Microbiology plays a vital role in groundwater quality. The bacterial distribution in groundwater mainly depends on various parameters like nutrient concentrations, availability of the organic matter, grazing pressure by protozoans and viral lysis. To examine the spatial distribution and the impact of biogeochemical parameters on bacterial distribution, groundwater samples are collected at 87 locations along the Indian coastal region during the monsoon. This study revealed E. coli O157:H7, Achromobacter sp., Pseudomonas aeruginosa are the most abundant bacteria. East coast groundwaters are more abundant with E. coli O157:H7 and west coast with Achromobacter sp. The total viable counts of bacteria showed significant linear positive relations with bacterial respiration rates pCO2 levels, TDCHO, TDFAA, and TDPRO and inverse relation with depth and DO saturation. Suggesting that the bacterial abundance and distribution mainly depending on organic matter availability. E. coli O157:H7 consumed more of TDCHO and TDFAA, and Achromobacter sp., more depend on DTPRO and Pseudomonas aeruginosa on TDCHO and TDPRO.
Keywords: Groundwater; Bacteria; E. coli; Monsoon; Carbohydrates; Amino acids; Proteins
Groundwater plays a vital role in human life, and it is one of the crucial sources of water for drinking and irrigation purposes. Since the last five decades, there have been drastic changes in the monsoonal pattern, and the precipitation decreases from year to year . Because of this, the usage of groundwater enhanced dramatically. It is essential to know the groundwater’s geological, chemical, and biological components, especially microbiology (bacteria, virus, and fungus), vital in groundwater health . According to the world health organization (WHO), nearly 3.4 million people die from water-related diseases . Microbial contamination is also happening at the location where the density of people is more . Some bacteria are noticed in groundwater, such as microbial contaminants, which are harmful to human health such as Escherichia coli, Clostridium, Campylobacter, Rhodococcus coprophilous, Enterococci, Arcobacter, Fecal streptococci, Archromobacter, Sulphite reducing Clostridia, Klebsiella, and Pseudomonas aeruginosa . In addition to this, other significant sources of pathogenic microorganisms into surface or groundwater are from agricultural lands, mixing of domestic sewage, underground storage tanks, and unauthorized dumpsites . Several factors influence the survival of microorganisms in groundwater, like pH, precipitation, soil moisture content, soil microflora, temperature, nature of organic carbon, amount of nutrients, dissolved oxygen levels . Diarrhoea, Cholera, Typhoid, and Hepatitis A, are the most common diseases found in many developed and developing countries because of groundwater contamination . From the above studies, it is clear that knowing about the microbiology of underground water is essential. However, very few studies are focused on microbial variability in Indian groundwater systems [9,10]. Therefore, we attempted to understand the qualitative structure of microorganisms (bacteria) in coastal groundwaters along the Indian coast and understand the impact of biogeochemical parameters on their distribution.
India has a very long coastline covering almost 7571km, including east and west coastal regions. There are nine major coastal states: West Bengal-WB, Odisha-OD, Andhra -Pradesh-AP, Tamil Nadu-TN, Kerala-KL, Karnataka-KA Goa-GA, Maharashtra-MH, and Gujarat- GJ, in which GJ has the highest coastline, 1256km. Groundwater samples have been collected from 87 wells along India’s east and west coast during the monsoon season (Figure 1).
Figure 1: Study area and Sample locations along the east and west coast of India.
Groundwater samples were collected from 87 wells along the east and west coast during the monsoon season, and the position of the sample collection locations are given in Table 1. The groundwater temperature was measured using a mercury thermometer, and the conductivity was measured with the help of a conductivity meter (H12003) with an accuracy of ±1%. Groundwater samples were collected with a 5L Niskin sampler and initially dissolved gas (dissolved oxygen-DO) samples were collected in 125ml glass bottles. DO was analyzed by the following method described by Carritt and Capenter . Bacterial Respiration (BR) is measured following the DO incubation method . The groundwater samples were collected in four 125ml bottles for DO analysis. The percentage of DO saturation is computed as
Table 1: Groundwater samples GPS locations of Indian coastal groundwaters during the monsoon period.
