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Two-stage removal of nitrate from groundwater using biological and chemical treatments
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 104, No. 2, 129–134. 2007 DOI: 10.1263/jbb.104.129
© 2007, The Society for Biotechnology, Japan
Two-Stage Removal of Nitrate from Groundwater Using Biological and Chemical Treatments Pudukadu Munusamy Ayyasamy,1 Kuppusamy Shanthi,2 Perumalsamy Lakshmanaperumalsamy,2 Soon-Jae Lee,1 Nag-Choul Choi,1 and Dong-Ju Kim1* Department of Earth and Environmental Sciences, Korea University, 5ka, Anam-Dong, Seoul 136-713, Republic of Korea1 and Department of Environmental Sciences, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India2 Received 12 March 2007/Accepted 18 May 2007
In this study, we attempted to treat groundwater contaminated with nitrate using a two-stage removal system: one is biological treatment using the nitrate-degrading bacteria Pseudomonas sp. RS-7 and the other is chemical treatment using a coagulant. For the biological system, the effect of carbon sources on nitrate removal was first investigated using mineral salt medium (MSM) containing 500 mg l–1 nitrate to select the most effective carbon source. Among three carbon sources, namely, glucose, starch and cellulose, starch at 1% was found to be the most effective. Thus, starch was used as a representative carbon source for the remaining part of the biological treatment where nitrate removal was carried out for MSM solution and groundwater samples containing 500 mg l–1 and 460 mg l–1 nitrate, respectively. About 86% and 89% of nitrate were removed from the MSM solution and groundwater samples, respectively at 72 h. Chemical coagulants such as alum, lime and poly aluminium chloride were tested for the removal of nitrate remaining in the samples. Among the coagulants, lime at 150 mg l–1 exhibited the highest nitrate removal efficiency with complete disappearance for the MSM solutions. Thus, a combined system of biological and chemical treatments was found to be more effective for the complete removal of nitrate from groundwater. [Key words: nitrate removal, chemical coagulants, Pseudomonas sp. RS-7, lime, groundwater]
most suitable carbon source. In another study conducted by Shanthi et al. (13), five different carbon sources, namely, glucose, glycerol, starch, methanol and acetic acid were investigated for their potential use in nitrate reduction from a synthetic solution with sewage and dairy sludge as inoculum. They found that there was almost no difference between these carbon sources except for methanol, yielding 95% to 100% nitrate reduction after 96 h. Rashid and Schaefer (14) reported that among various carbon sources, glucose and cellulose induced a very high degree of nitrate removal in a soil under anaerobic condition. Kim et al. (15) studied the denitrification of nitrate in contaminated groundwater supplemented with starch, and a nitrogen removal efficiency of 99.5% at a hydraulic residence time of 1 h was obtained with a C/N ratio of 2.58, corresponding to 4.3 g of soluble starch per 1 g of nitrate. Thus, in our study, commercially available and cheaper carbon sources such as glucose, starch and cellulose were selected as potential candidates for nitrate removal from groundwater. One of the major problems in biological methods used for nitrate removal from water is that secondary treatment is still required for bacterial removal. For the treatment of wastewater, coagulating agents have been widely applied to remove chemical ions and colloidal particles such as mineral colloids, microbial colloids and virus particles (16). Coagu-
The removal of nitrate is essential for water contaminated with nitrate before being utilized since a large amount of nitrate in drinking water often causes a disease called methemoglobinemia and other health disorders such as hypertension (1), increased infant mortality (2), goiter (3), stomach cancer (4), thyroid disorder (5), cytogenetic defects (6) and birth defects (7, 8). Physical and chemical processes such as reverse osmosis, ion exchange, electrodialysis and chemical denitrification have been developed for nitrate removal from water. Although these techniques are effective in removing nitrate from contaminated water, they are very expensive for pilot scale operation with a limited potential application (9, 10). Owing to these limitations in the removal of nitrate from water and/or wastewater, the most versatile and widely used technology is biological denitrification (10, 11). Several authors have studied the effects of carbon sources on the removal of nitrate from water and/or wastewater for biological processes. Gomez et al. (12) studied the effectiveness of three selected carbon sources, namely, sucrose, ethanol and methanol for the removal of nitrate from contaminated groundwater. They found that ethanol was the * Corresponding author. e-mail: [email protected] phone: +82-2-3290-3177 fax: +82-2-3290-3189 129
