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Reduction of indicator and pathogenic microorganisms in pig manure through fly ash and lime addition during alkaline stabilization
Journal of Hazardous Materials 169 (2009) 882–889
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Reduction of indicator and pathogenic microorganisms in pig manure through fly ash and lime addition during alkaline stabilization Jonathan W.C. Wong ∗ , Ammaiyappan Selvam Sino-Forest Applied Research Centre for Pearl River Delta Environment, Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR
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Article history: Received 22 October 2008 Received in revised form 9 April 2009 Accepted 9 April 2009 Available online 16 April 2009 Keywords: E. coli Fecal coliforms Fecal streptococcus Re-growth Salmonella
a b s t r a c t A pilot scale study was conducted to evaluate the effect of lime and alkaline coal fly ash (CFA) on the reduction of pathogens in pig manure during alkaline stabilization and suppression of re-growth during post-stabilization incubation. Pig manure was mixed with CFA at 25%, 33% and 50%, and a control without fly ash was maintained. To these manure–ash mixtures, lime was added at the rate of 2% or 4% and incubated for 8 days. During the incubation, the population of Salmonella, fecal coliforms, Escherichia coli, fecal Streptococcus and total bacteria were enumerated. After the alkaline stabilization process, the mixtures were incubated under green house condition to evaluate the re-growth of pathogens. During the 8-day alkaline stabilization, Salmonella, fecal coliforms, E. coli and fecal Streptococcus were completely devitalized in manure–ash–lime mixtures, whereas in the control, incubation reduced the pathogen and total bacterial population by 2–3 logs. Fecal streptococcus was destructed within 4 days of alkaline stabilization, whereas other pathogens needed 8 days for their destruction. During the incubation in green house, an increase in the population of the pathogens and total bacteria was observed. Results indicate that alkaline stabilization of pig manure with lime at 4% and CFA at 50% is effective in devitalizing the pathogens and reducing the post-stabilization re-growth. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Livestock manures are potential reservoirs of nutrients and are applied on the agricultural fields so as to manage the disposal as well as to recycle the nutrients. However, a wide variety of pathogenic bacteria may be found in the feces of animals posing threats to the public health. Land application of raw manure potentially spreads pathogens to a wider environment and as a consequence bacterial pollution of agricultural lands was demonstrated [1,2]. For example, contamination of the ground water with fecal coliforms and fecal streptococcus as a consequence of leaching from livestock manure was demonstrated [3,4]. When E. coli reached soil, via manure spreading or runoff from point source, it could survive, replicate, and move downward for up to 2 months, threatening non-target environments [5]. Biosolids should be treated to reduce the pathogenic potential to minimize the risks to the environment and public health. Fecal coliforms, most important subgroup of total coliforms, are thought to be a better indicator of fecal contamination, because they tolerate higher environmental temperatures [6]. Fecal streptococci, along with fecal coliforms have been used to differentiate
∗ Corresponding author. Tel.: +852 3411 7056; fax: +852 3411 5995. E-mail address: [email protected] (J.W.C. Wong). 0304-3894/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2009.04.033
human fecal contamination from that of other warm-blooded animals. Although fecal streptococci are not ideal as indicators of fecal contamination, these organisms are relatively easy to enumerate and survive longer than fecal coliforms [7]. In addition, Salmonella is of concern because of the potential exists for re-growth following treatments [8,9]. Salmonella can survive in manure for up to 3 weeks and in manure slurry for up to 5 weeks. In swine, the prevalence of Salmonella in the feces has been reported in Quebec to be between 8% and 25% [10]. Alkaline stabilization is commonly used to eliminate pathogens and reduce odors in biosolids. For example, lime stabilization of biosolids is one of the processes listed in the Part 503 regulations to significantly reduce pathogens to a level considered as Class B biosolids [11]. Pathogens such as fecal coliforms and fecal streptococcus, enriched in biosolids, were rapidly inactivated following treatment with calcium hydroxide or potassium hydroxide at a pH of 11 or 13 for a period of 2 weeks [12]. High pH following lime treatment was suggested as the major reason for destroying or inactivating the pathogens and microorganisms in biosolids [13]. Along with lime, alkaline coal fly ash (CFA) had also been employed in alkaline stabilization of sludge [14–19]. For example, in our earlier study [18], we found that alkaline stabilization of MSW sludge with 10% CFA and a minimum of 8.5% lime (dry weight basis) for at least 2 h resulted in acceptable levels of salmonella and coliforms. However, the re-growth of the pathogens was not evaluated
J.W.C. Wong, A. Selvam / Journal of Hazardous Materials 169 (2009) 882–889 Table 1 Selected physicochemical and microbiological properties of pig manure. Parameter
Pig manure
pH EC (dS m−1 ) NH4 –N (mg kg−1 ) PO4 –P (mg kg−1 ) Total organic carbon (%) Total N (% dry weight) Total P (% dry weight) Moisture content (%) Salmonella (log CFU g−1 ) Fecal coliform (log CFU g−1 ) E. coli (log CFU g−1 ) Fecal Streptococcus (log CFU g−1 ) Total bacteria (log CFU g−1 )
7.61 (0.05)a 6.83 (0.2) 1860 (46) 1434 (54) 36.6 (0.77) 3.03 (0.12) 1.02 (0.23) 75 (2.3) 7.32 (0.58) 6.77 (0.62) 6.65 (0.41) 6.69 (0.61) 7.61 (0.3)
a
Values in parentheses are standard deviation (n = 3).
in pig manure after alkaline stabilization. Although, many reports available on the MSW sludge, reports were not available on the pathogen elimination in pig manure using CFA–lime mixture during alkaline stabilization. Hence, the present study aims at evaluating the effectiveness of lime and CFA (a) as alkaline stabilizing agents for pig manure; (b) inactivation of fecal coliforms, fecal streptococci, Salmonella, and E. coli during alkaline stabilization in the pig manure; and (c) re-growth of the pathogens during post-alkaline stabilization incubation in the green house. 2. Materials and methods
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by a computer-controlled system during the whole process. Forced moistened ventilation at 1 L/kg dry weight/min was supplied to the reactors to provide oxygen to the stabilizing mass. The stabilizing mass in the incubators was turned on days 2, 4 and 6 to maintain the homogeneity of the composting mass and about 100 g of samples was collected from each treatment for analyses. 2.2. Chemical analysis The moisture content was determined by oven-drying at 105 ◦ C for 24 h while pH and EC were measured in water extracts [1:5, sample (w):deionized water (v)]. The total N and P were extracted by H2 SO4 acid digestion and then determined by using the Berthelot and molybdenum blue methods, respectively [20]. Soluble N (as NH4 –N) and P (as PO4 –P) were estimated using indophenol-blue and molybdenum blue methods, respectively [20]. Total organic carbon (TOC) was analyzed using the Walkley–Black method [20]. Cress seed germination test was conducted as per the standard methods for testing compost materials [21]. For the total heavy metal contents in CFA and composts, samples were subjected to mixed acid digestion (conc. HNO3 and conc. HClO4 ) and analyzed using atomic absorption spectrophotometer (Varian Techtron Model AA-10) and graphite furnace atomic absorption spectrophotometer (GFAAS) with deuterium background correction. For DTPA extractable Cu, Mn and Zn, soils were extracted with 1:5 (sample:extractant, w/v) diethylene triaminepentaacetic acid–triethanolamine (DTPA–TEA) [20], shaken at 200 rpm for 2 h; centrifuged at 8000 × g for 5 min and the supernatants were analyzed after filtration.
2.1. Alkaline stabilization process 2.3. Microbial population determination Fresh pig manure was collected from Kardoorie Farm & Botanic Garden and stored at 4 ◦ C until use. The coal fly ash was collected from the Castle Peak Power Station of China Light & Power Co. Ltd., Hong Kong and stored at room temperature until use. Selected properties of the pig manure and coal fly ash used in the study are presented in Tables 1 and 2, respectively. For the stabilization experiments, the pig manure was mixed with CFA at 1:1 (50%), 2:1 (33%) and 4:1 (25%) in dry weight basis in a closed blending mixer for 2 h. A control without the addition of CFA was maintained. Then, all the fly ash amended treatments were subjected to a lime treatment at two concentrations of 2 and 4% (w/w, dry weight basis) to check the additive effect of lime and CFA on pathogen elimination. Wood chips as bulking agent at 1:10 (woodchip:mixture, v/v) were added to all the treatments and then mixed using a concrete mixer. The mixtures, 4.5 kg per reactor, were incubated individually for 8 days for slow composting in a 20-L reactor with suitable insulation to prevent the heat loss. Temperature was monitored Table 2 Selected physicochemical characteristics of the coal fly ash. Parameter
Coal fly ash
pH EC (dS m−1 ) Moisture content (%) Organic carbon (%) N (%) P (%) Ca (%) Mg (%) K (%) Cadmium (mg kg−1 ) Copper (mg kg−1 ) Manganese (mg kg−1 ) Zinc (mg kg−1 )
12.4 (0.2)a 2.02 (0.03) 0.13 (0.02) 0.15 (0.012) 0.0047 (0.003) 0.33 (0.017) 5.9 (0.14) 0.51 (0.02) 0.14 (0.04) 3.51 (0.04) 37.9 (0.6) 293 (2.6) 34.2 (1.5)
a
Values in parentheses are standard deviation (n = 3).
