Reduction of indicator and pathogenic mi (1)
Journal of Hazardous Materials 169 (2009) 882–889
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Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
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
i n f o
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: jwcwong@hkbu.edu.hk (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
883
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.
884
J.W.C. Wong, A. Selvam / Journal of Hazardous Materials 169 (2009) 882–889
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
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.
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Contents lists available at ScienceDirect
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
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
i n f o
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: jwcwong@hkbu.edu.hk (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
883
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.
884
J.W.C. Wong, A. Selvam / Journal of Hazardous Materials 169 (2009) 882–889
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
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.
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