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lants used for wastewater treatment are predominantly inorganic salts of iron and aluminium (17). Some authors reported the use of aluminium sulfate (alum) as a coagulating agent for the treatment of steel mill wastewater (18), pulping effluents (19), phosphorous-containing water (20), and metal-contaminated wastewater (21). Jiang and Graham (17) reported that alum, ferric sulfate and ferric chloride are effective for phosphorous removal. Lime acts as a precipitant for phosphates, many trace metals and bacteria, and was used as a coagulant for the removal of suspended and colloidal materials in municipal wastewater (22–24). Poly aluminium chloride (PAC) is a water-soluble polymer that precipitates insoluble matter of aluminium poly hydroxide by absorbing suspended pollutants. It is used as a coagulant or flocculant during the treatments of drinking water and wastewater and is often used for pH adjustment (25). Although several authors used some precipitants alone for the removal of suspended and colloidal particles, a combined treatment using microorganisms and coagulants has not yet been conducted for the removal of nitrate. Therefore, in this study a two-stage treatment system is attempted using biological and chemical methods for a more efficient removal of nitrate from groundwater. MATERIALS AND METHODS Bacterial isolation Groundwater and soil samples contaminated with nitrate were collected under aseptic conditions using sterile bottles from Jodhpur and Poli districts in Rajasthan, India. Bacterial populations were estimated by the pour plate technique using nutrient agar (beef extract, 3 g l–1; yeast extract, 3 g l–1; peptone, 5 g l–1; sodium chloride, 5 g l–1; agar, 20 g l–1). Well-defined bacterial isolates were selected on the basis of colony and morphological characteristics and transferred to nutrient agar, and they were identified up to the genus level after purification (26). Nitrate reduction was tested on potassium nitrate broth (peptone, 5 g l–1; beef extract, 3 g l–1; sodium chloride, 5 g l–1; potassium nitrate, 5 g l–1). The ability of the isolates to reduce nitrate to nitrite and ammonia was determined by the addition of 1 ml Nessler’s reagent to the cultures. The appearance of yellow orange showed that nitrate and/or nitrite has been reduced to ammonia. Based on the intensity of the color, the isolates were grouped into low (+), moderate (++), and high (+++) nitrate reduction. Biological treatment Nitrate removal of the best isolate was first evaluated in a preliminary test by supplementing MSM (potassium dihydrogen phosphate, 0.1 g l–1; dipotassium hydrogen phosphate,1 g l–1; ammonium chloride, 0.5 g l–1; calcium chloride, 0.005 g l–1; magnesium sulphate, 0.1 g l–1; sodium silicate, 0.05 g l–1; pH 7) with three carbon sources (glucose, starch and cellulose) at various concentrations ranging from 0% to 3%. From the preliminary test, starch at 1% showed the highest removal efficiency of nitrate (Fig. 1); thus, it was selected as a representative carbon source for bacterial growth in the nitrate removal studies of MSM solution and groundwater samples. The bacterial isolate was inoculated to the nutrient broth and incubated at room temperature for 16 h. The cells were harvested at the stationary growth phase and the concentration of cells was adjusted to 0.5 OD using sterile distilled water. One milliliter of an inoculum with 0.5 OD (107 CFU ml–1) was added to 100 ml of MSM containing 500 mg l–1 nitrate and incubated for 72 h in a mechanical shaker (120 rpm). The control case was also maintained with the same concentration of nitrate but without a carbon source and a bacterial inoculum. Every 12 h, the utilization of nitrate was determined by the disulphonic
FIG. 1. Effect of carbon sources at different concentrations on nitrate removal in MSM containing 500 mg l–1 nitrate. (a) Glucose. (b) Starch. (c) Cellulose. Crosses, No carbon source and no bacteria; diamonds, 0%; squares, 1%; triangles, 2%; circles, 3%.
acid method (27). At the same time, the conversion to nitrite was also determined. Similarly, 1 ml of the cell suspension with 0.5 OD was inoculated to 100 ml of the groundwater sample collected from the Jodhpur district of Rajasthan containing 460 mg l–1 nitrate, which was supplemented with 1% starch. The flask was incubated at room temperature for 72 h with shaking. At 12 h interval, the utilization of nitrate was determined. At the same time, bacterial population and the amount of nitrite were also determined. The initial dissolved oxygen (DO) concentration of the groundwater sample was 8.2 mg l–1 and the final DO concentration after treatment was 2.7 mg l–1. Thus, the aerobic condition during denitrification (28–30) was maintained throughout the biological treat-
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ment. Chemical treatment A second set of tests was made to remove the remaining nitrate using chemical coagulants since the complete removal of nitrate was not attained with the bacterial treatment alone. Chemical coagulants such as alum, lime and PAC at various concentrations (50, 100, 150, 200, 250, 300, 350, and 400 mg l–1) were added to the MSM and groundwater samples containing 70 and 50 mg l–1 nitrate, respectively, after bacterial treatment. The flasks were incubated with agitation for 1 min at 120 rpm and 10 min at 15 rpm (31). Then, the samples were allowed to settle for 30 min and after sand filtration, nitrate concentration was determined.
