Disinfection of Effluents from UASB based Sewage Treatment Plants using Lime and Fenton s Reagent

Transcription

1 Synopsis on Disinfection of Effluents from UASB based Sewage Treatment Plants using Lime and Fenton s Reagent For the award of the degree of Doctor of Philosophy Submitted By Anju Pant 2002 RCE 007 Under the guidance of Dr. A.K. Mittal Department of Civil Engineering Indian Institute of Technology Delhi New Delhi, India July 2006

2 1. Introduction Microbiological quality is the most contentious issue linked to domestic wastewater disposal and its re-use. The presence of pathogens disease causing bacteria, viruses and other microorganisms, in the effluents discharged by the sewage treatment plants (STP) into a receiving water bodies can cause considerable threat to the public health. It has been observed that treated and untreated wastewater is a primary contributor of a variety of pathogenic microorganisms to the aquatic ecosystem (Vilanova et al., 2002). Human illness can result from drinking or swimming in water that contains pathogens or from eating shellfish harvested from such waters. It is also known that the most widespread contamination of water is from disease bearing human wastes (World Bank, 1992). Water-borne diseases cause more than 3 million causalities per year (World Bank, 1992). The treatment of such wastewaters using adequate process is essential to avoid the problem of waterborne diseases to human beings. Direct identification and monitoring for pathogens is very expensive and impractical, because pathogens are rarely detectable in water bodies. The presence of indicator microorganisms signifies the occurrence of fecal contamination and also indicates the presence of disease causing microorganisms. The most commonly used four indicator bacterias- total coliforms, fecal coliforms, E. coli, and enterococci - are normally prevalent in the intestines and feces of warm-blooded animals, including wildlife, farm animals, pets, and humans. Generally, the indicator bacteria are not pathogenic. Therefore, instead of monitoring pathogens, indicator" species are enumerated. Bacterial indicators of fecal contamination and enteric viruses are present in high concentrations in the raw wastewater. For example, typical abundance of total and fecal coliforms (FC) in raw sewage is, respectively, and per 100mL (Rose et al., 1996; George et al., 2002). There is a variation in the bacterial characteristics with the seasonal changes. Seidl et al., (1998) reported an increase in bacterial size and bacterial biomass in the wastewater during dry weather conditions along a combined sewer catchments. 1

3 Various processes like aerobic, anaerobic, membrane and hybrid are used for the wastewater treatment (Ng et al., 1993; Tare et al., 1993; Lettinga et al., 2001; Cornel et al., 2003; Ghosh and Philip, 2004; Mazumder and Dikshit, 2004). Conventional municipal sewage treatment plants, which generally do not include disinfection process, reduce fecal microorganism density by 1 to 3 logs (Miescer and Cabelli, 1982). World wide, 95% plants are still using chlorine as disinfectant (Kim et al, 2002). The scenario in India and most of other developing countries is starkly very different, where disinfection of wastewater even as a concept does not exist at large. Moreover, in India, most of the STPs are based on Up-Flow Anaerobic Sludge Blanket (UASB) process. The use of the UASB reactor has been successfully applied to treat municipal and industrial wastewater (Jawed and Tare, 1996; Prakash and Gupta, 2000; Lettinga et al., 2001; Sinha et al., 2002; Mahmoud et al., 2004). Additionally, this process requires low energy for operation and low initial capital. So, it becomes an attractive option for wastewater treatment in developing countries like India. Therefore, more than 20 STPs based on UASB process have been constructed. There has been a clear-cut shift in sewage treatment process from conventional aerobic process like Activated Sludge Process (ASP) to UASB based STPs. However, unlike ASP, UASB process does not effectively remove the biological pollution of the wastewater. ASP can remove BOD 5 up to 85-93%, while UASB removes 75-85% (Arceivala, 2002). Curtis (2003) reported that ASP could remove 60-90% microbial population, whereas almost zero in case of UASB. UASB is also ineffective in removing nutrients. Uemura et al. (2001) indicated that removal of indicator micro-organisms is not satisfactory in the UASB treatment. Thus, the effluents from such plants require posttreatment for the removal of pathogens, organics, and nutrients (Machdar et al., 1997). Thus, the attention has to be focused on the ability of STPs, especially based on UASB process, to reduce pathogenic microorganisms from the effluent. It is a common practice of disinfection to produce water of bacteriological acceptable standards (Venkobachar et al., 1975). Disinfection can be accomplished by the use of different disinfection technologies, viz., chlorination, UV irradiation, ozonation, photocatalysis, stabilization 2

