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TRANSCRIPT
REDUCTIONS OF INDICATOR ORGANISMS
IN RAW SLUDGE DURING
BIOLOGICAL SOLUBILIZATION OF METALS
Robert Hugh Major
A thesis submitted in conformity wiâh the requirements for the degree of Master of AppIied Science Graduate Department of Civif Engineering
University of Toronto
O Copyright by Robert Hugh Major, 1998
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Rednctions of Indicator Orguiisms in R.w Sludge During Biologid S d u b b t i o n of Metais Master of Applied Science, 1998 Robert Hugh Major Department of Civil Engineering University of Toronto
Abstract
The presence of toxic merals and pathogenic mimorganisms in muniapal sludges are
two limitilig factors for its reuse in agriculture. The present shidy was designeci to examine
the effects of bacterial leaching of municipal raw sludge on the survival of pathogenic
microorganisms during the process.
Experimental results from batch reactors and a continuous pilot plant
reactor showed significant reductions in indicator organism counts. Typical
results included 87 to 98% reductions in total heterotrophic counts, 5 log
reductions in total coliforms, 5 to 6 log reductions in fecal coliforms and 3 log
reductions in fecal streptococci counts. These results were dependent upon a
number of parameters, including the reactor pH, temperature, hydraulic retention
time and sludge type. E>cperiments also show4 that a sterile 1 mL pipette trader yielded
higher MPN total colifonn counts than the standard 3 loop trander in the analysis of
bioleached sludge.
ACKNOWLEDGEMENTS
The author wishes to thank Dr. Glynn Henry, professor of Civil Engineering at the
University of Toronto, for his assistance in this investigation and the preparation of this
report. Dr. Henry's experience, constructive cnticism and insightful input were al1 vital in
the production of this thesis.
In addition, the assistance of Durga Prasad, microbiologist, at the University of
Toronto, was essentiai in al1 aspects of this study. Also, the guidance of Rajesh Seth is to
be aclaiowledged, as is the assistance of Shaju Stephen, Arcot Shivalcumar and Asad Alam.
Finally, the author wishes to thank his parents for their support and encouragement
throughout his acadernic career and particularly during the writing of this thesis.
TABLE OF CON'rlmTs
. . ABSTRACT ............................................................................................ ll
......................................................................... ACKNOWLEDGEMENTS iii . . ................................................................................... LIST OF TABLES ~1. l
... IBT OF FIGURES ......... ........... ............................................................ m
.................................................. 1.0 NI'RODUCTION ...................... ... 1
1.1 Background ......................................................................... 1
1.1.1 SludgeDisposalOpti~~l~ .................................*................ 1
1.1 -2 Bacterial Leaching of Heavy Metals ..................................... 2 1.1 -3 Pathogenic Risk From Sludge Disposal .......................................... S
1 - 1 -4 RepreSenfative Pathogens and Indicator Organisms ......................... 6 . . 1 -2 Objectwes of This Study ........................................................................... -7
2.0 LITERATURE REVIE\iV .................................................................................... 10
................................................................. Indicator Orgaaisms 10
2.1.1 Total Heterotrophic Bacteria .............................................. 12 2.1.2 Total Colifonns ....................................... , ................................. -16
............................................................ 2.1.3 Fecal Cow0 rms 20
.......................................................... 2.1.4 Fecal Streptococci 24
Enumeration of Indicator Organisms ........................................................ -27
2.2.1 The Fate of Pathogens during Sewage Treatment ......................... -27
2.2.2 Enumeration Methods ............. .. ................................................... -33
Effects of Enviroqmental Conditions on Indicator Organisms and ............................................................................... Pathogem Bacteria.. -41
2.3.1 pH ................................................................................................. 42
2.3.2 Temperature ..................................................................................... 50
2.3.3 The Role of Solids in Microorganism Survival ............................... 53
2.3.4 Metals ........................................................................................... 56
2.3.5 AvailabilityofOxygen ............................................................... - 3 8
..................................................................... 2.3 -6 Microbial Interaction 61
Past Studies Cpaceming the Fate of Indicator Organisms and ........................................................ Pathogens Dunng Bacterial Leachmg -64
Health Aspects of On-Land Sludge Reuse ................................................. -68
................................................................................................. Summary -84
....................................................................... 3 -0 MATERIALS AND METHODS -9 1
................................................................................. 3.1 Experimental Setup 91
................................................................................... 3 -2 Source of Samples -93
......................................................................... 3 -3 Bacteriological Methods -93 . . ...................................................................... 3.3.1 Resusatabon Bro tk -94
........................................................... 3.3 -2 Total Heterotrophic Count -95
3.3.3 TotalColiforms ............................................................................. 95
.............................................................................. 3.3.4 FecalColiforms 97
........................................................................ 3 3 . 5 Fecal Streptococci -97
............................................................... 3.4 Physical and Chemical Methods 98
............................................................................... 3.4.1 pH 99
3 .4.2 Dissolved Oxygen ............. ... ................................... -99 ................................................................ 3.4.3 Temperature 99
....................................................................... 3 .4.4 Solids -100
.................................... 3 .4.5 Oxidation Reduction Potential -100
............................................................... 3 .4 .6 Sulp hate 100
...................................................... 4.0 RESULTS AND DISCUSSION 101
.................................... ........*. 4.1 Batch Reactors .. 10 1