DO saturation (%) = (DO measured / DO saturated) X 100
The total dissolved carbohydrates (TDCHO) were measured at 490nm using the spectrophotometric method described in Dubois et al. Dissolved amino acids were measured at 342nm excitation and 452nm emission using the spectrofluorometric method described in Parsons et al. The dissolved proteins were measured at 650nm, using the spectrophotometric method described in Lowry. CO2 was computed with the data of salinity, pH, alkalinity, silicate, phosphate, and dissociations constants from Millero et al. using the sys program . TVC method is used for the enumeration of pathogenic, indicator, and heterotrophic bacteria. R2A agar medium is used to enumerate heterotrophic bacteria and E. coli O157:H7 medium for the computation of pathogenic E. coli O157:H7 bacterium McConkey agar for total coliforms, cetrimide agar for enumeration of Pseudomonas sp. were used. 100μl of groundwater sample was transferred on the different agar plates, and then the selection was spread over the surface and allowed to be absorbed by the medium. Petri dishes were inverted and incubated in a BOD incubator at 35±5°C for two days. The colonies were quantified using a colony counter, and the numbers of colonies are represented in the colonyforming unit (CFU/ml).
Statistica program is used for the mathematical study of the rank correlation matrix (Spearman’s r) and the Student t-test measure. Arc GIS is used to visualize there-dimensional data plots.
The temperature in Indian coastal groundwater varied from 24.00 to 34.00 oC, with an average of 30.21±1.23 oC and higher temperatures are noticed in the east coast (30.92±1.35 oC) groundwater than west coast groundwater (29.61±1.19 oC; t = 4.75, p<0.001, n = 85; Table 2). Groundwater conductivity varied from 0.75 to 48 mS/cm, with an average 6.04±8.75 mS/cm and higher conductive waters are noticed along the east coast (8.84±9.74 mS/cm) water than west coast (3.67±7.04 mS/cm) and the conductivity difference between east and west coast groundwater is statistically significant (t = 2.86, p<0.001, n = 85; Table 2). The pH of coastal groundwater varied from 4.71 to 7.44, with an average of 6.53±0.73, and more neutral waters are on the east coast (6.95±0.30) water than on the west coast (6.17±0.80). The pH difference between east and west coast groundwater is statistically significant (t = 5.15, p<0.001, n = 85; Table 2). DO saturation of coastal groundwater varied from 26.5 to 101.2% with an average of 60.76±35.35%, and more saturated water is noticed along the west coast region (64.56±28.54%) than east coast (57.31±26.08%; Table 2). In contrast, to DO saturation, bacterial respiration (BR) rates are found to be high in the east coast region (4.33±3.14 mg/l/h) than the west coast region (2.87±2.93 mg/l/h; Table 2). Along with bacterial respiration rates, pCO2 concentrations were also found to be high along the east coast groundwaters (36232±19102 μatm) than west coast (26446±10361 μatm; Table 2). The concentrations of labile organic biochemical compounds such as TDCHO, TDFAA and TDPRO were high along with the east coast groundwater than west coast groundwater (Table 2).
DO Saturation (%)
Table 2: Mean (±SD) of groundwater properties along the Indian coast during the wet period.
The total viable heterotrophic bacterial counts in Indian coastal groundwater varied from 1.6x10² to 3x104 CFU/ml, and this is more in east coast groundwater than west coast (Table 2 and Figure 2a). The most common bacteria isolated from the Indian coastal groundwater are E. coli O157:H7 (Figure 2b), Achromobacter sp. (Figure 3a), and Pseudomonas aeruginosa (Figure 3b), Klebsiella sp. (Figure not shown). In the east coast groundwater, E. coli O157:H7, followed by Achromobacter sp., Pseudomonas aeruginosa, are more abundant types in which more dominant bacterium is E. coli O157:H7 (37%) followed by Achromobacter sp. (26%), and minimum Pseudomonas aeruginosa with 7% and other types of unidentified bacteria contribute 30%. In the west coast coastal groundwater, Achromobacter sp. with 40%, followed by E. coli O157:H7 (30%), Pseudomonas aeruginosa (10%), are dominant bacteria and in addition to this Klebsiella sp., also noticed with 7% distribution in this region and other unidentified bacteria contributed 13%. The spatial distribution of bacteria from state to state and from north to south in east and west coast varied significantly, the North East state of coastal India (West Bengal-WB) is equally shared with Achromobacter sp., and E. coli O157:H7 and Odisha state were dominated with E. coli O157:H7 and Pseudomonas aeruginosa, whereas in Andhra Pradesh Achromobacter sp., was dominated after E. coli O157:H7, and in Tamil Nadu, E. coli O157:H7was dominated next to Achromobacter sp., and Pseudomonas aeruginosa (Figure 4). Overall from north to south in the east coast region, E. coli O157:H7 was the dominant bacterium (Figure 4). However, on the west coast, the North West state (Gujarat-GJ) is dominated with Achromobacter sp., bacterium followed by E. coli O157:H7, in contrast to GJ, MH contains more number of E. coli O157:H7 followed by Achromobacter sp., and this state groundwater is contaminated with more unknown bacteria (64%; Figure 4). In GA and KA, E. coli O157:H7 is the dominant bacteria in groundwater, whereas in KL, Achromobacter sp. was predominant, followed by E. coli O157:H7. Overall in west coast groundwater most dominant bacterial type is Achromobacter sp., followed by E. coli O157:H7 (Figure 4).