4 ponds, other chemicals like lime, Fenton reagent, TiO 2 etc (Venkobachar et al., 1975; Warriner et al., 1985; Baron and Bourbigot, 1996; Christensen et al., 1997a, b; Davis- Colley et al., 1997). Chlorine is widely used for disinfecting wastewaters (Kim et al, 2002). However, this process is highly dependent on the quality of effluent. Typical chlorine dose for municipal wastewater disinfection is about 5-20 mg/l with a minutes contact time (Warriner et al., 1985; Lazarova et al., 1999). Several studies have been done on the inactivation of microbes by chlorination (Echelberger et al., 1971; Budde et al., 1977). Kampelmacher et al. (1977) observed a remarkable reduction of Salmonella, E. coli, fecal coliforms and fecal streptococci by chlorination with the total residual chlorine content of not less than 0.10 mg/l. Though chlorination is the most common method of disinfecting treated wastewater effluents (Buxton and Ross, 1979) but it is associated with the problem of formation of toxic disinfection- by-products (DBP). Ultraviolet (UV) is another choice of disinfection, which is increasingly recognized as a practical and effective alternative to chlorination for disinfection of treated wastewater (Baron and Bourbigot, 1996; Blatchley et al, 1997). Many studies have showed that UV disinfection can achieve the minimum disinfection value (WHO guidelines) of 10 3 FC/100ml (Baron and Bourbigot, 1996; Moreno et al., 1997; Liberti and Notarnicola, 1999; Hassen et al., 2000). UV irradiation has been widely used in wastewater treatment (Kundu et al., 2005) and disinfection because it is desirable to have no residual in treated wastewater causing no adverse impacts on aquatic life (Kim et al., 2002). Whitby et al. (1984) also reported that UV irradiation could reduce the bacterial concentrations with out affecting the aquatic biota of the receiving water body as compared to chlorination. Ozone is another disinfectant having strong biocidal characteristics due to a combination of its high oxidizing potential and its ability to diffuse through biological membranes (Hunt and Marinas, 1997). Ozone has been proved to be effective in the reduction of bacteria and virus (McBride and Taylor 1973; Farooq et al., 1977; Warriner et al., 1985; Hunt and Marinas, 1997; Hu et al, 1999) besides being effective in the oxidation of organics (Bose et al., 1994). According to Mara (1976), Waste Stabilization Pond (WSP) treatment technology is the most cost effective way of treating domestic wastewater. Several studies have shown that 3

5 a WSP system is very effective for the removal of fecal indicators (such as Escherichia coli, Streptococcus faecalis, Clostridium perfringens) and viruses like F +, somatic and Bacteroides fragilis phages (Davis-Colley et al., 1997). ph plays a very important role in the WSP treatment system. E.coli could not survive in wastewater due to high ph (Parhad and Rao, 1974). Low ph has more bactericidal effects on enterococci in domestic wastewater than in alkaline conditions (Awuah et al., 2002). Recently, the wetlands have also become the technology of choice to reduce the human pathogens in wastewater (Mandi et al., 1996; Gearheart, 1999; Karim et al., 2004). The reduction of Giardia, Cryptosporidium and enteric viruses in wetland system had been reported (Chendorain et al., 1998; Karpiscak et al., 1996). Tawfik et al. (2006) reported DHS-biotower system, composed of polyurethane material as the packed media, as a post-treatment unit for UASB based STP for the removal of pathogens. Several studies reported that the DHSbiotower, as a post treatment unit, is very effective improving the physicochemical as well as microbial quality of UASB effluent (Agrawal et al., 1997; Machdar et al., 1997; Uemura et al., 2001). Physicochemical treatments could be preferred in the case of combined wastewater (municipal and industrial wastewater) due to the unexpected variations in composition and to the eventual presence of toxic compounds (Moreno et al., 2003). Hydrogen peroxide has been widely used as an oxidant in advanced oxidation processes to decompose refractory or toxic organics present in the wastewater (Bowers et al., 1989; Kang and Chang, 1997). Ferrous ions react with hydrogen peroxide to generate strong oxidant hydroxyl radical (OH ). Several studies have shown that Fenton s oxidation effectively reduce refractory organics, such as color and COD in textile wastewater (Kuo, 1992; Lin and Peng, 1995). Ramirez-Zamora et al. (2002) have reported that Fenton s reagent efficiently removes most of the pathogens (fecal coliforms, Salmonella and Helminth eggs) from the sludge produced from the treatment of municipal wastewater. Peracetic acid (PAA) is another method of chemical disinfection of urban wastewater with an advantage of lack of adverse effects on the receiving environment (Sanchez-Ruiz et al., 1995). Veschetti et al. (2003) reported the effect of initial concentration of PAA and contact time on the reduction of indicator and pathogenic microorganisms. Another 4