............................................................. 4.1. 1 Experiment 1 101
............................................................. 4.1 -2 Experiment 2 -103
.............................................................. 4.1 -3 Experiment 3 104
............................................................. 4.1.4 Experiment 4 -107
............................................................ . 4.1 5 Experiment 5 -110
............................................................ 4.2 Continuous Reactors 113
.............................................. 4.3 MPN vs . Spread Plate Method -117
............. 4.4 Experiments Designed to Improve Microbial Counting 120 * * ............................................................... 4.4.1 Inltrai Trials 120
....................... 4.4.1.1 Pipette Transfer Technique 121
4.4.1.2 Broth/qouble Strength Broth/ .............................. Extende Recove3 Incubat~on -122
................................. 4.4.2 Recovery Broth . Detailed Study -128
......................... 4.4.3 1 mL Pipette Transfer - Detailed Study 134
.......................................................................... 5.0 CONCLUS IONS 13 7
6.0 RECOMMENDATIONS ................................................................. 139
....................................................................................... REFERENCES 140
........................................................................................... APPENDIX 149
LIST OF TABLES
Table
.............. Possible output of selecîed pathogens in sewage in a developing country.. 3 1
Removal of pat hogens by primary sedimentation tanks, trickling filters
...................................... and activated sludge processes =ch acting in isolation.. -3 2
Range of indicator O rganism concentrations in anaerobically digested
............................................................................................ sludge and raw sludge. -3 4
Effects of oxygen, pH and inoculum level on growth of Salmonella
.......................................................................................... senfrenberg at 3 0°C.. -59
SuMval of FC in aerated pond water in the light and the dark .............................. 62
Bacteria, viruses, protozoa and helminths in wastewater and sludges.. .................... -70
S u ~ v a i times of various excreted pathogens in soi1 and on crop
............................................................................................. surfàces at 20-3 0°C 73
SuMval times of vanous excreted pathogens in sludge at 20-30°C ....................... 74
Factors a f f i g the s u ~ v a l time of entenc bacteria in soi1 .................................. 76
Likelihood of exposure fiom pathogens to humans as refated to
the number of organisms potentially present in each pathway and
........................................................................................... the infectious dose.. .-77
Surnmary Table: Reductions in indicator organism counts during
two difTerent operating conditions of the continuous bacterial
................................................................................................. leaching process 1 15
Comparison of the MPN and the spread plate method for the
....................................................... enurneration of total coliforms in raw sludge 1 18
Comparison of the standard method with the recovery broth technique
in terms of the total number of positive presumptive, BGB and EC test
............................................................................. tubes producecl by each method 133
LIST OF FIGURES
Figure
Cornparison of the recovery of E-coli, E-aerogenrs and SJaecahs on
nonselective TGEY mediium after 24-hour exposure to an acid mine
....................................................... stream. ..................................................... ... -28
Components of a conventional sewage treatment system. ..................................... 29
Cornparison of the recovery of E coli and E. areogenes on nonselective
TGEY medium and selective DLA agar dunng a 9-hour exposure to the
environment of an acid mine stream.. ..................................................................... -44
Cornparison of the recovery of E. coli, E. aerogenes and S. faecalzs on
nonseledive and selective medium during a 24-hour exposure to the
environment ofan acid mine stream ............... .. ..................................................... -45
Universai flow diagram mode1 for the movement of pathogens ............................ -69
Effect of bacterial leaching on presumptive, total and fecal coliform counts - Expehent 1.. ................. .. .................................................................................. -102
Effect of bacterial leaching on presumptive, total and fecal coliform
counts - Expenment 2... ...................................................................................... 105
E f f i of bacterial leaching on fecal streptococci counts - Expenment 2 ............... 106
Effect of bacterial leaching on presumptive, total and fecal coliform
counts - Experiment 3. ....................................................................................... . 1 08
Effect of bacterial leaching on presurnptive, total and fecal coliform
counts - Experiment 4 ........................................................................................... 1 0 9
Effect of bacterial leaching on presumptive, total and fecal coliform
counts - Experiment 5 -.. ............. .... ................................................................. 1 1 1
Variation in indicator organism counts in raw sludge fiom the Main
WastewaterTreatment Plant in Toronto ............................................................. 114
Initial Trial : 3 loop versus 1 rnL, pipette transfer (pH= 1 -74). .............................. -123
Initial Trial : Single strength presumptive broth versus double strength
presumptive broth versus recovery broth in a sample analysis from the
.................... continuous bactend ieaching process (pH = 2.95, HRT = 10 days).. -125
Enlarged view of Figure 4.9 (fecal colifonn results ody) .................................... 126
4- 1 1 Comparison of the recovery broth technique and the standard method
in the analysis of a raw sludge sample for indicator organims ............................ 129
4.12 Comparison of the recovery broth technique with the standard method for
enumerating fecal coliforms during bacterial leaching in a batch reactor ............... 130
4.13 Cornparison of the 3 loop and 1 rnL pipeîte transfer in a batch reactor ................ 136
CHAPTER 1
INTRODUCTION
1.1 Background
1 . 1 1 Sludge Management Options
The individual who discovers an economical way to make municipal
sludge into a precious material wili probably win the Nobel prize and a m a s
great riches in the process. Until then however, the management of municipal
sludges is one of the greatest challenges facing environmental engineers today.