Figure 2: Distribution of a) TVC (CFU/ml) and b) E. coli O157:H7 in the east and west coast groundwaters of India.
Figure 3: Distribution of a) Achromobacter sp., and b) Pseudomonas aeruginosa in the east and west coast groundwaters of India.
Figure 4: Variability in the percentage of different bacteria in Indian coastal groundwater during the monsoon period.
During study period TVC showed a significant linear positive relation with temperature (r² =0.52, p<0.001; Table 3) and BR (r² =0.45, p<0.01; Table 3), pCO2 (r² =0.32, p<0.05; Table 3), and inverse relation with depth (r² =0.85, p<0.001; Table 3), DO saturation (r² =0.30, p<; Table 3), TDCHO (r² =0.45, p< 0.01; Table 3), TDFAA (r² =0.42, p<0.01; Table 3), and TDPRO (r² =0.35, p<0.05; Table 3). Achromobacter sp., also showed a linear significant positive relations with BR (r² =0.31, p<0.05; Table 3), pCO2 (r² =0.62, p<0.001; Table 3), and inverse relations with depth (r² = 0.86, p<0.001; Table 3), TDCHO (r² =0.30 , p<0.05; Table 3), TDFAA (r² =0.35, p<0.05; Table 3), TDPRO (r²=0.43, p<0.01; Table 3). E. coli O157:H7 also showed same relations with all these biogeochemical parameters as like above. E. coli O157:H7 showed a linear significant positive relations with BR (r²= 0.35, p<0.05; Table 3), pCO2 (r² = 0.30, p<0.05; Table 3), and inverse relations with depth (r² = 0.58, p<0.001; Table 3), DO saturation (r² = 0.30, p<0.05; Table 3), TDCHO (r² = 0.58, p<0.001; Table 3), TDFAA (r² = 0.54, p<0.001; Table 3), TDPRO (r² = 0.32, p<0.05; Table 3). Pseudomonas aeruginosa also showed a linear significant positive relations with BR (r² =0.32, p<0.05; Table 3), pCO2 (r² = 0.38, p< 0.05; Table 3), and inverse relations with depth (r² = 0.67 , p<0.001; Table 3), DO saturation (r² = 0.26; Table 3), TDCHO (r² = 0.55, p<0.001; Table 3), TDFAA (r² = 0.31, p<0.05; Table 3), TDPRO (r² = 0.56, p<0.001; Table 3). Even though the Klebsiella sp., noticed in only two states (KL and MH), but the relations with various biogeochemical parameters are same as like other bacterial groups. It showed a linear significant positive relations with BR (r² = 0.35, p<0.05; Table 3), pCO2 (r² = 0.42 p<0.01; Table 3), and inverse relations with depth (r² = 0.45, p<0.01; Table 3), DO saturation (r² = 0.57, p<0.001; Table 3), TDCHO (r² = 0.25, p<0.1; Table 3), TDFAA (r² = 0.61, p<0.001; Table 3), TDPRO (r² = 0.31, p<0.05; Table 3).
***p <0.001; **p <0.01; *p <0.05. #indicates the correlations taken for only two states (KL and MH).
Table 3: Rank and correlation matrix (Spearman’s, r) of different properties in Indian coastal ground waters during monsoon period.