On day 0, 2, 4, and 8, Salmonella, Escherichia coli, fecal coliforms, fecal Streptococcus and total bacterial populations were enumerated using XLD agar (CM 469), MacConkey Agar No. 3, mFC Agar, KF Streptococcus agar and nutrient agar, respectively. All the media were purchased from Oxoid (Hampshire, England), except mFC agar which was purchased from Difco, Sparks, MD, USA. Ten grams of sample were added aseptically to 90 ml of sterile water in a conical flask, and shaken for 1 h in an orbital shaker. Then the sample was allowed to settle for 20 min and the 100 l of the supernatant was used for culturing microbes after serial dilution. Plates were incubated at 35 ◦ C, except mFC agar for fecal coliforms, which was incubated at 44.5 ◦ C. The colonies were counted after 24 h incubation, except Salmonella, which was counted after 48 h. The results are presented as log CFU (colony forming units)/g sample. All microbial analyses were done within 24 h after sampling in triplicate samples. 2.4. Green house incubation and effect on soil properties After the 8-day composting treatment in the composters, the mixtures were stored in green house for 21 days to examine the re-growth of microbial population. In this storing phase, insulation and force ventilation was not provided and the moisture content of the mass was maintained at around 60% so as to simulate the field irrigation conditions as well as providing enough moisture to the microbes in the alkaline stabilized mass. The temperature range in the greenhouse is between 20 and 30 ◦ C. At days 0, 7, 14 and 21, samples were collected from each treatment and analyzed for bacterial population as described before. After greenhouse incubation, the alkaline stabilized pig manure products from different treatments were mixed with soil at 2%, 4% and 6% (w/w, dry basis) and the soils were analyzed for pH, EC, soluble NH4 –N, PO4 –P, DTPA extractable Cu, Mn and Zn.
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3. Results and discussion 3.1. Changes in physicochemical properties 3.1.1. Temperature The patterns of temperature changes for all the treatments were similar. Temperature increased from day 0 to day 2 (from 41–44 ◦ C to 50–55 ◦ C) (data not shown) and then declined to about 45 ◦ C. With the addition of CFA, the temperature was less than that in the control, mainly due to the dilution effect, which also reduced the microbial population. When comparing different rates of lime addition, higher temperature was observed in those with 4% lime treatment than in that with 2% lime treatment. The increase in temperature was mainly due to the exothermic reaction of calcium oxide hydration. 3.1.2. Moisture content, pH and electrical conductivity Changes in the moisture content, pH and electrical conductivity (EC) during the 8-day alkaline stabilization are presented through Fig. 1a–c. Initial moisture content of the manure mix was influenced by the addition of coal fly ash principally as the dilution effect (Fig. 1a). Moisture contents were decreased significantly for about 4 days in all the treatments, except control, where the reduction was
not as high as the lime amended treatments. Further, the increase in lime addition decreased the moisture content, might be due to the exothermic reaction of lime and consequent water loss. Addition of 2% lime with different concentrations of CFA increased the pH to >11; while, 4% lime addition increased the pH of the manure mass to >12 (Fig. 1b), which is recommended by the USEPA for the pathogen reduction in Class B certification [11]. The changes in the concentration of CFA did not influence the pH significantly. However, increasing CFA concentration in the mixture from 25% to 50% increased the pH from 12.08 to 12.27. Further, the pH of >12 was achieved and maintained for 4 days by the addition of lime at 4% and CFA at >33%. However, a pH of ∼12 can be achieved and maintained with 25% CFA and 4% lime. Maintaining a high pH can destroy pathogens and hence addition of lime at 4% along with 25% CFA is optimum to maintain the pH at about 12 for >2 days. The pH dropped gradually after 4 days in all the treatments. In contrast, in the control without lime and CFA, the pH increased gradually during the 8 days incubation. Increasing pH is the major factor in devitalizing the pathogens in sewage sludge alkaline stabilization with lime [13] as also observed in our study due to the addition of lime and CFA,. Lime and CFA amendment increased the EC to ∼11 dS m−1 (Fig. 1c) from 6.85 dS m−1 observed in the control. The EC increased
Fig. 1. Changes in the moisture content (a), pH (b), electrical conductivity (c), total organic carbon (d), soluble NH4 –N (e) and soluble PO4 –P (f) during alkaline stabilization of pig manure with coal fly ash and lime (CaO). ((䊉) control–pig manure without lime and fly ash, () pig manure + 50% ash + 2% CaO; () pig manure + 33% ash + 2% CaO; () pig manure + 25% ash + 2% CaO; () pig manure + 50% ash + 4% CaO; () pig manure + 33% ash + 4% CaO; and () pig manure + 25% ash + 4% CaO.)
J.W.C. Wong, A. Selvam / Journal of Hazardous Materials 169 (2009) 882–889
up to 2 days and gradually decreased to 5.27 dS m−1 in control, whereas in other treatments, the EC decreased gradually in the first 2 days; then sharply in the next 2 days and finally reached an EC of ∼6.3 dS m−1 . The increase in the addition of ash from 25% to 50% resulted in only a slight increase in the final EC. Similarly, the EC of treatments with 4% lime addition did not differ significantly from 2% lime addition. In the control and all treatments, the EC was higher than the phytotoxicity limit of 4 dS m−1 [22], which may potentially limit plant growth; however the phytotoxicity depends on the application rate. 3.1.3. Total organic carbon, NH4 –nitrogen and PO4 –phosphorus The changes in total organic carbon (TOC), soluble NH4 –N and PO4–P contents in the pig manure–CFA–lime mixtures are presented in Fig. 1d–f. Total organic carbon contents in the stabilizing mass decreased slowly (Fig. 1d). Generally, TOC reduction was higher with fly ash and lime amended treatments (10.7–16.7%) compared to control (7.4%), mainly due to the dilution effect of the ash contents and subsequent initial TOC contents. However, when the ash amendment was decreased from 50% to 25%, there was an increase in TOC reduction in both 2% and 4% lime treatments. Results indicate that the fly ash enhances the decomposition of organic matter and the efficiency was higher at 25% ash amendment. The NH4 –N concentrations decreased during the stabilization period of 8 days (Fig. 1e) mainly due to the volatilization loss of NH4 as NH3 gas at high pH [23]. High pH and the resulting ammonia had an enhanced disinfecting activity [24]. With an increase in ash content from 25% to 50%, the soluble NH4 –N concentration decreased due to the reduced amount of manure and volatilization due to higher pH. Since the soluble NH4 –N concentration in manure is 1.86 g/kg, the reduction due to CFA and lime would be beneficial in terms of treatment expenses to meet the standard value of <500 mg/kg. The soluble P contents decreased slightly during the incubation period in all the treatments. The reduction is likely due to the utilization of P by the inhabiting microbial population. Further, addition of CFA caused a reduction in the concentration of soluble P, attributed to the reduction in manure quantity and precipitation of PO4 –P to a less available form with CFA [18,25,26]. But in the present study, a significant reduction was not observed, indicating the interaction of organic rich pig manure with the CFA. In the alkaline treatment of the manure, the alkaline materials especially lime result in high concentrations of calcium at elevated pH, which favors the complexing of soluble phosphate in the manure with the calcium compounds to form insoluble complexes [25]. In the present study, only a marginal decrease in the PO4 –P was observed in all the treatments. This indicates the lack of interaction of PO4 –P in pig manure with lime or ash. 3.1.4. Changes in the pathogenic and total bacterial population The addition of CFA led to the reduction in Salmonella, fecal coliforms, E. coli and fecal Streptococcus populations (Fig. 2a–d). In control treatments, reduction of 2–3 log CFU in the pathogens was observed. Whereas in lime and CFA amended manures, complete destruction was observed within 8 days. Especially, E. coli and fecal Streptococcus were destructed within 4 days, indicating the efficiency of the alkaline treatments. When the lime addition increased to 4% with CFA, the populations of Salmonella and fecal coliforms were below the detection limit within 4 days, whereas, in 2% lime + CFA treatments, this was achieved in 8 days. However, E. coli and fecal streptococci populations were below detectable levels after 4 days even with 2% lime + CFA mixture. An increase in CFA content from 25% to 50% further resulted in faster destruction. For example, manure mixed with 4% lime and 25% CFA resulted in the below detectable levels of Salmonella, fecal coliforms, and
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fecal streptococcus within 4 days. But this below detectable level was achieved within 2 days when the concentration of CFA was increased to 50%, indicating the additional effect of CFA on pathogen reduction. Almost similar trends were observed with different CFA concentration with 2% lime. However, 25% CFA+ 2% lime resulted in below detectable levels of Salmonella and fecal coliforms within 4 days; whereas, in 33 and 50% CFA additions, below detectable level was achieved in 8 days. Venglovsky´ et al. [27] reported destruction of Salmonella within 60 min after the addition of 10% hydrated lime to MSW sludge. Similar effects of the alkaline materials were observed in our study also. Pathogenic populations were substantially (∼3–5 logs) destructed upon the addition of lime and CFA. Since the analysis was initiated within 24 h, at best this period could be <24 h. Aeration of slurries reduced the time required for salmonella destruction, and is accelerated by increasing temperatures [28]. In our study, the temperature was increased in the first 2 days which coincided with the maximum destruction of pathogens. Hence, increasing temperature along with an increase in pH due to the addition of CFA and lime led to the successful destruction of Salmonella and other pathogens. With the mixing of CFA up to 50% diluted the composted mass in terms of microbial populations and nutrients in relation with the control pig manure treatment. Although the reduction in control treatment would accounted for ∼3 log CFU/g composting mass, the reduction after day 4 is not as rapid as the initial days, indicating the persistence of the indicator and pathogenic microorganisms in considerable quantity. Although, there was a dilution due to the addition of CFA, the initial CFA and lime mixing led to the reduction of indicator and pathogenic microorganisms substantially, indicating the influence of these alkaline materials in pathogen reduction. Wong et al. [18] reported a slight increase in total coliforms in the sludge without lime and ash treatment, after 18 h of incubation. However such increases in coliforms were not observed in our study. Fecal coliforms are good indicators of pathogen density and are currently used as the microbiological parameter for Class B certification of USEPA. Farrell et al. [29] showed that if the fecal coliform reduction in treated sludge was 100-fold (2 logs), then a reduction of Salmonella and enteric viruses would be about 1.5 logs and 1.3 logs, respectively. However, Bean et al. [30] reported that fecal coliforms cannot be used as an indicator of treatment effectiveness for all pathogens, including Salmonella. But in our study, the patterns of reduction of fecal coliforms and Salmonella were similar indicating their applicability as indicator for Salmonella during the alkaline treatments. Most strains of E. coli inhabit the intestines of animals and humans are harmless and in many cases beneficial. But harmful enterotoxogenic strains such as E. coli O157:H7 produce potent toxins that can cause severe illness in humans. The prevalence of E. coli O157:H7 has been reported to be between 0.4% and 7.5% in healthy pigs and up to 1.5% of pork meat samples [31]. Turner [32] demonstrated the destruction of E. coli in pig feces after 2 h at 55 ◦ C. Similarly, Himathongkham et al. [28] reported a 105 fold reduction in E. coli O157:H7 after 45 days at 37 ◦ C. Although no attempts were made to identify the strains of E. coli in our study, generally population of E. coli, which includes the enterotoxogenic forms, can be a better indicator. Jepsen et al. [33] reported a reduction of fecal streptococci by 3 orders after 24 h stabilization after the addition of hydrated lime to bring the pH to >12, with Salmonella being undetectable. In our experiment, 48% reduction in fecal streptococci was observed in the treatment with 2% lime and 50% ash; and the percent reduction decreased to 14% with the reduction in CFA content to 25%, implying the effect of CFA. In treatment with 4% lime, no fecal streptococci were detectable after 4 days. Lewis-Jones and Winkler [7] reported that fecal streptococci can survive longer than fecal coliforms. In contrast, relatively shorter time was required to destruct
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Fig. 2. Changes in the population of Salmonella (a), fecal coliform (b), E. coli (c), fecal streptococci (d) and total bacteria (e) during alkaline stabilization of pig manure with coal fly ash and lime (CaO).