RESULTS AND DISCUSSION Bacterial isolation The bacteria isolates Bacillus sp., Micrococcus sp., Alcaligenes sp., Moroxella sp., Pseudomonas sp., Corynebacterium sp., Acinetobacter sp. and members of Enterobacteriaceae were isolated from the nitratecontaminated groundwater and soil samples. Among them, Pseudomonas sp. (RS-7) was found to be the most efficient bacteria for nitrate removal from the color intensity in the nitrate reduction test (Table 1). Therefore, we selected the bacterial species as the best isolate and used them for nitrate removal throughout this study. These species have been used as denitrifying bacteria for nitrate removal studies (32– 37). Effect of carbon sources on nitrate removal Nitrate reductions in MSM solutions containing 500 mg l–1 nitrate associated with the biological treatment system using RS-7 are shown in Fig. 1 for different carbon sources and concentrations. For all carbon sources, nitrate concentrations decreased rapidly up to 12 h and then decreased gradually thereafter. The rapid decrease is attributed to the active bacterial growth during the exponential stage using nitrate as a nutrient source since nitrate was the only nutrient in the MSM solutions when a carbon source was not supplemented. After 72 h, the initial nitrate concentration of 500 mg l–1 was reduced to about 100 mg l–1. However, the concentration of ammonia was relatively low (8 mg l–1). The formation of a small amount of ammonia is mainly due to the formation of nitrous oxide through hydroxylamine by dissimilatory nitrite reduction (38) or the incorporation of ammonia into cell materials since the products of the reductive pathway of nitrate to ammonia serve as a nutritional supplement for many bacteria if reduced nitrogen for assimilation is in short supply, and the assimilatory demands are readily met. From the four different concentrations tested, 1% carbon sources gave the highest denitrification rate regardless of the type of carbon sources, whereas 0% gave the lowest denitrification rate. Among the three candidate carbon sources, starch exhibited the highest nitrate reduction rate. This indicates that our denitrifier, RS-7, utilizes starch as a carbon source more than the other carbon sources such as glucose and cellulose. Our results are partly in contradiction with other findings in that glucose was found to be the best carbon source among glucose, cellulose and lignin (14), and that ethanol was reported as the best carbon source among sucrose, ethanol and methanol for enhancing bacterial denitrification (12). Our results, however, are in good agreement with the results of another study (15) where the use of starch as a carbon
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TABLE 1. Result of nitrate reduction test Nitrate/nitrite/ ammonia 1 RS-1 Bacillus + 2 RS-2 Micrococcus − 3 RS-3 Bacillus + 4 RS-4 Moroxella + 5 RS-5 Alcaligenes + 6 RS-6 Corynebacterium + 7 RS-7 Pseudomonas +++ 8 RW-1 Bacillus ++ 9 RW-2 Micrococcus + 10 RW-3 Bacillus ++ 11 RW-4 Alcaligenes ++ 12 RW-5 Pseudomonas + 13 RW-6 Corynebacterium + 14 RW-7 Acinetobacter + 15 RW-7 Enterobacteriaceae + RS, Rajasthan soil; RW, Rajasthan water; +++, high nitrate reduction; ++, moderate nitrate reduction; +, low nitrate reduction; −, no nitrate reduction. No.
Strain no.
Genus
source in the on-site biological treatment of nitrate in groundwater was successful with a nitrogen removal efficiency of 99.5% and a C/N ratio of 2.58 corresponding to 4.3 g of soluble starch per 1 g of NO3-N. The reason why starch exhibited the highest nitrate reduction rate can be explained by the fact that our bacterial culture is amylolitic (starch degraders) and is capable of utilizing starch as a carbon source. Nitrate removal in MSM and groundwater using bacteria The results of nitrate removal and the formation of nitrite along with bacterial growth for the MSM samples are shown in Fig. 2. In the presence of starch, a rapid decrease in nitrate concentration from 500 to 140 mg l–1 was observed in 12 h, after which a gradual decrease followed (Fig. 2a). When starch was not present in the MSM solution, the overall nitrate removal rate was relatively lower (≈ 55%) than in the case when starch was supplemented (≈ 86%). This was due to the faster bacterial growth corresponding to a population sevenfold larger than the original population at 72 h when starch was supplemented in the MSM solution as an additional carbon source. The maximum growth of 0.71 × 106 CFU ml–1 was attained in the MSM solution; however, the growth was much less and linear in the samples without starch. RS-7 reduced the maximum level of nitrate from 500 to 70 mg l–1 (86%) in MSM supplemented with starch at 1% after 72 h (Fig. 2a). The formation of nitrite is shown in Fig. 2b during nitrate reduction. The case with starch supplementation showed eightfold higher nitrite concentration than the case without starch supplementation with a concentration of about 0.8 NO2 mg l–1 after 36 h. For either case with or without starch supplementation, nitrite formation occurred during nitrate reduction in our study, suggesting that nitrite accumulation might have resulted from the difference in the rates of nitrate and nitrite reduction (39). However, nitrite accumulation during bacterial activity seems to depend on the carbon sources. For instance in the study of nitrate removal by Pinar and Ramos (40), nitrite accumulated when sucrose was used as the carbon source but it was not formed when glycerol was used. In another study (12), nitrite was formed when sucrose was used as the carbon source but it was not
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FIG. 2. Microbial reduction of nitrate in MSM in presence and absence of starch. (a) Squares, Nitrate reduction without starch; diamonds, nitrate reduction with 1% starch; triangles, bacterial growth without starch; crosses, bacterial growth with 1% starch. (b) Squares, Nitrite formation without starch; diamonds, nitrite formation with 1% starch.