6 chemical disinfectant, which is cost effective, is lime and it has been widely used in stabilization of sludge to reduce the pathogens and enabling lime-treated sludge to be safely disposed of in landfill (Farrell et al., 1974; Boost and Poon, 1998; Lim et al., 2002). The high ph ( 11) of lime/sludge mixture for sludge is to be maintained for the reduction of pathogens (Boost and Poon, 1998). Lime-applied biosolids effectively reduce fecal coliform, Salmonella bacteria and viruses levels well below those required to regard the biosolids as USEPA Class A within 1 day (Bujoczek et al., 2000). A high quality effluent with more than 99.9% removal of fecal coliforms, Salmonellae, total parasites and rotaviruses have been produced by lime treatment of raw domestic wastewater (Gambrill et al., 1989; Shanableh, 1998). High ph value (more than 11.2) has been shown to be effective in reducing number of enteric viruses, coliphages, enterococci and total plate and coliform bactetria for wastewater reclamation (Grabow et al., 1978; Chitranshi and Chaudhuri, 1983; Taylor et al., 1994). It is evident from the literature that there are a number of processes like physical, chemical, physicochemical and biological which could be used to kill/remove the microorganisms. However, several important issues are emerging from the literature as outlined below: 1. Though the chlorination is an established technology for the wastewater disinfection, still it is associated with the problem of formation of disinfection-byproducts (DBP). However, with increasing emphasis on promoting a sustainable ecological future and concern over introducing a toxic chemical into the water bodies, the design of disinfection process is increasingly leaning toward technologies that destroy the pathogens while balancing the effects of this disinfected wastewater on the receiving body of water's biota. 2. Ozonation and UV are very effective, but very much influenced by the effluent quality. Turbidity and high-suspended solids requires higher doses of these disinfectants (Whitby and Palmateer, 1993). Moreover, these technologies are relatively expensive and require skilled handling. 3. Operation and maintenance of WSP and constructed wetlands is cost effective; however, these technologies require large land area. 5

7 4. Another issue is that the anaerobic treatment does not produce good quality effluents and most of the STPs in India are based on UASB process. Thus, such technologies are needed, which are economical besides being effective in improving the microbial as well as physicochemical quality of the UASB effluent. 5. There are two classes of disinfectants. One is target oriented or microbe-targeting and other is non-microbe-targeting disinfectant. The chlorine, UV, ozonation and hydrogen peroxide are the example microbe-targeting disinfectant; and thermal process and lime are known as non-microbe-targeting disinfectants. In the present study, both type of disinfectant i.e., lime and Fenton s reagent have been employed. In India many sewage treatment plants (STP) under National River Action Plan are based on Up-Flow Anaerobic Sludge Blanket Reactor (UASB) technology. It is found that these plants could reduce the organic pollution load, but the bacteriological quality of the effluents from these plants is poor. Thus, it becomes imperative to disinfect the treated wastewater, so as to improve the water quality of receiving water bodies. It calls for the use of environmental friendly appropriate technologies for the wastewater disinfection. This study is directed towards development of a disinfection method, which could be effectively adapted in the context of developing world where maintenance and operation of intensively automated and mechanized technology may otherwise discourage its use. 2. Objective and Scope of the Study The objective of the present study was to develop a process for the removal of pathogens from the treated municipal wastewaters. The study was focused to address various issues related to the disinfection of wastewater using lime and Fenton s reagent. Application of lime would raise the ph of the wastewater to unacceptable levels. Fenton's reagent (hydrogen peroxide in conjugation with FeSO 4 at certain ratio) can effectively treat wastewater in the range of 3 to 4. So, use of either lime or Fenton s reagent will necessitates the ph adjustment before the final disposal of the disinfected wastewater. The addition of chemicals for ph adjustment could be avoided if both lime and Fenton s reagent are used simultaneously on separate portions of wastewater. These portions could 6

8 be mixed before the final discharge, which would bring the ph within normal range without addition of any chemical. The specific scope of the proposal shall be as follows: Investigation of microbial as well as others parameters of the UASB based sewage treatment plant. Selection of culture media for the isolation of Salmonella and Shigella. Improvement of enumeration methodology for the isolation of Salmonella and Shigella. Development of suitable alternative disinfection processes, both non-microbetargeting (lime) and microbe-targeting (Fenton s reagent). Evaluation of disinfectants like microbe-targeting and non-microbe-targeting. Study of parameters: o Microbes Fecal coliforms, Fecal streptococci, Salmonella and Shigella o Other parameters Chemical Oxygen Demand (COD), Biochemical Oxygen Demand (BOD) and Total Kjeldhal Nitrogen (TKN) Effect of dose of disinfectants (lime and Fenton s reagent) and kinetics on microbes and other parameters of municipal wastewater. Study of other parameters in context of reactor operation like sludge generation and ph change. Study of various existing kinetic models so as to develop better understanding of the mechanistic aspects. Finally, it is contemplated to evaluate the integrated approach i.e., use of disinfectants, lime and Fenton s reagent at the same time but to different portions of flow. 3. Materials and Methods 3.1 Experimental Methodology Present study is targeted towards improving the microbial quality of effluents from the UASB based sewage treatment plant (STP) using lime and Fenton s reagent. Accordingly, the methodology could be detailed as below: 7