fecal streptococci than fecal coliforms in our study may be due to the effect of CFA and lime. Although there was a reduction of pathogenic bacteria in the control treatment, high level of population was observed even after eight days of composting. This reduction may be attributed to the increase in temperature and increased pH driven NH3 produced during the decomposition of manure by microbes. The EPA Part 503 regulations require the fecal coliform density may not exceed 1000 CFU/g total solid and 3 CFU of Salmonella/4 g of total solid to qualify as Class A. Generally, alkaline treatment with lime and CFA resulted in stabilized products to meet the Class A standard. Further, increasing the CFA addition to 50% decreased the time required for the complete inactivation of pathogens. Total bacterial population was significantly reduced due to the addition of lime and CFA. Higher lime addition led to higher reduction in the population of bacteria. Even with the highest lime (4%) and ash addition (50%), the total bacterial population was nearly 105 log CFU/g after stabilization. During the alkaline stabilization process, E. coli and fecal streptococci were sensitive to lime and ash addition, due mainly to an increase in pH, NH3 and temperature, whereas, a relatively longer period of composting was required for the destruction of Salmonella and fecal coliforms. However, all
pathogens can be eliminated with an 8-day alkaline stabilization process involving ash and lime. Lime addition at 4% and CFA at 50% were satisfactory in pathogen reduction. Temperature, pH and ammonia are considered to be the major factors involved in pathogen reduction of biosolids. In our study, maximum temperature was observed on day 2 and the temperature declined thereafter. However the population of pathogenic organisms was detected even after 4 days, which indicates that the developed mesophilic temperature alone was not enough to devitalize all the pathogens. Muller [34] reported that the Salmonella survived less than 8 days at 37 ◦ C. In the present study, the temperature, through the 8-day stabilization period, did not fell below 45 ◦ C, which obviously should completely devitalize the pathogen population. The addition of 4% lime and 25% CFA was enough to achieve a pH of ∼12. The increased pH, which resulted in the release of NH3, played a significant role in pathogen reduction. In all the ash and lime amended treatments, the pathogens are completely eliminated. In the control, considerable population was observed even after the 8-day incubation period, which imply that increased pH due to the application of lime and CFA played a major role in devitalizing the pathogens, in addition to the mesophilic temperature.
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Fig. 3. Re-growth of Salmonella (a), fecal coliform (b), E. coli (c), fecal streptococci (d) and total bacteria (e) during incubation at green house after alkaline stabilization of pig manure with coal fly ash and lime (CaO).
3.2. Pathogens re-growth Bacterial population increased during the 21 days incubation period (Fig. 3). There was a marked increase in the population of Salmonella and fecal coliforms (Fig. 3a and b), which took relatively longer time to be eliminated during the alkaline stabilization. Whereas, E. coli and fecal streptococci increased marginally (Fig. 3c and d). After re-growth, the Salmonella population was above the limit of 3 CFU/4 g solid set out by the USEPA Part 5.3 in all the treatments, except the treatment with 50% CFA and 4% lime; whereas, the limit of 1000 CFU/g of fecal coliforms could be achieved with 4% lime and 25% CFA. Zaleski et al. [9] reported that re-growth of indicators and Salmonella was possible in biosolid under conditions of favorable moisture and temperature or substrate availability. Iranpour and Cox [35] also reported the recurrence of fecal coliforms and Salmonella sp. in the post-anaerobic digestion of biosolids. One of the suggested reasons was incomplete destruction of the fecal coliforms during thermophilic digestion. In a field study, Rufete et al. [36] reported the persistence of fecal and total coliforms due to the application of swine manure slurry. They also reported that the total coliforms increased when the soil amended with mineral fertilizer. Among the treatments, E. coli was detected only in 25% ash added treatments. Similarly, among the differ-
ent ash and lime treatments, fecal streptococci were detected only in 25% ash with 2% lime addition, which indicates that the alkaline CFA also involved in pathogen reduction. To conclude, Salmonella and fecal coliforms were completely devitalized in the 50% CFA and 4% lime addition enabling to reach the Class A biosolid standard. However, indigenous microbes present in soils are important for controlling the potential re-growth of bacterial pathogens [9]. Hence, if the manures are mixed with soil and incubated or establish with plant species, the antagonistic effects of indigenous microbes could be expected to suppress the re-growth of these pathogens. 3.3. Influence on soil properties After the greenhouse incubation, the alkaline stabilized products were mixed with soil at 2%, 4% and 6% (w/w, dry basis) so as to understand the influence on soil properties such as pH, EC, soluble NH4 –N, PO4 –P, DTPA extractable Cu, Mn and Zn and the results for the 6% application are presented in Table 3. The pH of the soils increased as the concentration of the lime and ash increased and ranged between 7.50 and 7.78 when compared to the 6.39 in the soil without any amendment. Similar trend was observed for the EC also as the amendment of alkaline substances increased the EC.
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Table 3 Selected chemical properties of the soil mixed with 6% of the alkaline stabilized pig manure (PM) with different treatments. Treatment
pH
EC (S/cm)
Soluble NH4 –N (mg/kg)
Soluble PO4 –P (mg/kg)
DTPA -Cu (mg/kg)
DTPA -Mn (mg/kg)
DTPA -Zn (mg/kg)
Soil
6.39 m (0.40)a
301 j (10.2)
1.30 j (0.35)
12.37 j (2.53)
0.15 l (0.03)
0.19 h (0.02)
1.46 ij (0.32)
PM Control
7.50 fg (0.02)
815 b (13)
138.0 a (5.5)
31.15 a (2.90)
5.76 a (0.19)
17.00 a (1.41)
31.31 a (2.86)
PM+ 50% ash +2% CaO
7.68 cd (0.01)
895 b (71)
72.3 d (18.3)
9.07 ghi (1.21)
4.60 ef (0.21)
10.88 de (1.16)
19.45 ef (2.47)
PM+ 33% ash +2% CaO
7.65 de (0.02)
878 b (76)
86.17 c (3.13)
13.9 cd (0.80)
5.07 cd (0.24)
14.49 bc (1.26)
23.55 c (2.52)
PM+ 25% ash +2% CaO
7.60 e (0.02)
834 b (27)
94.80 b (1.33)
18.19 b (1.74)
5.29 bc (0.19)
15.24 b (0.14)
26.87 b (1.67)
PM+ 50% ash +4% CaO
7.78 a (0.06)
1071 a (61)
65.88 d (3.02)
11.79 ef (0.48)
4.53 f (0.19)
10.50 de (0.23)
20.8 de (0.89)
PM+ 33% ash +4% CaO
7.74 ab (0.01)
1037 a (97)
83.48 c (4.39)
13.22 de (1.76)
4.85 de (0.18)
13.39 c (0.49)
21.03 de (2.95)
PM+ 25% ash +4% CaO
7.71 bc (0.02)
1020 a (61)
91.41 bc (6.86)
15.53 c (0.97)
5.09 bcd (0.15)
14.18 bc (0.40)
22.01 cd (1.63)
Values in parentheses are standard deviation (n = 3). Values followed by the same letter within a column do not differ significantly at 5% level according to the DMRT.