formed when ethanol or methanol was used. The results of changes in nitrate concentration and nitrite formation for the groundwater samples are shown in Fig. 3. Similar results were obtained for the trend of nitrate reduction (89%) and bacterial growth with the MSM solution except for a rather small difference in nitrite formation between the cases with and without starch supplementation. The small difference may be due to the presence of other microorganisms such as ammonifiers that prevent the formation of nitrite since the groundwater sample was not sterilized prior to the inoculation with RS-7. Nitrate removal in MSM and groundwater using coagulants The effect of adding 1% starch on nitrate removal using the combined system of biological and chemical treatments is shown in Fig. 4a and 4b. As can be observed from the figure, the addition of 1% starch to the groundwater sample during the first stage of biological treatment lowered the nitrate concentration from 460 to 50 mg l–1, whereas for the case without starch supplementation, it was lowered to only 108 mg l–1. During bacterial treatment of MSM containing 500 mg l–1 nitrate supplemented with 1% starch for 72 h, it was found that 70 mg l–1 nitrate remained in the samples. Among the various concentrations of coagu-
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FIG. 3. Microbial reduction of nitrate in groundwater in presence and absence of starch. (a) Squares, Nitrate reduction without starch; filled diamonds, nitrate reduction with 1% starch; triangles, bacterial growth without starch; crosses, bacterial growth with 1% starch. (b) Squares, Nitrite formation without starch; diamonds, nitrite formation with 1% starch.
lants used, lime at 150 mg l–1 reduced nitrate concentration from 70 to 0 mg l–1 (100% removal), followed by PAC from 70 to 4.8 mg l–1 (93.1% removal) and by alum from 70 to 8 mg l–1 nitrate (88.5% removal) (Fig. 4a). There was no further significant decrease in nitrate concentration when the dosage of the coagulants was increased above 150 mg l–1. Hence, the optimum dosage for the effective removal of nitrate from MSM and the groundwater samples was found to be 150 mg l–1 for lime and PAC. Similarly, in the groundwater samples the level of nitrate was reduced from 50 to 3.1 and 4.9 mg l–1, respectively, when lime and PAC were used at the concentration of 150 mg l–1 (Fig. 4b). The use of lime as a chemical coagulant gave the highest nitrate removal rate of 93.8%, whereas for alum, the nitrate removal rate was only 74.3%. The removal rate of nitrate was invariable as the concentrations of alum, lime and PAC were increased. The use of bacterial cultures appears to be efficient in reducing nitrate concentration prior to chemical treatment as well as in promoting the attachment of chemical coagulants onto biomass and thus achieving rapid precipitation of these biomass-coagulant complexes. Grabow et al. (22) and
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Pseudomonas sp. (RS-7) was found to be the most efficient in terms of nitrate reduction. From the above results, it could be concluded that nitrate reduction by RS-7 was affected by various carbon sources. The bacterial growth and nitrate reduction rates were high in MSM and groundwater supplemented with 1% starch as the sole carbon source compared with those in the media supplemented with glucose and cellulose under aerobic conditions at an optimum temperature of 30°C. Hence, 1% starch could be used as the best carbon source and concentration for nitrate removal in groundwater for the bacteria used in this study. According to the World Health Organization, the permissible limit of nitrate in drinking water is 45 mg l–1 (43). In this study using a bacterial inoculum, nitrate could not be reduced below the permissible limit. However, subsequent chemical treatment with lime at 150 mg l–1 decreased the concentration of nitrate below the permissible level. Therefore, it is suggested that bacterial treatment followed by chemical treatment will be more effective in treating nitrate-contaminated water. Lime was found to be an ideal material for the removal of remaining nitrate and also for elevating pH near neutral so that water could be directly used after disinfection. ACKNOWLEDGMENTS The authors are thankful to the Head of the Department of Environmental Sciences, Bharathiar University for providing facilities for the conduct of this research.
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Fig. 4. Nitrate removal by chemical coagulants from MSM and groundwater treated with bacteria. (a) Nitrate reduction in groundwater without starch. (b) Nitrate reduction in groundwater with 1% starch. (c) Nitrate reduction in MSM with 1% starch. Diamonds, Alum; squares, lime; triangles, PAC.