9 On-site Experimentation Microbial Profile of UASB based STP Microbial Parameters Fecal coliforms, Fecal streptococci, Salmonella Shigella Research Methodology Laboratory Experimentation Basic Microbial Assaying Applied Selection of Culture Media Improvement of Enumeration Methodology Dose (Lime and Fenton s Reagent) Kinetics (Lime and Fenton s Reagent) Microbial Parameters: Fecal coliforms, Fecal streptococci, Salmonella and Shigella Other Parameters: COD, BOD and TKN 8

10 3.1.1 Site Description and Sampling A full-scale STP was selected, which was based on UASB reactor. The capacity of the STP was 27 X 10 3 m 3 day -1, and is located in Noida a suburb of Delhi, India. Plant was commissioned in 1993, under Yamuna Action Plan by the National River Conservation Directorate, India. Wastewater is first passed through a grit chamber, and then it is treated in the UASB reactor, and the effluent from the UASB is passed through a polishing pond (Fig. 3.1). Sampling Point S1 Sampling Point S2 Sampling Point S3 Influent Screen and Grit Chamber UASB Reactor Polishing Pond Fig.3.1. Schematic of the Sewage Treatment Plant Collection of samples Effluent Wastewater samples were collected from three different stages of treatment viz., wastewater after grit chamber, after UASB reactor and after final polishing unit. These locations are shown as S1, S2 and S3 in Fig Samples were transported to the laboratory in an ice-box. In laboratory, samples were stored at less than 4 o C in a deep freezer. The microbial analysis was started within 4 h of sample collection, and microbial enumeration was carried for fecal coliforms, fecal streptococci, Salmonella and Shigella Microbial assay of field samples The fecal coliforms, fecal streptococci, Salmonella and Shigella were enumerated using multiple-tube fermentation technique to obtain MPN (Standard Methods, 1998). A number of factors viz. desiccation, low numbers of the organism and change in ph, influence the recovery of bacterial pathogens from fecal samples (Sewell, 2003). All these factors were taken into consideration. For the enumeration of fecal coliforms and fecal streptococci, samples were suitably diluted using sterile deionized water before inoculation in appropriate medium. Decimal dilutions of samples in triplicate were normally used throughout the study. 9

11 Fecal coliforms Enumeration of fecal coliforms was carried out by direct inoculation technique, using A1 broth (Difco) as per Standard Methods (1998). Microorganisms were recovered at the discriminating temperature of 44.5 C for 24 h Fecal streptococci Fecal streptococci were recovered on Azide dextrose broth (HiMedia) at an incubation temperature of 35 ± 0.5 C for 48 h. All positive tubes were subjected to the confirmation test using Pfizer selective enterococcus Agar (HiMedia) and were incubated at 35 ± 0.5 C, and brown colonies with black halos in the backside were observed Isolation of Salmonella and Shigella Sample concentration To isolate and enumerate Salmonella and Shigella, a five-tube multiple dilution technique was used. Prior to enrichment large volumes of wastewater samples were concentrated conveniently by filtering the samples through a membrane filter (HA 0.45µm pore size, Millipore Corp.). The filter paper was then placed into 100mL of sterilized peptone water and the flasks were gently shaken and mixed thoroughly Selective enrichment procedures Tetrathionate broth (HiMedia) and Nutrient broth with ph>8 (HiMedia) were used as an enrichment medium for Salmonella and Shigella respectively. The appropriate amounts or dilutions of the samples blended in sterilized peptone water were inoculated into tubes containing enrichment medium in three replicates.1ml samples and subsequent lower decimal dilutions were inoculated into 10mL of single-strength broth and 10mL of double-strength broth. All samples were then incubated at 35 ± 0.5 C for 24h Plating procedures After selective enrichment and incubation at 35 ± 0.5 C for 24h, a loopful of each tube was streaked on plates of selective culture media and incubated at 35 ± 0.5 C for 24h. 10

12 After incubation, two or more suspected colonies from each plate were inoculated into Triple sugar iron agar (TSI) slants and further incubated for 24h at 35 ± 0.5 C. Salmonella and Shigella isolates were identified by biochemical and serological methods (Collee et al., 1989) Other Parameters The physicochemical parameters, viz., COD, BOD, and TKN were determined as per Standard Methods (1998). 3.2 On-Site Experimentation Microbial Profiling of Sewage Treatment Plant Municipal sewage samples from different stages of treatment were analysed to study the microbiological profile of UASB based STP (Fig. 3.1). Samples were collected from different points, viz., after grit chamber, after UASB reactor and after final polishing unit to study the effect of individual treatment unit in UASB based STP on the microbiological quality. This study was also conducted to found out the relationship between the indicator microorganism and pathogens like Salmonella and Shigella within a STP. The other parameters viz., COD, BOD, TKN and turbidity were also determined as per Standard Methods (1998). 3.3 Laboratory Experimentation Basic Microbiology Selection of Culture Media for the Isolation of Salmonella and Shigella A number of culture media were evaluated to select the most suitable plating media for the isolation and enumeration of Salmonella and Shigella from the municipal wastewater and anaerobically treated wastewater. Xylose-lysine-desoxycholate agar (XLD), Brilliant green agar (BGA), Bismuth sulphite agar (BSA), Salmonella Shigella agar (SSA) and MacConkey agar (MAC) were selected for this purpose. Microbial protocol used is summarized in Fig

