 -1.283

Table 6.2: Selected parameters for highest nickel removal

Parameter Result
Bacteria D (Bacillus toyonensis)
Initial concentration (ppm) 25 ppm
Removal 55.35%

Bacteria vs percent removal plot at different initial nickel concentrations (ppm)

Figure 6: Bacteria vs percent removal plot at different initial nickel concentrations (ppm)

Zinc biosorption using bacterial isolates

All the four selected bacterial strains were found to remove zinc at the lower range concentrations of 25mg L-1 and 50 mgL-1 (Figure 7). Between the two levels, greater removal rates were observed at 75 mg-L (digested). Among the bacteria, isolate A ( Acenitobacter junii) and B (Acenitobacter tandoii ) showed removal capability for aqueous media containing 75 mg-L-1 to 100 mgL-1. On the other hand, no observed removal capacity was observed for isolates C (Bacillus cereus) and D (Bacillus toyonensis) at higher concentrations, from 75mg L-1 to 100 mg L-1. Maximum performance efficiency of 89.14% was attained by (Acenitobacter junii) at 75 mgL-1 comparable with Acenitobacter tandoii.

Table 7.1: Effect of sample digestion for the removal rate of zinc for 4 isolates

A) Acinetobacter sp. junii B) Acinetobacter sp. tandoii
Zn ppm Undigested (Zinc) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 53.772 76.998 -37.795 11.13 77.309 72.83
45.339 48.71 65.122 71.78 19.056 65.594
47.006 73.213 3.2 73.576
50 ppm 62.141 82.626 9.5123 9.89 83.066 81.57
58.879 58.87 77.969 81.1 10.481 78.527
55.583 82.692 9.7054 83.13
75 ppm 88.7 94.967 5.714 4.14 94.468 88.06
65.633 79.99 79.861 89.14 2.882 77.864
85.64 92.59 3.81 91.855
100 ppm 42.315 68.488 2.047 2.85 64.49 76.72
67.541 61.48 82.596 79.34 2.442 80.388
74.595 86.948 4.058 85.292

C) Bacillus cereus D) Bacillus toyonensis
Zn ppm Undigested (Zinc) Digested Undigested Digested
% removal Average of % removal % removal Average of % removal % removal Average of % removal % removal Average of % removal
25 ppm 31.755 -3.525 35.101 38.82 49.867 57.3
34.495 34.36 65.468 63.25 43.664 54.67
36.841 61.038 37.706 67.963
50 ppm 0.76 11.846 4.281 4.47 57.76 32.68
4.281 2.6 21.153 22.34 2.76 13.828
2.76 34.024 6.364 26.454
75 ppm -14.394 12.528 -26.145 6.4277 5.27
-18.791 24.084 15.38 24.753 1.6844
-18.02 9.796 -23.919 7.708
100 ppm -42.091 32.431 -40.17 30.064 27.41
-87.293 -1.778 23.12 -34.288 25.851
-30.278 13.81 -37.406 26.318

Table 7.2: Selected parameters for highest zinc removal

Parameter Result
Bacteria A(Acenitobacter sp. junii)
Initial concentration (ppm) 75 ppm
Removal 89.14%

Bacteria vs percent removal plot at different initial zinc concentrations (ppm)

Figure 7: Bacteria vs percent removal plot at different initial zinc concentrations (ppm)

Aerobic filter bed baffled bioreactor

Figure 8: Aerobic filter bed baffled bioreactor

Zinc Removal using Acenitobacter junii vs time, hrs

Figure 9: Zinc Removal using Acenitobacter junii vs time, hrs

The observed removal rates of Cu was 38.26% and Zn was 89.14% at a concentration of 75 ppm, Cr was 86.045% at 25 ppm, Ni was 36.42% at 50 ppm and Pb was consistently higher with acid digested waste water samples (using synthetic mine water), which was highest at the rate of 92.43% at 75 ppm.

On Table 1, except for sampling site 1 (Balatoc), the pH values obtained from the other areas were almost neutral and below the DENR standard. The color of the water samples appeared as dark brown, light brown and clear. Turbidity was highest at Balatoc site, a heavily silted and turbulent stream which registered a reading of 621 FAU. All sampling sites exhibited elevated levels of chromium, copper and lead that were way above the regulatory limits (DENR 2016). For other metals however, only site 1 (Balatoc), yielded Zn concentration that was higher than the DENR standard for water quality.

Conclusion

Analysis of untreated water samples collected from the six sites in Itogon, Benguet confirmed the presence of heavy metals at different concentrations. Moreover, all sites contain Cr (VI) with higher than the standard values set by DENR for inland waters. Further, high copper and lead concentrations were also present in some of these sites.

Preliminary screening and isolation carried-out with water samples that were collected from an abandoned mining site yielded high metal tolerant isolates. The selected microbes were molecularly identified as species of bacteria, yeast and fungi. The four identified isolates from Benguet mines contaminated wastewater were capable of removing heavy metals such as: copper, chromium, nickel, zinc and lead.

A total of 150 potential microbial strains were isolated from the wastewater collected from mine tailings in Itogon, Benguet, Mt Province, Philippines after 1 day incubation at room temperature in nutrient agar. After the incubation period, microbial growth was observed in 50 isolates. The fifty isolates were then grown for another 1 day in nutrient agar plate. The 4 promising heavy metals-resistant isolates were then identified at the Philippine Genome Center, UP Diliman, Quezon City. These were identified as isolates A) Acinetobacter sp. junii , B) Acinetobacter sp. tandoii, C) Bacillus cereus and D) Bacillus toyonensis. Experiments showed that the isolate Bacillus toyonensis had the highest capability of removing lead by 92.43% at 75 ppm. Isolate Acinetobacter sp. junii had the percentage removal of zinc by 89.14 % at 75 ppm.

Recommendation

i. Further optimization studies on the parameters for continuous and semi-continuous treatment system for heavy metal contaminated wastewater using the selected isolates.

ii. Further studies on the mechanism of heavy metal removal for the selected isolates in the study.

Acknowledgement

The authors would like to thank National Research Council of the Philippines (NRCP) Department of Science and Technology for funding this project, Benguet Mines in Itogon, Benguet for providing wastewater samples in this study. Department of Science and Technology Cordillera Administrative Region (CAR) for their help in this project. They also acknowledge the technical services of Cynthia Borromeo and Bernard Jude Gutierrez from the Waste Management Section, Environment and Biotechnology Division for the determination of heavy metals content of the samples and for the use of Atomic Absorption Spectrophotometer (AAS).