Dziubek and Kowal (24) have reported that lime acts as an effective precipitant for phosphates, many trace metals and bacteria and as a coagulant for the removal of suspended and colloidal materials in municipal wastewater. Coagulation removed 74–99.4% of E. coli and coliforms and the removal rate of protozoan cysts by coagulation and sedimentation exceeded 90% (41). Under laboratory conditions, coagulation and flocculation were effective in removing 90– 99% of viruses from water (42). In this study, the genera of Bacillus, Micrococcus, Alcaligenes, Moroxella, Pseudomonas, Corynebacterium, Acinetobacter and members of Enterobacteriaceae were isolated from groundwater and soil samples contaminated with nitrate and were found to be nitrate reducers. Among them,
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© 2007, The Society for Biotechnology, Japan
Two-Stage Removal of Nitrate from Groundwater Using Biological and Chemical Treatments Pudukadu Munusamy Ayyasamy,1 Kuppusamy Shanthi,2 Perumalsamy Lakshmanaperumalsamy,2 Soon-Jae Lee,1 Nag-Choul Choi,1 and Dong-Ju Kim1* Department of Earth and Environmental Sciences, Korea University, 5ka, Anam-Dong, Seoul 136-713, Republic of Korea1 and Department of Environmental Sciences, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India2 Received 12 March 2007/Accepted 18 May 2007
In this study, we attempted to treat groundwater contaminated with nitrate using a two-stage removal system: one is biological treatment using the nitrate-degrading bacteria Pseudomonas sp. RS-7 and the other is chemical treatment using a coagulant. For the biological system, the effect of carbon sources on nitrate removal was first investigated using mineral salt medium (MSM) containing 500 mg l–1 nitrate to select the most effective carbon source. Among three carbon sources, namely, glucose, starch and cellulose, starch at 1% was found to be the most effective. Thus, starch was used as a representative carbon source for the remaining part of the biological treatment where nitrate removal was carried out for MSM solution and groundwater samples containing 500 mg l–1 and 460 mg l–1 nitrate, respectively. About 86% and 89% of nitrate were removed from the MSM solution and groundwater samples, respectively at 72 h. Chemical coagulants such as alum, lime and poly aluminium chloride were tested for the removal of nitrate remaining in the samples. Among the coagulants, lime at 150 mg l–1 exhibited the highest nitrate removal efficiency with complete disappearance for the MSM solutions. Thus, a combined system of biological and chemical treatments was found to be more effective for the complete removal of nitrate from groundwater. [Key words: nitrate removal, chemical coagulants, Pseudomonas sp. RS-7, lime, groundwater]
most suitable carbon source. In another study conducted by Shanthi et al. (13), five different carbon sources, namely, glucose, glycerol, starch, methanol and acetic acid were investigated for their potential use in nitrate reduction from a synthetic solution with sewage and dairy sludge as inoculum. They found that there was almost no difference between these carbon sources except for methanol, yielding 95% to 100% nitrate reduction after 96 h. Rashid and Schaefer (14) reported that among various carbon sources, glucose and cellulose induced a very high degree of nitrate removal in a soil under anaerobic condition. Kim et al. (15) studied the denitrification of nitrate in contaminated groundwater supplemented with starch, and a nitrogen removal efficiency of 99.5% at a hydraulic residence time of 1 h was obtained with a C/N ratio of 2.58, corresponding to 4.3 g of soluble starch per 1 g of nitrate. Thus, in our study, commercially available and cheaper carbon sources such as glucose, starch and cellulose were selected as potential candidates for nitrate removal from groundwater. One of the major problems in biological methods used for nitrate removal from water is that secondary treatment is still required for bacterial removal. For the treatment of wastewater, coagulating agents have been widely applied to remove chemical ions and colloidal particles such as mineral colloids, microbial colloids and virus particles (16). Coagu-
The removal of nitrate is essential for water contaminated with nitrate before being utilized since a large amount of nitrate in drinking water often causes a disease called methemoglobinemia and other health disorders such as hypertension (1), increased infant mortality (2), goiter (3), stomach cancer (4), thyroid disorder (5), cytogenetic defects (6) and birth defects (7, 8). Physical and chemical processes such as reverse osmosis, ion exchange, electrodialysis and chemical denitrification have been developed for nitrate removal from water. Although these techniques are effective in removing nitrate from contaminated water, they are very expensive for pilot scale operation with a limited potential application (9, 10). Owing to these limitations in the removal of nitrate from water and/or wastewater, the most versatile and widely used technology is biological denitrification (10, 11). Several authors have studied the effects of carbon sources on the removal of nitrate from water and/or wastewater for biological processes. Gomez et al. (12) studied the effectiveness of three selected carbon sources, namely, sucrose, ethanol and methanol for the removal of nitrate from contaminated groundwater. They found that ethanol was the * Corresponding author. e-mail: [email protected] phone: +82-2-3290-3177 fax: +82-2-3290-3189 129