However, with a 6% application, the EC contents are within the limit and do not inhibit the plant growth. Both soluble NH4 –N and PO4 –P were higher with pig manure control product and decreased with addition of fly ash and lime. Further, increase in fly ash concentration decreased the concentrations of these nutrients, mainly due to the dilution effect. When considering the DTPA extractable metals, the concentrations of Cu, Mn and Zn were significantly lower for the fly ash amended treatments. Despite the high concentrations of the fly ash, when mixed with soils, the availability of the metals is controlled by the pH. Since the pH of the soil mixed with the alkaline stabilized product was not acidic, the availability of these heavy metals decreased in the soil mix as also reported earlier [15]. Further, increasing the lime concentrations from 2% to 4% reduced the availability of these metals in the soil mix. When the extracts of these soil mixes were tested for the cress seed germination, no inhibitory effect was observed and the germination was >90% for all the treatments (data not shown), indicating the suitability of the alkaline stabilized products in soil application. 4. Conclusions Temperature, pH and ammonia are the major factors involved in pathogen reduction of biosolids. The temperature through the 8-day stabilization period (did not fell below 45 ◦ C) together with the high pH and NH3 resulted in below detectable levels of indicator and pathogenic microorganisms. The addition of 4% lime and 25% CFA was enough to achieve a pH of ∼12 and the pH was maintained for about 4 days, which would be enough to inactivate the pathogens. High pH mediated release of NH3 also played a significant role in the pathogen reduction. Incidentally, the pH of the stabilized mass was alkaline after 8-day stabilization period. Either, this might be suitable for the application to acidic soils or a suitable length of curing phase may be employed to solve this problem. For all the pathogens tested, 4% lime with 25% ash addition to the manure resulted in below detectable levels within 4 days and this period was shortened to 2 days when the ash addition increased to 50%. The addition of 4% lime and 50% CFA to the manure was effective against the re-growth of pathogens after alkaline stabilization. However, the increase in EC should be considered, when a high concentration of lime and/or CFA is used. This situation could be overcome by the control of application rate of the stabilized product. On the basis of our results, 25% CFA and 4% lime could
destruct the indicator and pathogenic microbial population during alkaline stabilization; however, the post-stabilization re-growth of Salmonella above the standard limit was observed. Application of the alkaline stabilized product at ≤6% generally improved the soil properties and reduced the availability of metals. However, higher application may be employed in case of acidic soils, which needs further study. Hence, 4% lime and 50% coal fly ash addition to the pig manure during the alkaline stabilization is recommended to reduce the indicator microorganisms and reduce the post-stabilization regrowth below the detectable limit. References [1] T.Y. Guan, R.A. Holley, Pathogen survival in swine manure environments and transmission of human enteric illness—a review, J. Environ. Qual. 32 (2003) 383–392. [2] P.D. Gessel, N.C. Hansen, S.M. Goyal, L.J. Johnson, J. Webb, Persistence of zoonotic pathogens in surface soil treated with different rates of liquid pig manure, Appl. Soil Ecol. 25 (2004) 237–243. [3] J.A. Entry, N. Farmer, Movement of coliform bacteria and nutrients in ground water flowing through basalt and sand aquifers, J. Environ. Qual. 30 (2001) 1533–1539. [4] I.G. Krapac, W.S. Dey, W.R. Roy, C.A. Smyth, E. Storment, S.L. Sargent, J.D. Steele, Impacts of swine manure pits on groundwater quality, Environ. Pollut. 120 (2002) 475–492. [5] J.V. Gagliardi, J.S. Karns, Leaching of Escherichia coli O157:H7 in diverse soils under various agricultural management practices, Appl. Environ. Microbiol. 66 (2000) 877–883. [6] Y.N. Qi, S. Gillow, D.S. Herson, S.K. Dentel, Reactivation and/or growth of fecal coliform bacteria during dewatering of anaerobically digested biosolids, Water Sci. Technol. 50 (2004) 115–120. [7] R. Lewis-Jones, M. Winkler, Sludge Parasites and Other Pathogens, Ellis Horwood Limited, Chichester, West Sussex, England, 1991. [8] J. Sidhu, R.A. Gibbs, G.E. Ho, I. Unkovich, Selection of Salmonella typhimurium as an indicator for pathogen re-growth potential in composted biosolids, Lett. Appl. Microbiol. 29 (1999) 303–307. [9] K.J. Zaleski, K.L. Josephson, C.P. Gerba, I. Pepper, Potential regrowth and colonization of salmonellae and indicators in biosolids and biosolid-amended soil, Appl. Environ. Microbiol. 71 (2005) 597–602. [10] A. Letellier, Distribution of Salmonella in swine herds in Quebec, Vet. Microbiol. 67 (1999) 229–306. [11] USEPA, A plain English guide to the EPA Part 503 Biosolids rule, EPA/832/R93/003, Washington D.C., 1994. [12] L. Allievi, A. Colombi, E. Calcaterra, A. Ferrari, Inactivation of fecal bacteria in sewage sludge by alkaline treatment, Bioresour. Technol. 49 (1994) 5–30. [13] R.T. Haug, P.E. Kuchenrither, D. Oerke, T.B.S. Prakasam, S. Soszynski, D. Zenz, Sludge processing technology, in: C.L. Hing, D.R. Zenz, R. Kutchenrigher (Eds.), Municipal Sewage Sludge Management: Processing, Utilization and Disposal, vol. 4, Technomic Publishing Co. Inc., 1992, pp. 223–298. [14] M. Fang, J.W.C. Wong, G.X. Li, M.H. Wong, Changes in biological parameters during co-composting of sewage sludge and coal fly ash residues, Bioresour. Technol. 64 (1998) 55–61.
J.W.C. Wong, A. Selvam / Journal of Hazardous Materials 169 (2009) 882–889 [15] J.W.C. Wong, D.C. Su, The growth of Agropyron elongatum in an artificial soil mix from coal fly ash and sewage sludge, Bioresour. Technol. 59 (1997) 57–62. [16] T.J. Logan, B.J. Harrison, Physical characteristics of alkaline stabilized sewage sludge (N-viro soil) and their effects on soil physical properties, J. Environ. Qual. 24 (1995) 153–164. [17] J.W.C. Wong, S.W.Y. Li, M.H. Wong, Coal fly ash as a composting material for sewage sludge: effects on microbial activities, Environ. Technol. 16 (1995) 527–537. [18] J.W.C. Wong, M. Fang, R. Jiang, Persistency of bacterial indicators in biosolids stabilization with coal fly ash and lime, Water Environ. Res. 73 (2001) 607–611. [19] P. Samaras, C.A. Papadimitriou, I. Haritou, A.I. Zouboulis, Investigation of sewage sludge stabilization potential by the addition of fly ash and lime, J. Hazard. Mater. 154 (2008) 1052–1059. [20] A.L. Page, R.H. Miller, D.R. Kaeney, Methods of soil analysis. Part 2: chemical and microbial properties, second ed., American Society of Agronomy Inc., USA, 1982, pp. 435–676. [21] W. Thompson, P. Leege, P. Millner, M.E. Watson (Eds.), TMECC (Test Methods for the Examination of Composts and Composting), The US Composting Council, US Government Printing Office, 2003, http://tmecc.org/tmecc/index.html. [22] L.A. Richards, P.L. McCarty, Diagnosis and improvement of saline and alkaline soils, in: US Salinity Lab Agricultural Handbook, No. 60, 1954. [23] C.N. Sawyer, P.L. McCarty, Chemistry for Environmental Engineering, third ed., McGraw-Hill Book Company, NY, 1978. [24] B. Vinneras, Comparison of composting, storage and urea treatment for sanitizing of fecal matter and manure, Bioresour. Technol. 98 (2007) 3317–3321. [25] M. Fang, J.W.C. Wong, K.K. Ma, M.H. Wong, Co-composting of sewage sludge and coal fly ash: nutrient transformations, Bioresour. Technol. 67 (1999) 19–24. [26] J.W.C. Wong, K.M. Lai, M. Fang, K.K. Ma, Effect of sewage sludge amendment on soil microbial activity and nutrient mineralization, Environ. Int. 24 (1998) 935–943.
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´ I. Plachá, G. Gresérová, M. Fotta, N. Sasáková, Z. Pacajová, Aerobic [27] J. Venglovsky, ´ and hydrate lime stabilization of sewage sludge—comparison, in: J. Venglovsky, G. Gresérová (Eds.), Proceedings of the 10th International Conference ‘RAMIRAN 2002’, Strbske Pleso, High Tatras, Slovak Republic, May 14–18, 2002, pp. 59–63. [28] S. Himathongkham, S. Bahari, H. Riemann, D. Cliver, Survival of E. coli O157:H7 and Salmonella typhimurium in cow manure and cow slurry, FEMS Microbiol. Lett. 178 (1999) 251–257. [29] J.B. Farrell, G. Stern, A.D. Venosa, Microbial destructions achieved by full-scale anaerobic digestion, in: Workshop on Control of Sludge Pathogens, Series IV, Water Pollution Control Federation, Alexandria, VA, 1985. [30] C.L. Bean, J.J. Hansen, A.B. Margolin, H. Balkin, G. Batzer, G. Widmer, Class B stabilization to achieve pathogen inactivation, Int. J. Environ. Res. Public Health 4 (2007) 53–60. [31] S.C. Read, C.L. Gyles, R.C. Clarke, H. Lior, S. McEwen, Prevalence of verocytotoxigenic Escherichia coli in ground beef, pork, and chicken in southwestern Ontario, Epidemiol. Infect. 105 (1990) 11–20. [32] C. Turner, The thermal inactivation of E. coli in straw and pig manure, Bioresour. Technol. 84 (2002) 57–61. [33] S.E. Jepsen, M. Krause, H. Gruttner, Reduction of fecal Streptococcus and Salmonella by selected treatment methods for sludge and organic waste, Water Sci. Technol. 36 (1997) 203–210. [34] W. Muller, Die Lebensfahigkeit von salmonellen in beluftetum kommunalem Abwasser, Schlacht und Viehhof Zeitung 9 (1973) 332–336. [35] R. Iranpour, H.H.J. Cox, Recurrence of fecal coliforms and Salmonella species in biosolids following thermophilic anaerobic digestion, Water Environ. Res. 78 (2006) 1005–1012. [36] B. Rufete, M.D. Perez-Murcia, A. Perez-Espinosa, R. Moral, J. Moreno-Caselles, C. Paredes, Total and faecal coliform bacteria persistence in a pig slurry amended soil, Livestock Sci. 102 (2006) 211–215.