13 Improved Methodology for the Enumeration of Salmonella and Shigella A different protocol to improve the recovery of pathogens in addition to simplifying the whole methodology is proposed. Conventional method for the isolation of pathogens has been compared with the modified method so as to establish a reliable method for the maximum recovery of pathogens from the municipal wastewater. Wastewater sample Concentration on membrane filter paper Pre-enrichment (blending of filter paper into 0.1% sterile peptone water) For Salmonella Selective enrichment (Multiple tube serial dilution (MPN) in Tetrathionate broth) For Shigella Selective enrichment (Multiple tube serial dilution (MPN) in Nutrient broth) Incubation at 35 C for 24 hours Streaking on XLD, MAC, SSA, BGA and BSA Incubation at 35 C for 24 hours Transfer of suspected colonies onto TSI Incubation at 35 C for 24 hours Biochemical tests and Serological analysis Fig Protocol for enumeration of Salmonella and Shigella on different culture media 12

14 3.3.2 Applied Experimentations Experimental Setup A cylindrical bioreactor (1L) with mechanical stirrer was used for carrying disinfection kinetics using lime and Fenton s reagent. ph electrode was immersed in the bioreactor for continuous ph measurement. Lime (as calcium hydroxide) and Fenton s reagent (1:5::FeSO 4 :H 2 O 2 ) was used as disinfectant for the removal of indicator (fecal coliforms and fecal streptococci) and pathogenic (Salmonella and Shigella) microorganism. Samples after polishing pond have been used for these studies Experimental Study with Lime Effect of Dose on Microbes Effect of lime on fecal coliforms, fecal streptococci, Salmonella and Shigella removal was studied by conducting batch experiments at different doses i.e., 100, 300, 400, 500, 700 and 1000mg/L. 1L of wastewater sample was taken in cylindrical bioreactors with mechanical stirrer and different doses of lime was added to each reactor. Wastewater and lime was mixed rapidly with mechanical stirrer at 150 rpm for 60 seconds and the sample was left for a specific contact time (60 and 120 min for fecal coliforms, fecal streptococci; 30 and 45 min for Salmonella and Shigella) to observe the effect of different lime doses on the removal of these microorganisms. ph was also observed before the addition of lime and after the specific contact time. Lime was neutralized after the specified time using 0.1 N H 2 SO Kinetic Study for Microbes Kinetic experiments were conducted to study the effect of lime dose on the kinetics of the removal of fecal coliforms, fecal streptococci, Salmonella and Shigella. The experiments were carried out in cylindrical bioreactors; and the disinfectant and wastewater were mixed with mechanical stirrer at 150 rpm for 60 seconds. Samples were withdrawn after different contact time i.e., 2, 5, 10, 20, 30, 45 and 60 min. Lime was neutralized after the specified time using 0.1 N H 2 SO 4. 13

15 Effect of Dose on Other Parameters Batch experiments at different doses i.e., 100, 300, 400, 500, 700 and 1000mg/L of lime were conducted to study the effect of lime on COD, BOD, TKN. Same protocol was used as given in Section Kinetic Study for Physicochemical Parameters These experiments were conducted to study the effect of lime dose on the kinetics of the removal of COD, BOD, TKN using the same protocol as given in Section Experimental Study with Fenton s Reagent Effect of Dose on Microbes Effect of Fenton s reagent (Fe:H 2 O 2 :: 1:5) on fecal coliforms, fecal streptococci, Salmonella and Shigella removal was studied by conducting batch experiments at different doses i.e., 5, 15, 30, 60, 90, 150 and 300mg/L of hydrogen peroxide. The ph of sample was maintained between 3.5 and 4.0 with 0.1 N H 2 SO 4. Same protocol was used as given in Section In this study, samples were withdrawn after a contact time of 30 and 60 min for fecal coliforms, fecal streptococci; and after a contact time of 20 and 30 min for Salmonella and Shigella. Catalase has been used to neutralize the Fenton s reagent (Liu et al., 2003), so 10 mg/l catalase was added after the specified time Kinetic Study for Microbes These experiments were conducted to study the effect of Fenton s reagent dose on the kinetics of the removal of fecal coliforms, fecal streptococci, Salmonella and Shigella. The ph of sample was maintained between 3.5 and 4.0 with 0.1 N H 2 SO 4. Same protocol was used as given in Section mg/l catalase was added after the specified time. 14