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lants used for wastewater treatment are predominantly inorganic salts of iron and aluminium (17). Some authors reported the use of aluminium sulfate (alum) as a coagulating agent for the treatment of steel mill wastewater (18), pulping effluents (19), phosphorous-containing water (20), and metal-contaminated wastewater (21). Jiang and Graham (17) reported that alum, ferric sulfate and ferric chloride are effective for phosphorous removal. Lime acts as a precipitant for phosphates, many trace metals and bacteria, and was used as a coagulant for the removal of suspended and colloidal materials in municipal wastewater (22–24). Poly aluminium chloride (PAC) is a water-soluble polymer that precipitates insoluble matter of aluminium poly hydroxide by absorbing suspended pollutants. It is used as a coagulant or flocculant during the treatments of drinking water and wastewater and is often used for pH adjustment (25). Although several authors used some precipitants alone for the removal of suspended and colloidal particles, a combined treatment using microorganisms and coagulants has not yet been conducted for the removal of nitrate. Therefore, in this study a two-stage treatment system is attempted using biological and chemical methods for a more efficient removal of nitrate from groundwater. MATERIALS AND METHODS Bacterial isolation Groundwater and soil samples contaminated with nitrate were collected under aseptic conditions using sterile bottles from Jodhpur and Poli districts in Rajasthan, India. Bacterial populations were estimated by the pour plate technique using nutrient agar (beef extract, 3 g l–1; yeast extract, 3 g l–1; peptone, 5 g l–1; sodium chloride, 5 g l–1; agar, 20 g l–1). Well-defined bacterial isolates were selected on the basis of colony and morphological characteristics and transferred to nutrient agar, and they were identified up to the genus level after purification (26). Nitrate reduction was tested on potassium nitrate broth (peptone, 5 g l–1; beef extract, 3 g l–1; sodium chloride, 5 g l–1; potassium nitrate, 5 g l–1). The ability of the isolates to reduce nitrate to nitrite and ammonia was determined by the addition of 1 ml Nessler’s reagent to the cultures. The appearance of yellow orange showed that nitrate and/or nitrite has been reduced to ammonia. Based on the intensity of the color, the isolates were grouped into low (+), moderate (++), and high (+++) nitrate reduction. Biological treatment Nitrate removal of the best isolate was first evaluated in a preliminary test by supplementing MSM (potassium dihydrogen phosphate, 0.1 g l–1; dipotassium hydrogen phosphate,1 g l–1; ammonium chloride, 0.5 g l–1; calcium chloride, 0.005 g l–1; magnesium sulphate, 0.1 g l–1; sodium silicate, 0.05 g l–1; pH 7) with three carbon sources (glucose, starch and cellulose) at various concentrations ranging from 0% to 3%. From the preliminary test, starch at 1% showed the highest removal efficiency of nitrate (Fig. 1); thus, it was selected as a representative carbon source for bacterial growth in the nitrate removal studies of MSM solution and groundwater samples. The bacterial isolate was inoculated to the nutrient broth and incubated at room temperature for 16 h. The cells were harvested at the stationary growth phase and the concentration of cells was adjusted to 0.5 OD using sterile distilled water. One milliliter of an inoculum with 0.5 OD (107 CFU ml–1) was added to 100 ml of MSM containing 500 mg l–1 nitrate and incubated for 72 h in a mechanical shaker (120 rpm). The control case was also maintained with the same concentration of nitrate but without a carbon source and a bacterial inoculum. Every 12 h, the utilization of nitrate was determined by the disulphonic
FIG. 1. Effect of carbon sources at different concentrations on nitrate removal in MSM containing 500 mg l–1 nitrate. (a) Glucose. (b) Starch. (c) Cellulose. Crosses, No carbon source and no bacteria; diamonds, 0%; squares, 1%; triangles, 2%; circles, 3%.
acid method (27). At the same time, the conversion to nitrite was also determined. Similarly, 1 ml of the cell suspension with 0.5 OD was inoculated to 100 ml of the groundwater sample collected from the Jodhpur district of Rajasthan containing 460 mg l–1 nitrate, which was supplemented with 1% starch. The flask was incubated at room temperature for 72 h with shaking. At 12 h interval, the utilization of nitrate was determined. At the same time, bacterial population and the amount of nitrite were also determined. The initial dissolved oxygen (DO) concentration of the groundwater sample was 8.2 mg l–1 and the final DO concentration after treatment was 2.7 mg l–1. Thus, the aerobic condition during denitrification (28–30) was maintained throughout the biological treat-
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ment. Chemical treatment A second set of tests was made to remove the remaining nitrate using chemical coagulants since the complete removal of nitrate was not attained with the bacterial treatment alone. Chemical coagulants such as alum, lime and PAC at various concentrations (50, 100, 150, 200, 250, 300, 350, and 400 mg l–1) were added to the MSM and groundwater samples containing 70 and 50 mg l–1 nitrate, respectively, after bacterial treatment. The flasks were incubated with agitation for 1 min at 120 rpm and 10 min at 15 rpm (31). Then, the samples were allowed to settle for 30 min and after sand filtration, nitrate concentration was determined.