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Reduction of indicator and pathogenic microorganisms in pig manure through fly ash and lime addition during alkaline stabilization Jonathan W.C. Wong ∗ , Ammaiyappan Selvam Sino-Forest Applied Research Centre for Pearl River Delta Environment, Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong SAR
a r t i c l e
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Article history: Received 22 October 2008 Received in revised form 9 April 2009 Accepted 9 April 2009 Available online 16 April 2009 Keywords: E. coli Fecal coliforms Fecal streptococcus Re-growth Salmonella
a b s t r a c t A pilot scale study was conducted to evaluate the effect of lime and alkaline coal fly ash (CFA) on the reduction of pathogens in pig manure during alkaline stabilization and suppression of re-growth during post-stabilization incubation. Pig manure was mixed with CFA at 25%, 33% and 50%, and a control without fly ash was maintained. To these manure–ash mixtures, lime was added at the rate of 2% or 4% and incubated for 8 days. During the incubation, the population of Salmonella, fecal coliforms, Escherichia coli, fecal Streptococcus and total bacteria were enumerated. After the alkaline stabilization process, the mixtures were incubated under green house condition to evaluate the re-growth of pathogens. During the 8-day alkaline stabilization, Salmonella, fecal coliforms, E. coli and fecal Streptococcus were completely devitalized in manure–ash–lime mixtures, whereas in the control, incubation reduced the pathogen and total bacterial population by 2–3 logs. Fecal streptococcus was destructed within 4 days of alkaline stabilization, whereas other pathogens needed 8 days for their destruction. During the incubation in green house, an increase in the population of the pathogens and total bacteria was observed. Results indicate that alkaline stabilization of pig manure with lime at 4% and CFA at 50% is effective in devitalizing the pathogens and reducing the post-stabilization re-growth. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Livestock manures are potential reservoirs of nutrients and are applied on the agricultural fields so as to manage the disposal as well as to recycle the nutrients. However, a wide variety of pathogenic bacteria may be found in the feces of animals posing threats to the public health. Land application of raw manure potentially spreads pathogens to a wider environment and as a consequence bacterial pollution of agricultural lands was demonstrated [1,2]. For example, contamination of the ground water with fecal coliforms and fecal streptococcus as a consequence of leaching from livestock manure was demonstrated [3,4]. When E. coli reached soil, via manure spreading or runoff from point source, it could survive, replicate, and move downward for up to 2 months, threatening non-target environments [5]. Biosolids should be treated to reduce the pathogenic potential to minimize the risks to the environment and public health. Fecal coliforms, most important subgroup of total coliforms, are thought to be a better indicator of fecal contamination, because they tolerate higher environmental temperatures [6]. Fecal streptococci, along with fecal coliforms have been used to differentiate
∗ Corresponding author. Tel.: +852 3411 7056; fax: +852 3411 5995. E-mail address: [email protected] (J.W.C. Wong). 0304-3894/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2009.04.033
human fecal contamination from that of other warm-blooded animals. Although fecal streptococci are not ideal as indicators of fecal contamination, these organisms are relatively easy to enumerate and survive longer than fecal coliforms [7]. In addition, Salmonella is of concern because of the potential exists for re-growth following treatments [8,9]. Salmonella can survive in manure for up to 3 weeks and in manure slurry for up to 5 weeks. In swine, the prevalence of Salmonella in the feces has been reported in Quebec to be between 8% and 25% [10]. Alkaline stabilization is commonly used to eliminate pathogens and reduce odors in biosolids. For example, lime stabilization of biosolids is one of the processes listed in the Part 503 regulations to significantly reduce pathogens to a level considered as Class B biosolids [11]. Pathogens such as fecal coliforms and fecal streptococcus, enriched in biosolids, were rapidly inactivated following treatment with calcium hydroxide or potassium hydroxide at a pH of 11 or 13 for a period of 2 weeks [12]. High pH following lime treatment was suggested as the major reason for destroying or inactivating the pathogens and microorganisms in biosolids [13]. Along with lime, alkaline coal fly ash (CFA) had also been employed in alkaline stabilization of sludge [14–19]. For example, in our earlier study [18], we found that alkaline stabilization of MSW sludge with 10% CFA and a minimum of 8.5% lime (dry weight basis) for at least 2 h resulted in acceptable levels of salmonella and coliforms. However, the re-growth of the pathogens was not evaluated
J.W.C. Wong, A. Selvam / Journal of Hazardous Materials 169 (2009) 882–889 Table 1 Selected physicochemical and microbiological properties of pig manure. Parameter
Pig manure
pH EC (dS m−1 ) NH4 –N (mg kg−1 ) PO4 –P (mg kg−1 ) Total organic carbon (%) Total N (% dry weight) Total P (% dry weight) Moisture content (%) Salmonella (log CFU g−1 ) Fecal coliform (log CFU g−1 ) E. coli (log CFU g−1 ) Fecal Streptococcus (log CFU g−1 ) Total bacteria (log CFU g−1 )
7.61 (0.05)a 6.83 (0.2) 1860 (46) 1434 (54) 36.6 (0.77) 3.03 (0.12) 1.02 (0.23) 75 (2.3) 7.32 (0.58) 6.77 (0.62) 6.65 (0.41) 6.69 (0.61) 7.61 (0.3)
a
Values in parentheses are standard deviation (n = 3).
in pig manure after alkaline stabilization. Although, many reports available on the MSW sludge, reports were not available on the pathogen elimination in pig manure using CFA–lime mixture during alkaline stabilization. Hence, the present study aims at evaluating the effectiveness of lime and CFA (a) as alkaline stabilizing agents for pig manure; (b) inactivation of fecal coliforms, fecal streptococci, Salmonella, and E. coli during alkaline stabilization in the pig manure; and (c) re-growth of the pathogens during post-alkaline stabilization incubation in the green house. 2. Materials and methods
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by a computer-controlled system during the whole process. Forced moistened ventilation at 1 L/kg dry weight/min was supplied to the reactors to provide oxygen to the stabilizing mass. The stabilizing mass in the incubators was turned on days 2, 4 and 6 to maintain the homogeneity of the composting mass and about 100 g of samples was collected from each treatment for analyses. 2.2. Chemical analysis The moisture content was determined by oven-drying at 105 ◦ C for 24 h while pH and EC were measured in water extracts [1:5, sample (w):deionized water (v)]. The total N and P were extracted by H2 SO4 acid digestion and then determined by using the Berthelot and molybdenum blue methods, respectively [20]. Soluble N (as NH4 –N) and P (as PO4 –P) were estimated using indophenol-blue and molybdenum blue methods, respectively [20]. Total organic carbon (TOC) was analyzed using the Walkley–Black method [20]. Cress seed germination test was conducted as per the standard methods for testing compost materials [21]. For the total heavy metal contents in CFA and composts, samples were subjected to mixed acid digestion (conc. HNO3 and conc. HClO4 ) and analyzed using atomic absorption spectrophotometer (Varian Techtron Model AA-10) and graphite furnace atomic absorption spectrophotometer (GFAAS) with deuterium background correction. For DTPA extractable Cu, Mn and Zn, soils were extracted with 1:5 (sample:extractant, w/v) diethylene triaminepentaacetic acid–triethanolamine (DTPA–TEA) [20], shaken at 200 rpm for 2 h; centrifuged at 8000 × g for 5 min and the supernatants were analyzed after filtration.
2.1. Alkaline stabilization process 2.3. Microbial population determination Fresh pig manure was collected from Kardoorie Farm & Botanic Garden and stored at 4 ◦ C until use. The coal fly ash was collected from the Castle Peak Power Station of China Light & Power Co. Ltd., Hong Kong and stored at room temperature until use. Selected properties of the pig manure and coal fly ash used in the study are presented in Tables 1 and 2, respectively. For the stabilization experiments, the pig manure was mixed with CFA at 1:1 (50%), 2:1 (33%) and 4:1 (25%) in dry weight basis in a closed blending mixer for 2 h. A control without the addition of CFA was maintained. Then, all the fly ash amended treatments were subjected to a lime treatment at two concentrations of 2 and 4% (w/w, dry weight basis) to check the additive effect of lime and CFA on pathogen elimination. Wood chips as bulking agent at 1:10 (woodchip:mixture, v/v) were added to all the treatments and then mixed using a concrete mixer. The mixtures, 4.5 kg per reactor, were incubated individually for 8 days for slow composting in a 20-L reactor with suitable insulation to prevent the heat loss. Temperature was monitored Table 2 Selected physicochemical characteristics of the coal fly ash. Parameter
Coal fly ash
pH EC (dS m−1 ) Moisture content (%) Organic carbon (%) N (%) P (%) Ca (%) Mg (%) K (%) Cadmium (mg kg−1 ) Copper (mg kg−1 ) Manganese (mg kg−1 ) Zinc (mg kg−1 )
12.4 (0.2)a 2.02 (0.03) 0.13 (0.02) 0.15 (0.012) 0.0047 (0.003) 0.33 (0.017) 5.9 (0.14) 0.51 (0.02) 0.14 (0.04) 3.51 (0.04) 37.9 (0.6) 293 (2.6) 34.2 (1.5)
a
Values in parentheses are standard deviation (n = 3).