16 Effect of Dose on Other Parameters Study was carried out to evaluate the effect of Fenton s reagent (Fe:H 2 O 2 :: 1:5) on COD, BOD and TKN removal at different doses i.e., 5, 15, 30, 60, 90, 150 and 300mg/L of hydrogen peroxide. Same protocol was used as given in Section Kinetic Study for Other Parameters This study was carried out to investigate the effect of Fenton s reagent (Fe:H 2 O 2 :: 1:5) on the kinetics of the removal of COD, BOD and TKN using the same protocol as given in Section mg/l catalase was added after the specified time 4. Results and Discussion 4.1 Selection of Culture Media for the Isolation of Salmonella and Shigella This study is carried out to compare the performance of different plating media for the isolation and enumeration of Salmonella and Shigella from the municipal wastewater and anaerobically treated wastewater with an objective to select the specific media for the enumeration of these pathogens for further study. The culture media compared in the study were selected on the basis of previous studies cited in the literature (Taylor and Schelhart, 1967; Dunn and Martin, 1971; Pollock and Dahlgren, 1974; Pignato et al., 1995; Sherrod et al., 1995; Uyttendaele et al., 2001). Xylose-lysine-desoxycholate agar (XLD), Brilliant green agar (BGA), Bismuth sulphite agar (BSA), Salmonella Shigella agar (SSA) and MacConkey agar (MAC) were compared. The rate of isolation of pathogens on different media was observed. Table 4.1 presents the relative recovery of Salmonella and Shigella by different culture media. It is evident from Table 4.1 that XLD and MAC are the most suitable media for the isolation of Salmonella and Shigella respectively from the municipal wastewater. These two media were used for all further studies. 15

17 Table 4.1. Recovery of Salmonella and Shigella by different plating media Plating Media a Salmonella recovery (%) b Shigella recovery (%) XLD SSA MAC BGA 35 0 BSA 32 0 a Abbreviations: XLD - Xylose-lysine-desoxycholate agar, SSA - Salmonella Shigella agar, MAC - MacConkey agar, BGA - Brilliant green agar and BSA - Bismuth sulphite agar b All percentage are based on the number of positives 4.2 Improved Methodology for the Isolation of Salmonella and Shigella In the present study, a different protocol, which may improve the recovery of pathogens in addition to simplifying the whole methodology, is proposed. Conventional method (Standard Methods, 1998) for the isolation of pathogens has been compared with the modified method so as to establish a reliable method for the maximum recovery of pathogens from the municipal wastewater. All the wastewater samples were analysed for the recovery of Salmonella and Shigella, both by Conventional (Standard Methods, 1998) and the proposed Direct method. Fig. 4.1 presents both the methodologies. Recovery of both Salmonella and Shigella were observed to be high in Direct method as compared to Conventional method. The respective average recovery of Salmonella and Shigella by Direct method was approximately 105 and 276% higher than by Conventional method. Student s t-test was also applied to determine if the difference between Direct method and conventional method is significant or not. There was statistically significant difference between the results obtained with the Direct method and conventional method. In all the cases, the t-test values were higher than the corresponding critical t-test values obtained from the same data set, which show a significant difference between data obtained by Direct method and conventional method. 16

18 4.3 Microbial Profiling of STP Density of indicator microorganisms (fecal coliforms and fecal streptococci) and pathogens (Salmonella and Shigella) at various stages of treatment in the STP have been determined. Other parameters like COD, BOD, TKN and turbidity were also considered. It has been found that the concentrations of fecal coliforms (FC) and fecal streptococci (FS) in the raw sewage were very high. Salmonella spp. and Shigella spp. were also recovered from the almost all the wastewater samples. The indicator microorganism as well as the pathogens could not be reduced considerably by UASB. FC to FS ratio indicates that the source of the microbial contamination was human. The ratio of pathogenic microbes to indicator microbes has been evaluated during the profiling of UASB based STP. Student s t-test was also applied to determine if the relationship between indicators and selected pathogens is significant or not. In all the cases, the t-test values were lower than the corresponding critical t-test values obtained from the same data set, which show a significant relationship between indicators and selected pathogens. For both Salmonella and Shigella, the Student s t-test is significantly related with the indicators at all the stages of treatment, though the absolute number of micro-organisms at different stages of treatment are different by more than one order but the relationship between the indicators and the pathogens remain significant. 17

19 Proposed Direct Method Wastewater sample Concentration on membrane filter paper Pre-enrichment (blending of filter paper into 0.1% sterile peptone water) Selective enrichment (Multiple tube serial dilution (MPN) in Enrichment media) Conventional Method Incubation at 37 C for 24 hours Sub culture (Streaking on growth/ culture media) Incubation at 37 C for 24 hours Transfer of suspected colonies onto TSI, LIA Incubation at 37 C for 24 hours Biochemical tests and Serological analysis Fig Schematic of the proposed Direct and Conventional methods for enumeration of Salmonella and Shigella. 4.4 Experimental Study with Lime Effect of Dose on Microbes These experiments were conducted to evaluate the effect of lime on the removal of fecal coliforms, fecal streptococci, Salmonella and Shigella from wastewater. Results indicate that at a lime dose of 1000 mg/l, the ph rises to 10.83, which is very effective to kill indicator microorganisms. The average reduction of FC, at 1000 mg/l of lime dose and a contact time of 60 and 120 min, was 2.46 and 3.51 Log respectively. The average reduction of FS, at the same lime dose and contact time, was 2.30 and 3.00 Log 18

