Municipal Wastewater Treatment by Indigenous

International Conference on “Recent Trends on Environment Sustainable Development “(RTESD-2018) February 23-25, 2018 at Vivekananda Global University, Jaipur

Municipal Wastewater Treatment by Indigenous Bacterial Isolates Kanhaiya Kumar Singh1, a), R. C. Vaishya1, b) and Arnav Gupta2, c) 1

Department of Civil Engineering, Motilal Nehru National Institute of Technology-Allahabad, 211004, U.P., India, 2 Department of Civil Engineering, Birla Institute of Technology & Science, Pilani-333031, Rajasthan, India a) Corresponding author: [email protected], [email protected] b) [email protected] c) [email protected]

Abstract. Allowing municipal wastewater to be released in the environment can make a lot of problems. The wastewater may be treated by physicochemical or biological processes. Biological treatment is preferred solution for improving the municipal wastewater treatment. The investigation of bacterial potential for the municipal wastewater treatment was the purpose of this study. Total seven bacterial isolates showed growth on wastewater agar medium. These isolates were identified on the basis of morphological and biochemical test. These isolates were identified as Bacillus Anthracis A9, Serratia Marcescens B7, Listeria Monocytogenes C3, Klebsiella Pneumonia D13, Streptococcus Pneumonia R5, Enterococcus Faecalis S2 and Staphylococcus Aureus T6. The B. Anthracis A9 showed most quiescent to decrease all parameter under study than other isolates exempting from Ammonical nitrogen. Bacillus Anthracis A9 and Staphylococcus Aureus T6 illustrated maximum reduction (41.86%) in BOD each. Bacillus Anthracis A9 and Enterococcus Faecalis S2 implied 83.76% and 82.61% reduction in COD respectively. All the isolates have quiescent to removed phosphate from 18.55% -72.35%. Bacillus Anthracis A9, Serratia Marcescens B7, Listeria Monocytogenes C3 and Enterococcus Faecalis S2 showed diminution in TSS ranging from 43.69%-79.84%. Bacillus Anthracis A9, Serratia Marcescens B7, Listeria Monocytogenes C3, Streptococcus Pneumonia R5 and Enterococcus Faecalis S2 actively exhibited reduction in TDS ranging from 14.4%-81.4%. Keywords: Physicochemical processes, Municipal wastewater, Wastewater agar medium, Bioremediation.

INTRODUCTION All living organisms need water to survive their life; water is an important natural resource. It is variegated application include cooking, drinking, irrigation, washing and industrial enterprises (1). Newly, water contamination is major difficulty as a consequence of unrestrained urbanization which is due to effluent of sewage biased into water bodies and edges to the negative effect on living organism (2). Due to such problems the main global agenda is environmental management, treatment and disposal, wastes recycling, pollution control and prevention and reuse of the wastewater (3, 4). Sewage liquid is a multifarious pattern. These include high concentration of BOD, COD and high dissolved solid. The quality of wastewater is determined by analyzing parameters such as COD, BOD, nitrate, TSS, TDS etc. These parameters implement vital data of the wastewater quality (5, 6). Wastewater is generated by residential, institutional, commercial and industrial establishments and includes household liquid wastes from baths, toilets, kitchens and sinks that are disposed of via sewers. The composition of sewage is differ widely but may contain more than 95% water with pathogens i.e. bacteria, viruses and parasitic worms and nonpathogenic bacteria. Chemical contaminants include organic particles, inorganics particles (7-10).Global attention has been drawn on ways to sustain the environment using microorganism to remediate environmental pollutants because physical and chemical treatment are costly and can lead to production of toxic substance (11-13). Bioremediation involves the use of microorganism to reduce or remove the pollutants from contaminated area which may lead to restoration of the original natural substance without further disruption to the local environment (14-17). Mostly, the oxidized products of organic materials are CO 2 and new microbial cells. The organic matter provides energy and carbon as a nutrient source for cell growth (18, 19). Bioremediation is an efficient, ecological and requires less fancy techniques for water contamination. But the correct microbe should be utilized in the appropriate


place with the precise environmental factors (20, 21). Therefore, the main goal of present study was to examine the ability of indigenous bacteria for bioremediation of the municipal wastewater.