On day 0, 2, 4, and 8, Salmonella, Escherichia coli, fecal coliforms, fecal Streptococcus and total bacterial populations were enumerated using XLD agar (CM 469), MacConkey Agar No. 3, mFC Agar, KF Streptococcus agar and nutrient agar, respectively. All the media were purchased from Oxoid (Hampshire, England), except mFC agar which was purchased from Difco, Sparks, MD, USA. Ten grams of sample were added aseptically to 90 ml of sterile water in a conical flask, and shaken for 1 h in an orbital shaker. Then the sample was allowed to settle for 20 min and the 100 l of the supernatant was used for culturing microbes after serial dilution. Plates were incubated at 35 ◦ C, except mFC agar for fecal coliforms, which was incubated at 44.5 ◦ C. The colonies were counted after 24 h incubation, except Salmonella, which was counted after 48 h. The results are presented as log CFU (colony forming units)/g sample. All microbial analyses were done within 24 h after sampling in triplicate samples. 2.4. Green house incubation and effect on soil properties After the 8-day composting treatment in the composters, the mixtures were stored in green house for 21 days to examine the re-growth of microbial population. In this storing phase, insulation and force ventilation was not provided and the moisture content of the mass was maintained at around 60% so as to simulate the field irrigation conditions as well as providing enough moisture to the microbes in the alkaline stabilized mass. The temperature range in the greenhouse is between 20 and 30 ◦ C. At days 0, 7, 14 and 21, samples were collected from each treatment and analyzed for bacterial population as described before. After greenhouse incubation, the alkaline stabilized pig manure products from different treatments were mixed with soil at 2%, 4% and 6% (w/w, dry basis) and the soils were analyzed for pH, EC, soluble NH4 –N, PO4 –P, DTPA extractable Cu, Mn and Zn.
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3. Results and discussion 3.1. Changes in physicochemical properties 3.1.1. Temperature The patterns of temperature changes for all the treatments were similar. Temperature increased from day 0 to day 2 (from 41–44 ◦ C to 50–55 ◦ C) (data not shown) and then declined to about 45 ◦ C. With the addition of CFA, the temperature was less than that in the control, mainly due to the dilution effect, which also reduced the microbial population. When comparing different rates of lime addition, higher temperature was observed in those with 4% lime treatment than in that with 2% lime treatment. The increase in temperature was mainly due to the exothermic reaction of calcium oxide hydration. 3.1.2. Moisture content, pH and electrical conductivity Changes in the moisture content, pH and electrical conductivity (EC) during the 8-day alkaline stabilization are presented through Fig. 1a–c. Initial moisture content of the manure mix was influenced by the addition of coal fly ash principally as the dilution effect (Fig. 1a). Moisture contents were decreased significantly for about 4 days in all the treatments, except control, where the reduction was
not as high as the lime amended treatments. Further, the increase in lime addition decreased the moisture content, might be due to the exothermic reaction of lime and consequent water loss. Addition of 2% lime with different concentrations of CFA increased the pH to >11; while, 4% lime addition increased the pH of the manure mass to >12 (Fig. 1b), which is recommended by the USEPA for the pathogen reduction in Class B certification [11]. The changes in the concentration of CFA did not influence the pH significantly. However, increasing CFA concentration in the mixture from 25% to 50% increased the pH from 12.08 to 12.27. Further, the pH of >12 was achieved and maintained for 4 days by the addition of lime at 4% and CFA at >33%. However, a pH of ∼12 can be achieved and maintained with 25% CFA and 4% lime. Maintaining a high pH can destroy pathogens and hence addition of lime at 4% along with 25% CFA is optimum to maintain the pH at about 12 for >2 days. The pH dropped gradually after 4 days in all the treatments. In contrast, in the control without lime and CFA, the pH increased gradually during the 8 days incubation. Increasing pH is the major factor in devitalizing the pathogens in sewage sludge alkaline stabilization with lime [13] as also observed in our study due to the addition of lime and CFA,. Lime and CFA amendment increased the EC to ∼11 dS m−1 (Fig. 1c) from 6.85 dS m−1 observed in the control. The EC increased
Fig. 1. Changes in the moisture content (a), pH (b), electrical conductivity (c), total organic carbon (d), soluble NH4 –N (e) and soluble PO4 –P (f) during alkaline stabilization of pig manure with coal fly ash and lime (CaO). ((䊉) control–pig manure without lime and fly ash, () pig manure + 50% ash + 2% CaO; () pig manure + 33% ash + 2% CaO; () pig manure + 25% ash + 2% CaO; () pig manure + 50% ash + 4% CaO; () pig manure + 33% ash + 4% CaO; and () pig manure + 25% ash + 4% CaO.)
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up to 2 days and gradually decreased to 5.27 dS m−1 in control, whereas in other treatments, the EC decreased gradually in the first 2 days; then sharply in the next 2 days and finally reached an EC of ∼6.3 dS m−1 . The increase in the addition of ash from 25% to 50% resulted in only a slight increase in the final EC. Similarly, the EC of treatments with 4% lime addition did not differ significantly from 2% lime addition. In the control and all treatments, the EC was higher than the phytotoxicity limit of 4 dS m−1 [22], which may potentially limit plant growth; however the phytotoxicity depends on the application rate. 3.1.3. Total organic carbon, NH4 –nitrogen and PO4 –phosphorus The changes in total organic carbon (TOC), soluble NH4 –N and PO4–P contents in the pig manure–CFA–lime mixtures are presented in Fig. 1d–f. Total organic carbon contents in the stabilizing mass decreased slowly (Fig. 1d). Generally, TOC reduction was higher with fly ash and lime amended treatments (10.7–16.7%) compared to control (7.4%), mainly due to the dilution effect of the ash contents and subsequent initial TOC contents. However, when the ash amendment was decreased from 50% to 25%, there was an increase in TOC reduction in both 2% and 4% lime treatments. Results indicate that the fly ash enhances the decomposition of organic matter and the efficiency was higher at 25% ash amendment. The NH4 –N concentrations decreased during the stabilization period of 8 days (Fig. 1e) mainly due to the volatilization loss of NH4 as NH3 gas at high pH [23]. High pH and the resulting ammonia had an enhanced disinfecting activity [24]. With an increase in ash content from 25% to 50%, the soluble NH4 –N concentration decreased due to the reduced amount of manure and volatilization due to higher pH. Since the soluble NH4 –N concentration in manure is 1.86 g/kg, the reduction due to CFA and lime would be beneficial in terms of treatment expenses to meet the standard value of <500 mg/kg. The soluble P contents decreased slightly during the incubation period in all the treatments. The reduction is likely due to the utilization of P by the inhabiting microbial population. Further, addition of CFA caused a reduction in the concentration of soluble P, attributed to the reduction in manure quantity and precipitation of PO4 –P to a less available form with CFA [18,25,26]. But in the present study, a significant reduction was not observed, indicating the interaction of organic rich pig manure with the CFA. In the alkaline treatment of the manure, the alkaline materials especially lime result in high concentrations of calcium at elevated pH, which favors the complexing of soluble phosphate in the manure with the calcium compounds to form insoluble complexes [25]. In the present study, only a marginal decrease in the PO4 –P was observed in all the treatments. This indicates the lack of interaction of PO4 –P in pig manure with lime or ash. 3.1.4. Changes in the pathogenic and total bacterial population The addition of CFA led to the reduction in Salmonella, fecal coliforms, E. coli and fecal Streptococcus populations (Fig. 2a–d). In control treatments, reduction of 2–3 log CFU in the pathogens was observed. Whereas in lime and CFA amended manures, complete destruction was observed within 8 days. Especially, E. coli and fecal Streptococcus were destructed within 4 days, indicating the efficiency of the alkaline treatments. When the lime addition increased to 4% with CFA, the populations of Salmonella and fecal coliforms were below the detection limit within 4 days, whereas, in 2% lime + CFA treatments, this was achieved in 8 days. However, E. coli and fecal streptococci populations were below detectable levels after 4 days even with 2% lime + CFA mixture. An increase in CFA content from 25% to 50% further resulted in faster destruction. For example, manure mixed with 4% lime and 25% CFA resulted in the below detectable levels of Salmonella, fecal coliforms, and
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fecal streptococcus within 4 days. But this below detectable level was achieved within 2 days when the concentration of CFA was increased to 50%, indicating the additional effect of CFA on pathogen reduction. Almost similar trends were observed with different CFA concentration with 2% lime. However, 25% CFA+ 2% lime resulted in below detectable levels of Salmonella and fecal coliforms within 4 days; whereas, in 33 and 50% CFA additions, below detectable level was achieved in 8 days. Venglovsky´ et al. [27] reported destruction of Salmonella within 60 min after the addition of 10% hydrated lime to MSW sludge. Similar effects of the alkaline materials were observed in our study also. Pathogenic populations were substantially (∼3–5 logs) destructed upon the addition of lime and CFA. Since the analysis was initiated within 24 h, at best this period could be <24 h. Aeration of slurries reduced the time required for salmonella destruction, and is accelerated by increasing temperatures [28]. In our study, the temperature was increased in the first 2 days which coincided with the maximum destruction of pathogens. Hence, increasing temperature along with an increase in pH due to the addition of CFA and lime led to the successful destruction of Salmonella and other pathogens. With the mixing of CFA up to 50% diluted the composted mass in terms of microbial populations and nutrients in relation with the control pig manure treatment. Although the reduction in control treatment would accounted for ∼3 log CFU/g composting mass, the reduction after day 4 is not as rapid as the initial days, indicating the persistence of the indicator and pathogenic microorganisms in considerable quantity. Although, there was a dilution due to the addition of CFA, the initial CFA and lime mixing led to the reduction of indicator and pathogenic microorganisms substantially, indicating the influence of these alkaline materials in pathogen reduction. Wong et al. [18] reported a slight increase in total coliforms in the sludge without lime and ash treatment, after 18 h of incubation. However such increases in coliforms were not observed in our study. Fecal coliforms are good indicators of pathogen density and are currently used as the microbiological parameter for Class B certification of USEPA. Farrell et al. [29] showed that if the fecal coliform reduction in treated sludge was 100-fold (2 logs), then a reduction of Salmonella and enteric viruses would be about 1.5 logs and 1.3 logs, respectively. However, Bean et al. [30] reported that fecal coliforms cannot be used as an indicator of treatment effectiveness for all pathogens, including Salmonella. But in our study, the patterns of reduction of fecal coliforms and Salmonella were similar indicating their applicability as indicator for Salmonella during the alkaline treatments. Most strains of E. coli inhabit the intestines of animals and humans are harmless and in many cases beneficial. But harmful enterotoxogenic strains such as E. coli O157:H7 produce potent toxins that can cause severe illness in humans. The prevalence of E. coli O157:H7 has been reported to be between 0.4% and 7.5% in healthy pigs and up to 1.5% of pork meat samples [31]. Turner [32] demonstrated the destruction of E. coli in pig feces after 2 h at 55 ◦ C. Similarly, Himathongkham et al. [28] reported a 105 fold reduction in E. coli O157:H7 after 45 days at 37 ◦ C. Although no attempts were made to identify the strains of E. coli in our study, generally population of E. coli, which includes the enterotoxogenic forms, can be a better indicator. Jepsen et al. [33] reported a reduction of fecal streptococci by 3 orders after 24 h stabilization after the addition of hydrated lime to bring the pH to >12, with Salmonella being undetectable. In our experiment, 48% reduction in fecal streptococci was observed in the treatment with 2% lime and 50% ash; and the percent reduction decreased to 14% with the reduction in CFA content to 25%, implying the effect of CFA. In treatment with 4% lime, no fecal streptococci were detectable after 4 days. Lewis-Jones and Winkler [7] reported that fecal streptococci can survive longer than fecal coliforms. In contrast, relatively shorter time was required to destruct
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Fig. 2. Changes in the population of Salmonella (a), fecal coliform (b), E. coli (c), fecal streptococci (d) and total bacteria (e) during alkaline stabilization of pig manure with coal fly ash and lime (CaO).
