Transcript E. coli - York College of Pennsylvania

Factors Affecting E. coli Growth in Limed Versus Anaerobically Digested Sludge
Justin Hayes and Dr. Carol Bair, Dept. of Biological Sciences, York College of PA
Anaerobically Digested
Limed
DISCUSSION
Indigenous Bacteria
ABSTRACT
METHODS
A useful method for disposing of stabilized sewage sludge
(“biosolids”) is field application, particularly agricultural fields, where
it can be used as a fertilizer. It has been demonstrated that
pathogens such as Salmonella or E. coli can grow in biosolids,
especially when inoculated by animals (Sidhu 2001). Alkali (lime)
addition and anaerobic digestion are two different techniques used to
stabilize Class B sludge before field application, which suggests that
growth of inoculated bacteria could differ substantially between
samples. The objective of this study was to analyze bacterial growth
in limed and anaerobically digested biosolids, in relation to nutrients,
pH and indigenous microflora. The pH of the limed samples was
found to be significantly higher (p=0.029) compared to the non-limed
(anaerobically digested) samples for the 3 trials. Indigenous
populations, on the other hand, were generally steady during the 3
trials, with no significant differences found between limed and nonlimed samples. Nutrient analysis is planned for future studies. While
seeded E. coliR grew well (107-1010/g dry wt) in non-limed samples
over six-hours, its growth in limed samples was minimal (105 max/g
dry wt), and mostly in the first hour of seeding. Due to the fact that
pre-limed (samples from the alkali plant not containing lime) control
samples allowed extensive growth (108-1011/g dry wt) of seeded E.
coliR, it was concluded that the pH level is likely the main factor in
affecting bacterial growth in these types of biosolids, no matter what
the nutrient content may have been. The results suggest that
bacteria could potentially grow to notably higher levels in
anaerobically digested biosolids compared to limed biosolids, which
may raise some safety concerns.
Sample Collection: Samples were obtained from two different wastewater
treatment plants, one that uses anaerobic digestion and the other using
alkali addition for stabilization. For the 2nd and 3rd trials, additional samples
containing no lime (“pre-limed”) were obtained from the alkali addition plant.
Samples were transported to the lab on ice and large materials were
removed by sieving.
We hypothesized that limed biosolids would encourage less
bacterial growth than anaerobically digested biosolids,
predominantly because of pH, and this was supported.
These preliminary results are based on 3 trials conducted within
a small period of time.
CFU/g dry weight
8
(x 10 )
20
15
10
5
Considering the extreme pH differences, some conclusions can
be made:
• Although it is unlikely that nutrient content (Carbon, Nitrogen)
would have been a factor, these studies remain to be
completed.
• Since the limed and non-limed indigenous populations were
generally steady throughout the trials, they would probably not
impact growth to the same degree as the pH differences.
0
Limed
Moisture Content: Dry weight was determined for each sample.
Non-Limed
Type of Biosolid
pH analysis: Each sample was diluted 1:10 and the pH was recorded.
Indigenous Fungi
7.5
5.0
The fact that pre-limed control samples allowed extensive growth
of seeded E. coliR suggested that the addition of lime, which
raises the pH, provided a hostile environment for bacterial
growth and was likely the reason why seeded E. coliR levels
were more inhibited in limed samples.
2.5
0.0
Limed
Non-Limed
Type of Biosolid
It would seem that additional treatment should be performed on
the anaerobically digested sludge to prevent the possibility of
potentially high bacterial growth, and subsequent possible
danger to the public. In actual practice, pH of soil is considered
in choice of fields where sludge is applied. Although PADEP
has not established fecal coliform limits in Class B limed and
anaerobically digested biosolids, it does mandate that fields
receiving Class B biosolids must not be open to the public or
cultivated for an extensive period of time, which also lessens any
safety risks.
Indigenous Actinomycetes
Viability plating: A standard E. coli lab strain was selected for Rifampicin
resistance, maintained at –90°C, and grown in peptone water. Fifty grams of
autoclaved biosolids were mixed with 45 mL of H2O and 5 mL (~1.5 x 109
cells) of an overnight E. coliR culture, followed by incubation in a 37°C
shaking water bath. Using timed intervals, the samples were plated on
nutrient agar containing Rifampicin (50 g/mL), followed by incubation at
37°C for 24 hours and colony counting (Sidhu 2001).
15
8
CFU/g dry weight
20
(x 10 )
Enumeration of indigenous bacteria, fungi, and actinomycetes: The standard
spread plate count procedure was used as described in APHA (1995). Serial
ten-fold dilutions were prepared in phosphate buffer (0.25M) for all 3 types of
indigenous organisms. All plating was in duplicate.
• Bacteria were isolated on R2A agar (Difco).
• Fungi were isolated on streptomycin-terramycin-malt
extract agar (STMEA).
• Actinomycetes were isolated on double-layer plates of
starch-casein-cycloheximide agar (SCA).
CFU/g dry weight
8
(x 10 )
10.0
10
5
0
These processes also help to reduce the attraction of vectors
(animals that may carry pathogens) to biosolids by significantly
reducing bacterial levels. If indigenous bacterial levels are not
reduced, organic matter could be broken down and vector-attracting
odors subsequently released. Vectors could inoculate biosolids with
potentially pathogenic organisms after land application, or could
transmit pathogens from sewage sludge to humans (PADEP 2000).