Groundwater can be contaminated in several ways, natural and anthropogenic. In the natural processes, groundwater is contaminated because of rock weathering, organic matter decomposition, and leaching of inorganic compounds, organic compounds, and microorganisms during the monsoon season. Anthropogenic sources contain mainly septic sewage disposal, agricultural waste, and industrial and domestic waste in open lands. Several studies on groundwater quality noticed that groundwater’s biological contamination mostly occurs through septic sewage and open dump yards [14,15]. India is the second-largest country in the population (1,390,508,600) after China. In the last decade, the Indian population has increased from 123.1 crores to 137.5 crores; concerning enhancement in population, the amount of sewage production also increased. Central pollution control board (https://cpcb.nic.in/status-of-stps) reported that sewage treatment plants have increased from 450 to 1760 in the last ten years all over India (http://www.sulabhenvis.nic.in/Database/ STST_wastewater_2090.aspx). However, most of the rural and urban regions are still using septic tanks at homes. The sewage produced from these is not included in the plants as mentioned above count. A few research studies reported that the sewage from the septic tank could also be used for recycling of water for irrigation and the sludge used as fertilizer for the crop, in such cases, there are many chances for groundwater to be contaminated in more porous regions [16,17]. In some areas, this septic tank sewage is dumping in the ponds and lagoons, which has a significant effect on the groundwater contamination with microbes . Along with this, septic tanks and soakaways increased drastically in housing, public apartments, and industries as the population increased day by day. This may lead to an increase in the seepage of sewage into groundwater through soil , so all these practices are contaminating the groundwater chemically and biologically. Al-Bahry et al.  noticed that bacteria in sewage sludge remain viable for several days to weeks if that kind of material is used as fertilizers; consequently, microbial contamination increases in the environment.
E. coli is one of the most commonly found bacteria in any aquatic system, especially in groundwater, and it is a good indicator of the presence of pathogens in groundwater. In Indian coastal groundwater on the east coast, 40% and 30% of groundwater are contaminated with these bacterial groups on the west coast. OD and WB have the highest E. coil count on the east coast, and KA and GA have the highest count on the west coast. Not only in Indian groundwater but also there are several countries facing problems with E. coli contamination in groundwater, USA , UK, Finland) , Ireland , Canada . E. coli or coliform bacteria is not identified in any 100mL of drinking water sample  standards for microbiological quality of drinking water . E. coli O157:H7 is a faecal pathogen that is regularly isolated from water  and identified as the primary cause of gastrointestinal illness outbreaks . Several studies noticed that these bacteria could also survive in warm and cold and nutrient starving conditions .
Achromobacter sp. are found in the groundwater of WB, AP, GJ, KA, KL, TN states and these bacteria are well known for oxidation of arsenite to arsenate in the groundwater . This bacterium was found more in the west coast groundwater (40%) than in the east (25%). Fertilizers, pesticides, and herbicides, which are used for agricultural purposes, contain a high percentage of arsenic it may contaminate the groundwater with arsenic in these regions. Shahina et al.  noticed that majority of the area in Indian states are significantly contaminated with arsenic (As) pollution and also found the highest contamination in WB (20%), GJ (5%), KA (0.5%), KL (0.5%), and OD (0.4%), AP, TN, and MH with (0.1%). The two most prevalent clinical manifestations of Achromobacter sp. infections in non-CF hosts are pneumonia and bacteremia . Skin and soft tissue infections, urinary tract infections, intra-abdominal organ infections, Central Nervous System (CNS), eye and ear infections are less common, with endocarditis and bone infections being extremely rare .
Pseudomonas aeruginosa is more common in the west coast region (20%), especially in Karnataka and Goa state groundwater and on the east coast (8%) in Odisha and Tamil Nadu state groundwater. Pseudomonas aeruginosa is a prevalent pathogen found in faeces, soil, water, and sewage. It can multiply in water settings and on the surface of appropriate organic materials in contact with water. Pseudomonas aeruginosa is a well-known source of hospital-acquired infections that can lead to significant consequences. It was insulated from various wet surroundings, including sinks, water baths, hot water systems, showers, and spa pools . Pseudomonas aeruginosa can cause deadly progressive lung infections. Warm, wet surroundings, such as swimming pools and spas, are connected with water-related folliculitis and ear infections. Many strains are resistant to various antimicrobial treatments, which increases the organism’s importance in medical settings . Shahina et al.  noticed that most southeast coast groundwater is contaminated with Pseudomonas aeruginosa. This could be possible due to the open dumping of waste, which causes groundwater contamination with microbes.
Klebsiella sp. is the least abundant bacterial group found in Indian coastal groundwater. This is only noticed in west coast groundwater and two states, Kerala (12%) and Maharashtra (14%). This bacterium is the natural occupant of many aquatic settings. They may reproduce rapidly in nutrient-rich fluids such as pulp mill effluent, textile finishing factories, and sugar-cane processing facilities. They are known to colonize tap washers in drinking-water distribution systems. Organisms can develop in water distribution networks. Klebsiella sp. are also found in the faeces of many healthy animals, and they can be found in sewage-contaminated water . Bartram  noticed that Klebsiella sp. was infecting hospital patients, with dissemination linked to frequent patient handling (e.g., in intensive care units). Patients with compromised immune systems, such as the elderly or very young, patients with burns or extensive wounds, those on immunosuppressive medicine, or those infected with HIV/AIDS, are at the greatest danger.