MATERIALS AND METHODS Collection of Municipal Wastewater Sample: Municipal Wastewater sample was collected from railway pulliya MNNIT Campus Allahabad district, India, in pre-uncontaminated bottle as reported by approved procedures from American Public Health Association and reassigned immediately to the testing room.

Identification of Bacterial Strains: Wastewater sample was gradually adulterated and inoculated on the mineral salt agar medium distinctly. Morphologically contrasting colonies were deserted and preserved at 40C on mineral salt agar slants. The deodorized strains were identified by morphological and biochemical characteristics based on Bergey’s Manual of Determinative Bacteriology (22).

Initially Screening of Effective Bacterial Strains for Biodegradation Study : All bacterial strains were injected on wastewater agar medium. The content of the medium per 100 ml was 100 ml uncontaminated wastewater with 2% agar. All plates were injected for 48 hours at 350C. Those strains which illustrated germination on wastewater agar medium were utilized for biodegradation studies.

Analysis of Wastewater Samples: Wastewater samples were analyzed earlier and later the treatment. The parameters contain pH, Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Ammoniacal Nitrogen, Phosphate, Total Suspended Solid (TSS) and Total Dissolved Solid (TDS) under this study. All parameters were measured by using Standard Methods for the Examination of Water and Wastewater (23). Removal efficiencies of all parameters were analyzed according to the following equation: Removal Efficiency (RE %) = (C0-RC)/C0× 100 Where, C0=Initial Concentration before Treatment, RC= Final Concentration after Treatment

Bioremediation Study under Batch Experiments: Every bacterial culture were injected separately in pre-axenic 50 ml wastewater broth i, e. only wastewater, in it 1% peptone were supplemented to optimize the rise of bacterial strains. The beaker was preserved in a shaker at 120 rpm for 48 h at 35 0C. The Optical density of cell suspension was adapted to 0.5 by employing barren saline solution (0.85%) at 600 nm. Inoculum of each strain (10%) was placed in 250 ml beaker including 90 ml contaminated wastewater separately. Together with this, one control beaker was kept containing contaminated wastewater alone. These beakers were placed in shaker at 120 rpm for 72 hours (33). Subsequently after treatment, all illustrations were centrifuged at 10,000 rpm for 20 minutes at 100C and supernatants were preserved for another determination.

RESULTS AND DISCUSSION Identification of Bacterial Strains: All seven bacterial strains were identified through morphological and biochemical characteristics. These strains were identified as Bacillus Anthracis A9, Serratia Marcescens B7, Listeria Monocytogenes C3, Klebsiella Pneumonia D13, Streptococcus Pneumonia R5, Enterococcus Faecalis S2 and Staphylococcus Aureus T6.

Bioremediation Study under Batch Experiments: Total 108 bacterial strains were segregated on mineral salt agar medium. Out of these, 7 bacterial strains displayed rise on wastewater agar medium. The organic and inorganic substances in the wastewater contribute substrate and mineral salt for these strains. These strains were passed down for biodegradation under batch experiments. The introductory pH of water sample was partially acidic while it was marginally alkaline after biological treatment. The control displayed insignificant change in pH.