RESULTS AND DISCUSSION Bacterial isolation The bacteria isolates Bacillus sp., Micrococcus sp., Alcaligenes sp., Moroxella sp., Pseudomonas sp., Corynebacterium sp., Acinetobacter sp. and members of Enterobacteriaceae were isolated from the nitratecontaminated groundwater and soil samples. Among them, Pseudomonas sp. (RS-7) was found to be the most efficient bacteria for nitrate removal from the color intensity in the nitrate reduction test (Table 1). Therefore, we selected the bacterial species as the best isolate and used them for nitrate removal throughout this study. These species have been used as denitrifying bacteria for nitrate removal studies (32– 37). Effect of carbon sources on nitrate removal Nitrate reductions in MSM solutions containing 500 mg l–1 nitrate associated with the biological treatment system using RS-7 are shown in Fig. 1 for different carbon sources and concentrations. For all carbon sources, nitrate concentrations decreased rapidly up to 12 h and then decreased gradually thereafter. The rapid decrease is attributed to the active bacterial growth during the exponential stage using nitrate as a nutrient source since nitrate was the only nutrient in the MSM solutions when a carbon source was not supplemented. After 72 h, the initial nitrate concentration of 500 mg l–1 was reduced to about 100 mg l–1. However, the concentration of ammonia was relatively low (8 mg l–1). The formation of a small amount of ammonia is mainly due to the formation of nitrous oxide through hydroxylamine by dissimilatory nitrite reduction (38) or the incorporation of ammonia into cell materials since the products of the reductive pathway of nitrate to ammonia serve as a nutritional supplement for many bacteria if reduced nitrogen for assimilation is in short supply, and the assimilatory demands are readily met. From the four different concentrations tested, 1% carbon sources gave the highest denitrification rate regardless of the type of carbon sources, whereas 0% gave the lowest denitrification rate. Among the three candidate carbon sources, starch exhibited the highest nitrate reduction rate. This indicates that our denitrifier, RS-7, utilizes starch as a carbon source more than the other carbon sources such as glucose and cellulose. Our results are partly in contradiction with other findings in that glucose was found to be the best carbon source among glucose, cellulose and lignin (14), and that ethanol was reported as the best carbon source among sucrose, ethanol and methanol for enhancing bacterial denitrification (12). Our results, however, are in good agreement with the results of another study (15) where the use of starch as a carbon
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TABLE 1. Result of nitrate reduction test Nitrate/nitrite/ ammonia 1 RS-1 Bacillus + 2 RS-2 Micrococcus − 3 RS-3 Bacillus + 4 RS-4 Moroxella + 5 RS-5 Alcaligenes + 6 RS-6 Corynebacterium + 7 RS-7 Pseudomonas +++ 8 RW-1 Bacillus ++ 9 RW-2 Micrococcus + 10 RW-3 Bacillus ++ 11 RW-4 Alcaligenes ++ 12 RW-5 Pseudomonas + 13 RW-6 Corynebacterium + 14 RW-7 Acinetobacter + 15 RW-7 Enterobacteriaceae + RS, Rajasthan soil; RW, Rajasthan water; +++, high nitrate reduction; ++, moderate nitrate reduction; +, low nitrate reduction; −, no nitrate reduction. No.
Strain no.
Genus
source in the on-site biological treatment of nitrate in groundwater was successful with a nitrogen removal efficiency of 99.5% and a C/N ratio of 2.58 corresponding to 4.3 g of soluble starch per 1 g of NO3-N. The reason why starch exhibited the highest nitrate reduction rate can be explained by the fact that our bacterial culture is amylolitic (starch degraders) and is capable of utilizing starch as a carbon source. Nitrate removal in MSM and groundwater using bacteria The results of nitrate removal and the formation of nitrite along with bacterial growth for the MSM samples are shown in Fig. 2. In the presence of starch, a rapid decrease in nitrate concentration from 500 to 140 mg l–1 was observed in 12 h, after which a gradual decrease followed (Fig. 2a). When starch was not present in the MSM solution, the overall nitrate removal rate was relatively lower (≈ 55%) than in the case when starch was supplemented (≈ 86%). This was due to the faster bacterial growth corresponding to a population sevenfold larger than the original population at 72 h when starch was supplemented in the MSM solution as an additional carbon source. The maximum growth of 0.71 × 106 CFU ml–1 was attained in the MSM solution; however, the growth was much less and linear in the samples without starch. RS-7 reduced the maximum level of nitrate from 500 to 70 mg l–1 (86%) in MSM supplemented with starch at 1% after 72 h (Fig. 2a). The formation of nitrite is shown in Fig. 2b during nitrate reduction. The case with starch supplementation showed eightfold higher nitrite concentration than the case without starch supplementation with a concentration of about 0.8 NO2 mg l–1 after 36 h. For either case with or without starch supplementation, nitrite formation occurred during nitrate reduction in our study, suggesting that nitrite accumulation might have resulted from the difference in the rates of nitrate and nitrite reduction (39). However, nitrite accumulation during bacterial activity seems to depend on the carbon sources. For instance in the study of nitrate removal by Pinar and Ramos (40), nitrite accumulated when sucrose was used as the carbon source but it was not formed when glycerol was used. In another study (12), nitrite was formed when sucrose was used as the carbon source but it was not
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FIG. 2. Microbial reduction of nitrate in MSM in presence and absence of starch. (a) Squares, Nitrate reduction without starch; diamonds, nitrate reduction with 1% starch; triangles, bacterial growth without starch; crosses, bacterial growth with 1% starch. (b) Squares, Nitrite formation without starch; diamonds, nitrite formation with 1% starch.