Several biogeochemical properties significantly impact the distribution of bacterial count in the coastal groundwater along the Indian coast. A significant inverse linear relation between Total Viable Count (TVC) to depth (r² = 0.85, p<0.001; Table 3), and a significant linear positive relation between TVC and temperature (r² =0.52, p<0.001; Table 3), which is suggesting that the bacterial number decreasing with depth and warm groundwater has more bacterial count than low-temperature waters. East coast groundwater has warm temperatures and high TVC (Table 1). TVC also shows a significant positive relations with bacterial respiration (r² = 0.45, p<0.01; Table 3) and pCO2 (r² =0.32, p<0.05; Table 3), and inverse relation with labile organic compounds such as total dissolved carbohydrates (TDCHO; r² = 0.45, p<0.01), total dissolved amino acids (TDFAA; r² = 0.42, p<0.01; Table 3), total dissolved proteins (TDPRO; r² = 0.35, p<0.05; Table 3). The linear positive relations with BR and pCO2 and inverse relations with labile organic compounds of TVC, suggesting that bacteria utilized the available labile organic compounds significantly for their growth in coastal groundwater (Figure 5). The presence of high bacteria (TVC (CFU/ml): 7.1x10³±6.6x10³; Table 2) along the east coast groundwater is may be due to the high availability of labile organic compounds such as TDCHO (1.16±0.10 mg/l; Table 2), TDFAA (0.30±0.12 mg/l; Table 2), and TDPRO (0.90±0.3mg/l; Table 2), which also associated with high bacterial respiration rates in the east coast (4.33±3.14mg/l/h; Table 2) than west coast (2.87±2.93 mg/l/h; Table 2) groundwater along the Indian coast. Achromobacter sp. showed a significant relation with TDPRO than TDCHO and TDFAA, suggesting that these bacteria utilizing more proteinaceous food than TDCHO and TDFAA (Table 3; Figure 5). E. coli O157:H7 showed significant relations with TDCHO and TDFAA among three compounds, indicating that these bacteria may be more dependent on these two compounds for their growth (Table 3; Figure 5). Pseudomonas aeruginosa bacteria showed significant relation with TDCHO and TDPRO compounds, suggesting that these bacteria may consume them to meet their growth requirements (Table 3; Figure 5). (Any references supporting this will be useful).
Figure 5: Principal component analysis of different controlling factors on bacterial distribution in Indian coastal ground waters during the monsoon period.
Groundwater is one of the vital water sources for drinking and irrigation in many countries, especially in India. The quality of groundwater is mainly depending on the composition of the chemical ions and biological groups present. The microbiology of the groundwater is another essential tool to assess the health of the water body. In Indian coastal groundwater, the total viable count of bacteria varied from 1.6x10² to 3.0x104 CFU/ml with a high abundance in groundwater along the east coast of India. However, more bacterial diversity is noticed along with the west coast groundwater. The most dominant bacteria type in east coast groundwater is E. coli with 37% and in west coast Achromobacter sp., with 40%. All these bacteria are significantly influenced by biogeochemical properties of groundwater, the inverse linear relations with TDCHO, TDFAA, TDPRO, BR, pCO2 suggesting that the growth of these bacteria depends on the biochemical, organic compounds available in the groundwater along the coastal regions of India. In addition to this, specific bacteria have shown good correlations with specific biochemical compounds, indicating that the growth also depends on the nutrition specificity. Further, more experimental studies are required for better understating the bacteria food selection and chemistry of groundwater.
The senior author (BSK) gratefully acknowledges the SERB - Department of Science and Technology (DST), New Delhi, for fellowship and financial support in the form of SERB-DST-National Post Doctoral Fellowship (Ref No. PDF/2017/001451) for carrying this work. I want to thank the Head of the department, Department of Environmental Science, GITAM (Deemed to be University), for his encouragement and support. I would also like to thank Dr. VVSS Sarma, Deputy Director, and TNR Srinivas, Senior Scientist, CSIRNational Institute of Oceanography, RC- Visakhapatnam, for their continuous support from sampling to manuscript preparation. I am also thankful to Mrs. Vara Lakshmi, Sri Lakshmi and Mr. V. Srinivas, D.N. Rao, B. Karteek, Drs. R. Viswanadham, VR Kumari, DB Rao, G.D Rao, V.V. Ramana and S.A Naidu for their support during the sampling and preparation of the manuscript.
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