Biodegradation of amino acids and its polymers existing in municipal wastewater was transformed into ammonia that raises the pH of water sample to somewhat alkaline. The pH changes in wastewater pose that microorganisms have performed activity consequently degrading organic matter. As definition of Biochemical oxygen demand is referred to a measure of the oxygen needed to microbes for the degradation of organic materials. The opening biochemical oxygen demand of water samples was greater than the acceptable limit. An eminent biochemical oxygen demand reveals abundant amount of organic matter, it sparks to oxygen deficiency and initiates anaerobic conditions which would get in reduction of diversity and heterogeneity of aquatic animal kingdom. Anaerobic action will be stilted by organic matter leading to the gathering of toxic substances in water bodies (24-27). The highest percentage ejection of biochemical oxygen demand was noticed by Bacillus Anthracis A9 and Staphylococcus Aureus T6 i.e. 41.86% each followed by Klebsiella Pneumonia D13, Serratia Marcescens B7, Streptococcus Pneumonia R5, Listeria Monocytogenes C3, and Enterococcus Faecalis S2 with 28.94%, 28.51%, 25.36%, 21.91%, and 14.39% respectively whereas control showed only 8.92% in 72 hours (Fig. 1). Related results were recognized by Shrivastava et al., (2013) and Prasad and Manjunath (2011) and initiated that Bacillus Anthracis and Staphylococcus Aureus has organic and inorganic reduction potential whereas analyzing on biodegradation of lipid rich wastewater and Yamuna water correspondingly. Vasconcellos et al., (2009) stated Bacillus Anthracis for bioremediation of cassava processing wastewater while Ravi Kumar et al., (2013) stated removal efficiency of 36.41% biochemical oxygen demand by Bacillus Anthracis for biodegradation of sewage.

Percentage Removal of Biochemical Oxygen Demand in 72 Hours 45

41.86

41.86

40 35

28.94

28.51

30

25.36 21.91

25 20

14.39

15

8.92

10 5 0 A9

B7

C3

D13

R5

S2

T6

Control

Bacterial Strains FIGURE 1.Percentage removal of Biochemical Oxygen Demand in 72 hours by bacterial strains

Chemical Oxygen Demand analysis is the most excellent and very quick procedure for evaluation of organic substances existing in the municipal wastewater sample. In this experiment it was recognized that all bacterial strains exhibiting reduction in chemical oxygen demand after 72 hours (Fig. 2). Bacillus Anthracis A9 demonstrated highest reduction in Chemical Oxygen Demand (83.76%) followed by Enterococcus Faecalis S2, Listeria Monocytogenes C3, Staphylococcus Aureus T6, Klebsiella Pneumonia D13, Serratia Marcescens B7 and Streptococcus Pneumonia R5 with 82.61%, 60.12%, 55.34%, 22.57%, 21.37% and 20.51% respectively and distinguished with control which exhibited 14.91% reduction in Chemical Oxygen Demand. In bioremediation, bacterial strains utilize organic substances as a substrate for their improvement and progress (28-30). These bacterial strains are efficient of generating a large diversity of enzymes that can mineralize complicated organic substances into CO2 and water existing in the municipal wastewater (31, 32). The bacterial strains existing in the municipal wastewater has no momentous effect on removal of organic and inorganic substances as recognized for control. Nevertheless, our strains illustrated auspicious results. Analogous


results were recognized by Gaikwad et al., (2014) and Zhao et al., (2009) raised that Bacillus Anthracis and Staphylococcus Aureus were capable to reduced organic and inorganic substances existing in the wastewater. Mazzucotelli et al., (2014) stated application of Stenotrophomonas for bioremediation of dairy wastewater and Jihong et al., (2008) also stated chemical oxygen demand reducer Aeromonas sp.

Percentage Removal of Chemical Oxygen Demand in 72 Hours 14.91 55.34

83.76

Babterial Strains A9 21.37

B7 C3

82.61

D13 60.12 20.51

22.57

R5 S2 T6 Control

FIGURE 2.Percentage removal of Chemical Oxygen Demand in 72 hours by bacterial strains