formed when ethanol or methanol was used. The results of changes in nitrate concentration and nitrite formation for the groundwater samples are shown in Fig. 3. Similar results were obtained for the trend of nitrate reduction (89%) and bacterial growth with the MSM solution except for a rather small difference in nitrite formation between the cases with and without starch supplementation. The small difference may be due to the presence of other microorganisms such as ammonifiers that prevent the formation of nitrite since the groundwater sample was not sterilized prior to the inoculation with RS-7. Nitrate removal in MSM and groundwater using coagulants The effect of adding 1% starch on nitrate removal using the combined system of biological and chemical treatments is shown in Fig. 4a and 4b. As can be observed from the figure, the addition of 1% starch to the groundwater sample during the first stage of biological treatment lowered the nitrate concentration from 460 to 50 mg l–1, whereas for the case without starch supplementation, it was lowered to only 108 mg l–1. During bacterial treatment of MSM containing 500 mg l–1 nitrate supplemented with 1% starch for 72 h, it was found that 70 mg l–1 nitrate remained in the samples. Among the various concentrations of coagu-
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FIG. 3. Microbial reduction of nitrate in groundwater in presence and absence of starch. (a) Squares, Nitrate reduction without starch; filled diamonds, nitrate reduction with 1% starch; triangles, bacterial growth without starch; crosses, bacterial growth with 1% starch. (b) Squares, Nitrite formation without starch; diamonds, nitrite formation with 1% starch.
lants used, lime at 150 mg l–1 reduced nitrate concentration from 70 to 0 mg l–1 (100% removal), followed by PAC from 70 to 4.8 mg l–1 (93.1% removal) and by alum from 70 to 8 mg l–1 nitrate (88.5% removal) (Fig. 4a). There was no further significant decrease in nitrate concentration when the dosage of the coagulants was increased above 150 mg l–1. Hence, the optimum dosage for the effective removal of nitrate from MSM and the groundwater samples was found to be 150 mg l–1 for lime and PAC. Similarly, in the groundwater samples the level of nitrate was reduced from 50 to 3.1 and 4.9 mg l–1, respectively, when lime and PAC were used at the concentration of 150 mg l–1 (Fig. 4b). The use of lime as a chemical coagulant gave the highest nitrate removal rate of 93.8%, whereas for alum, the nitrate removal rate was only 74.3%. The removal rate of nitrate was invariable as the concentrations of alum, lime and PAC were increased. The use of bacterial cultures appears to be efficient in reducing nitrate concentration prior to chemical treatment as well as in promoting the attachment of chemical coagulants onto biomass and thus achieving rapid precipitation of these biomass-coagulant complexes. Grabow et al. (22) and
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Pseudomonas sp. (RS-7) was found to be the most efficient in terms of nitrate reduction. From the above results, it could be concluded that nitrate reduction by RS-7 was affected by various carbon sources. The bacterial growth and nitrate reduction rates were high in MSM and groundwater supplemented with 1% starch as the sole carbon source compared with those in the media supplemented with glucose and cellulose under aerobic conditions at an optimum temperature of 30°C. Hence, 1% starch could be used as the best carbon source and concentration for nitrate removal in groundwater for the bacteria used in this study. According to the World Health Organization, the permissible limit of nitrate in drinking water is 45 mg l–1 (43). In this study using a bacterial inoculum, nitrate could not be reduced below the permissible limit. However, subsequent chemical treatment with lime at 150 mg l–1 decreased the concentration of nitrate below the permissible level. Therefore, it is suggested that bacterial treatment followed by chemical treatment will be more effective in treating nitrate-contaminated water. Lime was found to be an ideal material for the removal of remaining nitrate and also for elevating pH near neutral so that water could be directly used after disinfection. ACKNOWLEDGMENTS The authors are thankful to the Head of the Department of Environmental Sciences, Bharathiar University for providing facilities for the conduct of this research.
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Fig. 4. Nitrate removal by chemical coagulants from MSM and groundwater treated with bacteria. (a) Nitrate reduction in groundwater without starch. (b) Nitrate reduction in groundwater with 1% starch. (c) Nitrate reduction in MSM with 1% starch. Diamonds, Alum; squares, lime; triangles, PAC.
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