RESULTS
The median pH value for the limed biosolids was 12.19, while the non-limed
and pre-limed values were 8.53 and 7.93, respectively (Figure 1). The
differences in pH were significant between limed biosolids and both non-limed
and pre-limed biosolids.
pH values for the 3 Trials
15
*
10
Figure 2. Indigenous microflora counts for the 3 trials. The results of the MannWhitney tests are that variation in median population levels was not significant
between limed and non-limed samples (p values of 0.400, 0.826, and 0.700 for
bacteria, fungi and actinomycetes, respectively). The 95% confidence interval is
represented by the bars.
2.5
LITERATURE CITED
-2.5
0
1
2
3
4
5
6
Time (Hours)
0
Non-Limed
Pre-Limed
Trial 3 Viability Counts
Type of Biosolid
12.5
Non-Limed
Limed
Pre-Limed
R
10.0
E. coli
Figure 1. pH values for the 3 trials. Variation among column medians was significant between limed (*)
and pre-limed biosolids (p=0.016), and between limed and non-limed biosolids (p=0.029). The 95%
confidence interval is represented by the bars.
Log Growth of
For this study, it was expected that existing microbes, pH levels
and nutrient levels would affect bacterial growth in both
samples. It was expected that the limed sample would support
less growth predominantly because of pH, making it extremely
difficult for survival or growth of any organism to occur.
5.0
0.0
Limed
7.5
5.0
2.5
0.0
Figure 2 displays results of the indigenous microflora counts. No significant
differences were found among median population levels of limed and nonlimed samples.
It is also important to point out that there is variability among
samples from each plant, and from the same plant. The fact that
each treatment facility has different industrial sources implies that
each sample may have contained different varieties and
concentrations of metals. Therefore, heavy metals could
have played a significant role in affecting bacterial growth in
either sample. Each treatment plant also has different domestic
and commercial sources that would likely cause variability in
organic matter composition of each set of samples. Neither of
these variables has been addressed.
During viability plating, seeded E. coliR grew well in non-limed
samples, with values ranging from 3.1 x 107 – 8.5 x 1010/g dry
weight. Similar growth values (1.5 x 108 – 1.4 x 1011/g dry weight)
were found for the pre-limed samples. However, the limed samples
allowed minimal growth (1.6 x 105 maximum/g dry weight), occurring
mostly within the first hour, with almost all of the E. coliR falling
Future studies will address the following:
below detectable levels afterwards (Figure 3).
1. Investigate the role of nutrient content in relation to pH levels
Trial 2 Viability Counts
2. Conduct heavy metal analysis of each sample as an
12.5
additional factor affecting growth.
Non-Limed
10.0
Limed
3. Compare E. coliR growth in non-sterilized biosolids to that of
Pre-Limed
7.5
sterilized biosolids used in this study.
5
In past studies, bacteria such as Salmonella were used to test growth
potential in biosolids that were composted following anaerobic
digestion. A Salmonella typhimurium strain (Sidhu 1999) was found
to grow as high as 106/g, and the indigenous microbial population
played a significant role in controlling Salmonella growth (Sidhu
2001).
Non-Limed
Type of Biosolid
Log Growth of
R
E. coli
Sewage sludge is a solid, semi-solid, or liquid residue generated
from the treatment of municipal sewage, and can be classified as
Class A or B following treatment. Class B biosolids, used in this
study, may contain some pathogens, and are therefore applied only
to farmland, reclamation sites, forests, or public contact sites where
crop harvesting, animal grazing, and public contact is restricted for a
certain period of time. Alkali addition and anaerobic digestion are
two common methods used to stabilize sewage sludge.
•Anaerobic digestion breaks down most of the
biodegradable material, which helps to reduce the mass
of volatile solids in sewage sludge by at least 38 percent.
•Alkali (lime) addition raises the pH of sludge
to 12 or higher, which is maintained for 2 hours, along
with a pH of 11.5 or higher for an additional 22 hours.
Statistical Analyses: To control for variability of different moisture levels on
different days, data were compared as to dry weight (per gram).
All statistical analyses were calculated at the 0.05 level using non-parametric
Mann-Whitney tests (Instat).
pH
INTRODUCTION
Limed
-2.5
0
1
2
3
4
5
6
APHA, AWWA, and WEF. 1995. Standard methods for the examination of
water and wastewater. 19th ed. American Public Health
Association Washington, DC.
Pennsylvania Department of Environmental Protection [PADEP]. 2000
December. Sampling manual for pollutant limits, pathogen and
vector attraction reductions in sewage sludge. Available from:
http://www.dep.state.pa.us/. Accessed 2001 July 2.
Sidhu, J., Gibbs, R.A., Ho, G.E. and Unkovich, I. 1999. Selection of
Salmonella typhimurium as an indicator for pathogen regrowth
potential in composted biosolids. Letters in Applied Microbiology
29:303-307.
Sidhu, J., Gibbs, R.A., Ho, G.E. and Unkovich, I. 2001. The role of
indigenous microorganisms in suppression of Salmonella regrowth
in composted biosolids. Water Research 35:913-920.
Time (Hours)
Figure 3. Comparison of E. coliR viability in limed, non-limed and pre-limed biosolids.
ACKNOWLEDMENTS
Thanks to Dr. Carol Bair for her guidance and to the wastewater treatment
plants for allowing me to obtain samples. Thanks to Mrs.Taylor for lab
assistance.