In this experiment, none of the bacterial strains demonstrated rebate in Ammoniacal nitrogen. The cause of rising nitrogen concentration was biodegradation of amino acids and nitrogenous substances into ammonia. The phosphate is most deliberated environmental problems because of its donation to the eutrophication process of ponds and other natural waters. It happens in sediments, natural water, wastewater and sludge. The attainable entry of phosphate and nitrogen ions into amphibious environment is through domestic sewage. The devaluation demonstrated by Staphylococcus Aureus T6 (72.35%), Serratia Marcescens B7 and Bacillus Anthracis A9 (58.98%) each, Enterococcus Faecalis S2 (55.06%), Listeria Monocytogenes C3 (47.69%), Streptococcus Pneumonia R5 (41.54%) and Klebsiella Pneumonia D13 (18.55%). The control illustrated no elimination in phosphate concentration (Fig. 3). Analogous results were recognized by Krishnaswamy et al., (2011) through Bacillus Anthracis RS-1 and Serratia Marcescens YLW-7 was analyzed to be effective in phosphate contraction.


Percentage Removal of Phosphate in 72 Hours

0

Bacterial Strains

58.98

72.35

A9 B7 C3 58.98

D13

55.06

R5 S2 T6

47.69

41.54 18.55

Control

FIGURE 3.Percentage Removal of Phosphate in 72 hours by bacterial strains

A huge amount of suspended particles has pernicious effects on floating animal and plant kingdom to reduce the assortment of life in water system and build up oxygen depletion (33-37). The results from Fig.4 advised that the highest reduction in total suspended solids was illustrated by Serratia Marcescens B7 (79.84%) followed by Bacillus Anthracis A9 (67.88%), Enterococcus Faecalis S2 (54.15%), Listeria Monocytogenes C3 (43.69%) whereas Staphylococcus Aureus T6, Klebsiella Pneumonia D13, Streptococcus Pneumonia R5 and control were inadequate to reduce total suspended solid.

Percentage Removal of Total Suspended Solid in 72 Hours 80 60 40

67.88

79.84

20

43.69 54.15

0 A9

B7

0 C3

D13

0 R5

0 S2

0

T6 Control

FIGURE 4.Percentage removal in Total Suspended Solids by bacterial strains

Total dissolved solid (TDS) indicates to all dissolved elements existing in the wastewater. The clearance of wastewater with a greater total dissolve solid level would have conflicting brunt on aquatic life and decreases crop profits if it utilized for irrigation. In present experiment most of the strains implied attrition in total dissolves solid.


Percentage Removal of Total Dissolve Solid in 72 Hours A9 90 80 71.08 70 60 50 40 30 12 20 10 0 0

Control

T6

B7 68.6

C3

14.4 0

81.4

S2

D13 76.74

R5 FIGURE 5.Percentage reduction in Total Dissolve Solids in 72 hours by bacterial strains

The highest reduction in total dissolve solid was exhibited by Enterococcus Faecalis S2 (81.40 %,), Streptococcus Pneumonia R5 (76.74%), Bacillus Anthracis A9 (71.08%), Serratia Marcescens B7 (68.6%), control (12%), Listeria Monocytogenes C3 (14.40%) whereas Staphylococcus Aureus T6 and Klebsiella Pneumonia D13were inadequate to reduce total dissolve solid (Fig. 5). Tomar and Mittal (2014) also invested that Enterococcus Faecalis is capable for degradation of total dissolve solid and total suspended solid.

CONCLUSION In this experiment, total seven bacterial strains namely Bacillus Anthracis A9, Serratia Marcescens B7, Listeria Monocytogenes C3, Klebsiella Pneumonia D13, Streptococcus Pneumonia R5, Enterococcus Faecalis S2 and Staphylococcus Aureus T6 were isolated from municipal wastewater and further utilized for bioremediation study. These strains were exhibited biodegradation of organic materials in term of six different parameters biological oxygen demand, chemical oxygen demand, phosphate, nitrate, total suspended solid and total dissolve solid. Hence, these strains should be utilized for biodegradation of municipal wastewater to prevent water pollution.

ACKNOWLEDGMENTS This research was supported by Environmental Laboratory (Department of Civil Engineering), Biotechnology Laboratory and Centre for Interdisciplinary Research, Motilal Nehru National Institute of Technology Allahabad21104 (India).

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