fecal streptococci than fecal coliforms in our study may be due to the effect of CFA and lime. Although there was a reduction of pathogenic bacteria in the control treatment, high level of population was observed even after eight days of composting. This reduction may be attributed to the increase in temperature and increased pH driven NH3 produced during the decomposition of manure by microbes. The EPA Part 503 regulations require the fecal coliform density may not exceed 1000 CFU/g total solid and 3 CFU of Salmonella/4 g of total solid to qualify as Class A. Generally, alkaline treatment with lime and CFA resulted in stabilized products to meet the Class A standard. Further, increasing the CFA addition to 50% decreased the time required for the complete inactivation of pathogens. Total bacterial population was significantly reduced due to the addition of lime and CFA. Higher lime addition led to higher reduction in the population of bacteria. Even with the highest lime (4%) and ash addition (50%), the total bacterial population was nearly 105 log CFU/g after stabilization. During the alkaline stabilization process, E. coli and fecal streptococci were sensitive to lime and ash addition, due mainly to an increase in pH, NH3 and temperature, whereas, a relatively longer period of composting was required for the destruction of Salmonella and fecal coliforms. However, all
pathogens can be eliminated with an 8-day alkaline stabilization process involving ash and lime. Lime addition at 4% and CFA at 50% were satisfactory in pathogen reduction. Temperature, pH and ammonia are considered to be the major factors involved in pathogen reduction of biosolids. In our study, maximum temperature was observed on day 2 and the temperature declined thereafter. However the population of pathogenic organisms was detected even after 4 days, which indicates that the developed mesophilic temperature alone was not enough to devitalize all the pathogens. Muller [34] reported that the Salmonella survived less than 8 days at 37 ◦ C. In the present study, the temperature, through the 8-day stabilization period, did not fell below 45 ◦ C, which obviously should completely devitalize the pathogen population. The addition of 4% lime and 25% CFA was enough to achieve a pH of ∼12. The increased pH, which resulted in the release of NH3, played a significant role in pathogen reduction. In all the ash and lime amended treatments, the pathogens are completely eliminated. In the control, considerable population was observed even after the 8-day incubation period, which imply that increased pH due to the application of lime and CFA played a major role in devitalizing the pathogens, in addition to the mesophilic temperature.
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Fig. 3. Re-growth of Salmonella (a), fecal coliform (b), E. coli (c), fecal streptococci (d) and total bacteria (e) during incubation at green house after alkaline stabilization of pig manure with coal fly ash and lime (CaO).
3.2. Pathogens re-growth Bacterial population increased during the 21 days incubation period (Fig. 3). There was a marked increase in the population of Salmonella and fecal coliforms (Fig. 3a and b), which took relatively longer time to be eliminated during the alkaline stabilization. Whereas, E. coli and fecal streptococci increased marginally (Fig. 3c and d). After re-growth, the Salmonella population was above the limit of 3 CFU/4 g solid set out by the USEPA Part 5.3 in all the treatments, except the treatment with 50% CFA and 4% lime; whereas, the limit of 1000 CFU/g of fecal coliforms could be achieved with 4% lime and 25% CFA. Zaleski et al. [9] reported that re-growth of indicators and Salmonella was possible in biosolid under conditions of favorable moisture and temperature or substrate availability. Iranpour and Cox [35] also reported the recurrence of fecal coliforms and Salmonella sp. in the post-anaerobic digestion of biosolids. One of the suggested reasons was incomplete destruction of the fecal coliforms during thermophilic digestion. In a field study, Rufete et al. [36] reported the persistence of fecal and total coliforms due to the application of swine manure slurry. They also reported that the total coliforms increased when the soil amended with mineral fertilizer. Among the treatments, E. coli was detected only in 25% ash added treatments. Similarly, among the differ-
ent ash and lime treatments, fecal streptococci were detected only in 25% ash with 2% lime addition, which indicates that the alkaline CFA also involved in pathogen reduction. To conclude, Salmonella and fecal coliforms were completely devitalized in the 50% CFA and 4% lime addition enabling to reach the Class A biosolid standard. However, indigenous microbes present in soils are important for controlling the potential re-growth of bacterial pathogens [9]. Hence, if the manures are mixed with soil and incubated or establish with plant species, the antagonistic effects of indigenous microbes could be expected to suppress the re-growth of these pathogens. 3.3. Influence on soil properties After the greenhouse incubation, the alkaline stabilized products were mixed with soil at 2%, 4% and 6% (w/w, dry basis) so as to understand the influence on soil properties such as pH, EC, soluble NH4 –N, PO4 –P, DTPA extractable Cu, Mn and Zn and the results for the 6% application are presented in Table 3. The pH of the soils increased as the concentration of the lime and ash increased and ranged between 7.50 and 7.78 when compared to the 6.39 in the soil without any amendment. Similar trend was observed for the EC also as the amendment of alkaline substances increased the EC.
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Table 3 Selected chemical properties of the soil mixed with 6% of the alkaline stabilized pig manure (PM) with different treatments. Treatment
pH
EC (S/cm)
Soluble NH4 –N (mg/kg)
Soluble PO4 –P (mg/kg)
DTPA -Cu (mg/kg)
DTPA -Mn (mg/kg)
DTPA -Zn (mg/kg)
Soil
6.39 m (0.40)a
301 j (10.2)
1.30 j (0.35)
12.37 j (2.53)
0.15 l (0.03)
0.19 h (0.02)
1.46 ij (0.32)
PM Control
7.50 fg (0.02)
815 b (13)
138.0 a (5.5)
31.15 a (2.90)
5.76 a (0.19)
17.00 a (1.41)
31.31 a (2.86)
PM+ 50% ash +2% CaO
7.68 cd (0.01)
895 b (71)
72.3 d (18.3)
9.07 ghi (1.21)
4.60 ef (0.21)
10.88 de (1.16)
19.45 ef (2.47)
PM+ 33% ash +2% CaO
7.65 de (0.02)
878 b (76)
86.17 c (3.13)
13.9 cd (0.80)
5.07 cd (0.24)
14.49 bc (1.26)
23.55 c (2.52)
PM+ 25% ash +2% CaO
7.60 e (0.02)
834 b (27)
94.80 b (1.33)
18.19 b (1.74)
5.29 bc (0.19)
15.24 b (0.14)
26.87 b (1.67)
PM+ 50% ash +4% CaO
7.78 a (0.06)
1071 a (61)
65.88 d (3.02)
11.79 ef (0.48)
4.53 f (0.19)
10.50 de (0.23)
20.8 de (0.89)
PM+ 33% ash +4% CaO
7.74 ab (0.01)
1037 a (97)
83.48 c (4.39)
13.22 de (1.76)
4.85 de (0.18)
13.39 c (0.49)
21.03 de (2.95)
PM+ 25% ash +4% CaO
7.71 bc (0.02)
1020 a (61)
91.41 bc (6.86)
15.53 c (0.97)
5.09 bcd (0.15)
14.18 bc (0.40)
22.01 cd (1.63)
Values in parentheses are standard deviation (n = 3). Values followed by the same letter within a column do not differ significantly at 5% level according to the DMRT.