20 respectively. The log reduction of FC and FS was found to decrease with decrease in dose concentration. In the present study, the concentration of pathogenic microorganism was very low as compared to indicator microorganism, thus at 1000 mg/l of lime dose, both the pathogens (Salmonella and Shigella) were found to be removed completely. Thus lower doses (500, 400, 300 and 100 mg/l) of lime were considered in case of Salmonella and Shigella. The ph rises to 9.89 and 9.05 at a 500 mg/l and 400 mg/l respectively. At 500 and 400 mg/l of lime and a contact time of 30 min, the average reduction of Salmonella was 1.31 and 0.78 Log respectively. Similarly, at same dose of lime and contact time, the average reduction of Shigella was 1.16 and 0.96 Log respectively. The log reduction of Salmonella and Shigella was also found to decrease with decrease in dose concentration Kinetic Study for Microbes Kinetics of disinfection using lime dose for the survival of fecal coliforms, fecal streptococci, Salmonella and Shigella from wastewater was studied to relate the effect of dose with time. A number of kinetic models have been proposed in past to describe the kinetics of chemical disinfection. Microbial inactivation has been described as a pseudo first-order chemical reaction (Gyurek and Finch, 1998). Inactivation curves indicated a tailing-off mode of inactivation and characteristic biphasic mode. The kinetics with lime dose typically found the exponential kinetics following an initial shoulder or lag that has been attributed to inadequate mixing, delays in diffusion of the disinfectant to sites of action, and/or multiple targets necessary for inactivation. On the basis of survival curves obtained from the experimental data, it was found that the Selleck model was the best to describe kinetics for all the microorganisms, i.e., the fecal coliforms (Fig. 4.1), fecal streptococci, Salmonella and Shigella. 19

21 0.00 Contact Time (min) Log N/No Experimental Data Chick Watson Modified Collin Selleck Collin Selleck Multiple Target Selleck Model Fig Fecal coliform survival curve for different kinetic models at 500 mg/l of lime dose Effect of Dose on Other Parameters This study was conducted to evaluate the effect of lime dose on the removal of COD, BOD and TKN from wastewater. At 1000 mg/l of lime dose and contact time of 60 and 120 min, the average reduction of COD, BOD and TKN was 53, 56 and 52% respectively. Similarly, when the contact time was 120 min, the average reduction of COD, BOD and TKN was found to increase as 62, 64 and 61% respectively. The reduction (%) of COD, BOD, TKN was found to decrease with decrease in dose concentration Kinetic Study for Other Parameters Kinetics of disinfection using lime to remove COD, BOD, and TKN from wastewater was carried out to evaluate the effect of lime dose with time. Kinetic study showed that for all the parameters, reduction (%) increases with increase in contact time for different doses of lime. At 1000 mg/l of lime dose, COD reduction was increased from 11 to 52% when the contact time was increased from 2 min to 60 min. Similar results were observed for BOD, TKN. 20

22 4.5 Experimental Study with Fenton s Reagent Effect of Dose on Microbes This study was carried out to investigate the effect of Fenton s reagent on the removal of fecal coliforms, fecal streptococci, Salmonella and Shigella from wastewater. It was found that at a dose of 300 mg/l of hydrogen peroxide and contact time of 30 min, average reduction of FC was Similarly, at the same dose and contact time, average reduction of FS was 1.76 Log. The log reduction of FC and FS was found to decrease with decrease in dose concentration. The concentration of pathogenic microorganism was very low as compared to indicator microorganism, so at 300 mg/l of hydrogen peroxide, both the pathogens (Salmonella and Shigella) were found to be removed completely. Thus, study was carried out at lower doses (5, 15, 30, 60 and 90 mg/l) to evaluate the effect of lime dose on Salmonella and Shigella. At 90mg/L and 60mg/L of hydrogen peroxide and contact time of 20 min, average reduction of Salmonella was 1.15 and 0.85 Log respectively. At the same dose and contact time, average reduction of Shigella was 1.18 and 1.04 Log respectively. The log reduction of Salmonella and Shigella was found to decrease with decrease in dose concentration Kinetic Study for Microbes Kinetics of disinfection using Fenton s reagent for the survival of fecal coliforms, fecal streptococci, Salmonella and Shigella from wastewater was carried out to investigate the effect of dose with time. Different kinetic models (Gyurek and Finch, 1998) have been studied to determine the kinetics of chemical disinfection for the inactivation of indicator and pathogenic microorganisms. Survival curves obtained from the experimental data indicated a tailing-off mode of inactivation and characteristic biphasic mode, which was best described by Chick Watson model for all the microorganisms, i.e., the fecal coliforms (Fig. 4.2), fecal streptococci, Salmonella and Shigella. 21

23 Contact Time (min) Log N/No Experimental Data Chick Watson Modified Collin Selleck Collin Selleck Multiple Target Modified Hom Model Fig Fecal coliform survival curve for different kinetic models at 30 mg/l of Fenton s reagent dose Effect of Dose on Other Parameters The experiments were performed to determine the effect of Fenton s reagent on the removal of COD, BOD and TKN from wastewater. At 300 mg/l of hydrogen peroxide and contact time of 60 min, average reduction of COD, BOD and TKN was 77, 79 and 82% respectively. When contact time was 120 min at same dose, COD, BOD and TKN reduction was found to increase as 80, 81and 86% respectively. The reduction (%) of COD, BOD and TKN was found to decrease with decrease in dose concentration Kinetic Study for Other Parameters To evaluate the effect of dose with time, kinetics of disinfection using Fenton s reagent for the removal of COD, BOD and TKN from wastewater have been carried out. Results of kinetics indicate that for all the parameters, reduction (%) increases with increase in contact time. At a dose 300 mg/l of hydrogen peroxide, COD reduction was increased from 62 to 77% when the contact time was increased from 2 to 60 min. Similar results were observed for BOD and TKN. 22

24 4.6 Integrated Approach: Parallel Treatment with Lime and Fenton s Reagent The Volumetric ratio of lime added effluent and Fenton s reagent added effluent was determined in order to neutralize ph without addition of any other chemical. At a lime dose of 500 mg/l, ph of the wastewater rises to 9.89, whereas Fenton s reagent reduces ph to ph of the 200 ml lime added wastewater could be brought to 7.33 by adding 100 ml of wastewater treated with Fenton s reagent. Thus, if the ratio of wastewater treated with lime (ph~9.89) and Fenton s reagent (ph~3.41) is kept 2, the ph of the wastewater obtained after mixing these two effluents would be around Conclusions On the basis of the present study, the following conclusions can be drawn. 1. XLD and MAC are the most suitable media for the isolation of Salmonella and Shigella respectively from the municipal wastewater. 2. The recovery of both Salmonella and Shigella were observed to be high in proposed Direct method as compared to Conventional method. 3. The ph of the lime treated wastewater increases with increase in lime dose from 100 to 1000 mg/l. At a dose of 1000 and 500 mg/l of lime, ph rises to and 9.89 respectively, which is very effective for the killing of indicator as well as pathogenic microorganisms. 4. At 1000 mg/l of lime and contact time of 60 min, a log reduction of fecal coliforms was higher as compared to fecal streptococci. 5. Lower doses of lime and lesser contact time were required for the reduction of pathogenic microorganisms as compared to indicator microbes. 6. At 500 mg/l of lime and contact time of 30 min, log reduction of Salmonella was higher than Shigella. 7. The log reduction of indicator and pathogenic microbes was found to decrease with decrease in lime dose concentration. 8. On the basis of survival curves obtained from the experimental data, the Selleck model was found to be the best to describe disinfection kinetics using lime for all 23

25 the microorganisms, i.e., the fecal coliforms, fecal streptococci, Salmonella and Shigella. 9. Reduction of COD, BOD and TKN were found to increase with increase in lime dose from 100 to 1000 mg/l. 10. Kinetics of disinfection using lime showed increase in COD, BOD and TKN reduction with increase in contact time from 2 to 60 min. 11. At 300 mg/l of Fenton s reagent and contact time of 60 min, a log reduction of fecal coliforms was higher as compared to fecal streptococci. 12. Lower doses of Fenton s reagent and lesser contact time were required for the reduction of pathogenic microorganisms as compared to indicator microbes. 13. At 90mg/L of Fenton s reagent and a contact time of 20 min, log reduction of Salmonella was higher than Shigella. 14. The log reduction of indicator and pathogenic microbes was found to decrease with decrease in Fenton s reagent dose concentration. 15. On the basis of survival curves obtained from the experimental data, the Chick Watson model was found to be the best to describe disinfection kinetics using Fenton s reagent for all the microorganisms, i.e., the fecal coliforms, fecal streptococci, Salmonella and Shigella. 16. With the increase in Fenton s reagent dose from 5 to 300 mg/l, reduction in COD, BOD and TKN increases. 17. Kinetics of disinfection using Fenton s reagent showed the increase in reduction of COD, BOD and TKN from wastewater when the contact time was increased from 2 to 60 min. 18. The ph of the wastewater could be neutralized (ph~7.33), when the wastewater treated with lime (ph~9.89) and Fenton s reagent (ph~3.41) was mixed in ratio 2:1. 24

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