However, with a 6% application, the EC contents are within the limit and do not inhibit the plant growth. Both soluble NH4 –N and PO4 –P were higher with pig manure control product and decreased with addition of fly ash and lime. Further, increase in fly ash concentration decreased the concentrations of these nutrients, mainly due to the dilution effect. When considering the DTPA extractable metals, the concentrations of Cu, Mn and Zn were significantly lower for the fly ash amended treatments. Despite the high concentrations of the fly ash, when mixed with soils, the availability of the metals is controlled by the pH. Since the pH of the soil mixed with the alkaline stabilized product was not acidic, the availability of these heavy metals decreased in the soil mix as also reported earlier [15]. Further, increasing the lime concentrations from 2% to 4% reduced the availability of these metals in the soil mix. When the extracts of these soil mixes were tested for the cress seed germination, no inhibitory effect was observed and the germination was >90% for all the treatments (data not shown), indicating the suitability of the alkaline stabilized products in soil application. 4. Conclusions Temperature, pH and ammonia are the major factors involved in pathogen reduction of biosolids. The temperature through the 8-day stabilization period (did not fell below 45 ◦ C) together with the high pH and NH3 resulted in below detectable levels of indicator and pathogenic microorganisms. The addition of 4% lime and 25% CFA was enough to achieve a pH of ∼12 and the pH was maintained for about 4 days, which would be enough to inactivate the pathogens. High pH mediated release of NH3 also played a significant role in the pathogen reduction. Incidentally, the pH of the stabilized mass was alkaline after 8-day stabilization period. Either, this might be suitable for the application to acidic soils or a suitable length of curing phase may be employed to solve this problem. For all the pathogens tested, 4% lime with 25% ash addition to the manure resulted in below detectable levels within 4 days and this period was shortened to 2 days when the ash addition increased to 50%. The addition of 4% lime and 50% CFA to the manure was effective against the re-growth of pathogens after alkaline stabilization. However, the increase in EC should be considered, when a high concentration of lime and/or CFA is used. This situation could be overcome by the control of application rate of the stabilized product. On the basis of our results, 25% CFA and 4% lime could
destruct the indicator and pathogenic microbial population during alkaline stabilization; however, the post-stabilization re-growth of Salmonella above the standard limit was observed. Application of the alkaline stabilized product at ≤6% generally improved the soil properties and reduced the availability of metals. However, higher application may be employed in case of acidic soils, which needs further study. Hence, 4% lime and 50% coal fly ash addition to the pig manure during the alkaline stabilization is recommended to reduce the indicator microorganisms and reduce the post-stabilization regrowth below the detectable limit. References [1] T.Y. Guan, R.A. Holley, Pathogen survival in swine manure environments and transmission of human enteric illness—a review, J. Environ. Qual. 32 (2003) 383–392. [2] P.D. Gessel, N.C. Hansen, S.M. Goyal, L.J. Johnson, J. Webb, Persistence of zoonotic pathogens in surface soil treated with different rates of liquid pig manure, Appl. Soil Ecol. 25 (2004) 237–243. [3] J.A. Entry, N. Farmer, Movement of coliform bacteria and nutrients in ground water flowing through basalt and sand aquifers, J. Environ. Qual. 30 (2001) 1533–1539. [4] I.G. Krapac, W.S. Dey, W.R. Roy, C.A. Smyth, E. Storment, S.L. Sargent, J.D. Steele, Impacts of swine manure pits on groundwater quality, Environ. Pollut. 120 (2002) 475–492. [5] J.V. Gagliardi, J.S. Karns, Leaching of Escherichia coli O157:H7 in diverse soils under various agricultural management practices, Appl. Environ. Microbiol. 66 (2000) 877–883. [6] Y.N. Qi, S. Gillow, D.S. Herson, S.K. Dentel, Reactivation and/or growth of fecal coliform bacteria during dewatering of anaerobically digested biosolids, Water Sci. Technol. 50 (2004) 115–120. [7] R. Lewis-Jones, M. Winkler, Sludge Parasites and Other Pathogens, Ellis Horwood Limited, Chichester, West Sussex, England, 1991. [8] J. Sidhu, R.A. Gibbs, G.E. Ho, I. Unkovich, Selection of Salmonella typhimurium as an indicator for pathogen re-growth potential in composted biosolids, Lett. Appl. Microbiol. 29 (1999) 303–307. [9] K.J. Zaleski, K.L. Josephson, C.P. Gerba, I. Pepper, Potential regrowth and colonization of salmonellae and indicators in biosolids and biosolid-amended soil, Appl. Environ. Microbiol. 71 (2005) 597–602. [10] A. Letellier, Distribution of Salmonella in swine herds in Quebec, Vet. Microbiol. 67 (1999) 229–306. [11] USEPA, A plain English guide to the EPA Part 503 Biosolids rule, EPA/832/R93/003, Washington D.C., 1994. [12] L. Allievi, A. Colombi, E. Calcaterra, A. Ferrari, Inactivation of fecal bacteria in sewage sludge by alkaline treatment, Bioresour. Technol. 49 (1994) 5–30. [13] R.T. Haug, P.E. Kuchenrither, D. Oerke, T.B.S. Prakasam, S. Soszynski, D. Zenz, Sludge processing technology, in: C.L. Hing, D.R. Zenz, R. Kutchenrigher (Eds.), Municipal Sewage Sludge Management: Processing, Utilization and Disposal, vol. 4, Technomic Publishing Co. Inc., 1992, pp. 223–298. [14] M. Fang, J.W.C. Wong, G.X. Li, M.H. Wong, Changes in biological parameters during co-composting of sewage sludge and coal fly ash residues, Bioresour. Technol. 64 (1998) 55–61.
J.W.C. Wong, A. Selvam / Journal of Hazardous Materials 169 (2009) 882–889 [15] J.W.C. Wong, D.C. Su, The growth of Agropyron elongatum in an artificial soil mix from coal fly ash and sewage sludge, Bioresour. Technol. 59 (1997) 57–62. [16] T.J. Logan, B.J. Harrison, Physical characteristics of alkaline stabilized sewage sludge (N-viro soil) and their effects on soil physical properties, J. Environ. Qual. 24 (1995) 153–164. [17] J.W.C. Wong, S.W.Y. Li, M.H. Wong, Coal fly ash as a composting material for sewage sludge: effects on microbial activities, Environ. Technol. 16 (1995) 527–537. [18] J.W.C. Wong, M. Fang, R. Jiang, Persistency of bacterial indicators in biosolids stabilization with coal fly ash and lime, Water Environ. Res. 73 (2001) 607–611. [19] P. Samaras, C.A. Papadimitriou, I. Haritou, A.I. Zouboulis, Investigation of sewage sludge stabilization potential by the addition of fly ash and lime, J. Hazard. Mater. 154 (2008) 1052–1059. [20] A.L. Page, R.H. Miller, D.R. Kaeney, Methods of soil analysis. Part 2: chemical and microbial properties, second ed., American Society of Agronomy Inc., USA, 1982, pp. 435–676. [21] W. Thompson, P. Leege, P. Millner, M.E. Watson (Eds.), TMECC (Test Methods for the Examination of Composts and Composting), The US Composting Council, US Government Printing Office, 2003, http://tmecc.org/tmecc/index.html. [22] L.A. Richards, P.L. McCarty, Diagnosis and improvement of saline and alkaline soils, in: US Salinity Lab Agricultural Handbook, No. 60, 1954. [23] C.N. Sawyer, P.L. McCarty, Chemistry for Environmental Engineering, third ed., McGraw-Hill Book Company, NY, 1978. [24] B. Vinneras, Comparison of composting, storage and urea treatment for sanitizing of fecal matter and manure, Bioresour. Technol. 98 (2007) 3317–3321. [25] M. Fang, J.W.C. Wong, K.K. Ma, M.H. Wong, Co-composting of sewage sludge and coal fly ash: nutrient transformations, Bioresour. Technol. 67 (1999) 19–24. [26] J.W.C. Wong, K.M. Lai, M. Fang, K.K. Ma, Effect of sewage sludge amendment on soil microbial activity and nutrient mineralization, Environ. Int. 24 (1998) 935–943.
889
´ I. Plachá, G. Gresérová, M. Fotta, N. Sasáková, Z. Pacajová, Aerobic [27] J. Venglovsky, ´ and hydrate lime stabilization of sewage sludge—comparison, in: J. Venglovsky, G. Gresérová (Eds.), Proceedings of the 10th International Conference ‘RAMIRAN 2002’, Strbske Pleso, High Tatras, Slovak Republic, May 14–18, 2002, pp. 59–63. [28] S. Himathongkham, S. Bahari, H. Riemann, D. Cliver, Survival of E. coli O157:H7 and Salmonella typhimurium in cow manure and cow slurry, FEMS Microbiol. Lett. 178 (1999) 251–257. [29] J.B. Farrell, G. Stern, A.D. Venosa, Microbial destructions achieved by full-scale anaerobic digestion, in: Workshop on Control of Sludge Pathogens, Series IV, Water Pollution Control Federation, Alexandria, VA, 1985. [30] C.L. Bean, J.J. Hansen, A.B. Margolin, H. Balkin, G. Batzer, G. Widmer, Class B stabilization to achieve pathogen inactivation, Int. J. Environ. Res. Public Health 4 (2007) 53–60. [31] S.C. Read, C.L. Gyles, R.C. Clarke, H. Lior, S. McEwen, Prevalence of verocytotoxigenic Escherichia coli in ground beef, pork, and chicken in southwestern Ontario, Epidemiol. Infect. 105 (1990) 11–20. [32] C. Turner, The thermal inactivation of E. coli in straw and pig manure, Bioresour. Technol. 84 (2002) 57–61. [33] S.E. Jepsen, M. Krause, H. Gruttner, Reduction of fecal Streptococcus and Salmonella by selected treatment methods for sludge and organic waste, Water Sci. Technol. 36 (1997) 203–210. [34] W. Muller, Die Lebensfahigkeit von salmonellen in beluftetum kommunalem Abwasser, Schlacht und Viehhof Zeitung 9 (1973) 332–336. [35] R. Iranpour, H.H.J. Cox, Recurrence of fecal coliforms and Salmonella species in biosolids following thermophilic anaerobic digestion, Water Environ. Res. 78 (2006) 1005–1012. [36] B. Rufete, M.D. Perez-Murcia, A. Perez-Espinosa, R. Moral, J. Moreno-Caselles, C. Paredes, Total and faecal coliform bacteria persistence in a pig slurry amended soil, Livestock Sci. 102 (2006) 211–215.