It is primarily an economic problem, since the construction cost of a sludge
processing facility can be one-third of the total wastewater treatment plant
price-tag (Viessman and Hammer, 1993).
The conventional methods of sludge disposal include burial in landfill,
incineration, production of soi1 conditioner (biosolids) and ocean dumping
(Viessman and Hammer, 1993). For coastal cities ocean dumping, where not
prohibited, is often the least expensive (Viessman and Hammer, 1993),
however, this practice is often closely controlled by government agencies and
may be disallowed depending on the coastal area (Bruce and Davis, 1989). It
has been observed that for inland works in the UK, the application of sludge
on farmland is usually the least cost disposa1 route (Davis, 1987). However,
sludge applied to agricultural land (grassland or arable) is subject to soi1 metal
limits, organic compound lirnits and to reguiations intended to minimize the
risk of disease transmission (Bruce and Davis, 1989). Burial of sludge is often
practised if landfill area is available, however, it is also subject to certain
constraints, particularly concerning the physical stability of t h e sludge
constituents (Bruce and Davies, 1989; Viessman and Hammer, 1993).
incineration, although costly, may b e the only feasible method of disposa1 in
some urbanized areas (Viessman and Hammer, 1993). Minor outiets such as
protein extraction and oii production are currently only at the experimental
level (Bruce and Davis, 1989).
1.1.2 Bacterial Leaching of Heavy MetaIs
Two major difficulties associated with the land spreading of sewage
sludges are: (1) unacceptably high levels of heavy metals cari be present in the
sludges; and (2) pathogenic microorganisms in t he sludges can pose health
risks to humans and livestock.
Heavy metals can accumulate in the food chain if spread on land and
thus cause harmful health effects in humans. Two general rnethods to obtain
sludges with acceptable heavy metal contents for land application are:
(1) impose and enforce regulations to prevent industries from dumping heavy
metals into the municipal sewerage system; and (2) treat the heavy metal laden
sludge to remove the heavy metals. The first method may not always produce
acceptable results and thus option number two may have to be employed. In
addition, even if the untreated sludge has an acceptably low heavy metal
content, further rnetal reductions through sludge treatment could increase the
"useable lifetime" of the dumping site. As well, environmental regulations
have a tendency to become more stringent as time goes on and method (2) may
become the only feasible way for a municipality to meet extremely stringent
environmental guidelines regarding the heavy metal content o f its wastewater
sludge.
Two possible techniques for implementing method (2) are acid treatment
and a biological process called bacterial leaching. Treatment of sludge with
acid for metal removal is not practical since lead (Pb) and copper (Cu) are not
significantly rernoved and also because a costly amount of acid is required
(Wong and Henry, 1983). The biological process of bacterial leaching was
first proposed i n an article by Wong and Henry (1983) and since then
numerous studies have been conducted, to develop t h e technology (Blais et
al., 1992; Singh, 1997; Seth, 1997). Some investigators have suggested that
bacterial leaching could replace the conventional aerobic sludge digestion
process (Blais et al., 1992).
Two methods for bioleaching are: (1 ) bio-solubilization using
acidification to pH 4 plus Thiobaciilrts ferrooxidans and ferrous sulphate as
substrate; and (2) bio-solubilization using adapted strains of Thiobaci lhs
with elemental sulpur as substrate. Method (2) has been shown to be the most
effective method for bioleaching to date. In method (2) the bacterial oxidation
of added elemental sulfur and subsequent acidification (pHC2.5) of the sludge
results in metal solubilization according to equation ( 1 - 1 ) (Blais et a!, 1992):
The solubilization of metals from reduced sulphur compounds can also
occur according to equation (1-2) (Blais et al., 1992). Although this process
renders the sludge safe for application to agricultural land with regard to its
heavy metal content the possibility exists that it still may contain pathogens.
The present study was designed to examine the effects of bacterial leaching of
municipal raw sludge on the suwival of pathogenic microorganisms during the
process.
1.1.3 Pathogenic Risk From Sludge Disposa1
Pathogens are microorganisms that are potentially disease-causing to
humans and livestock. Pathogenic organisms present in sludge of human origin
include certain bacteria, viruses, fungi, protozoa and helminths (Lohaza,
1985).
The number and types of microorganisms present in municipal
wastewater sludges depend on a number of factors. These include such things
as the degree of urbanization, population chemistry and sanitary habits, season
of the year and the rate of disease in the community (Fradkin et al., 1985).
Although extensive literature exists regarding pathogen survival and the
potential health hazards, there is a lack of conclusive evidence (case histories)
of diseases resulting from pathogen contact from sludge from any of the
aforementioned disposa1 methods (Fradkin et al., 1985). According to
Schwartzbrod et al. ( 1 987) the literature is very scarce on the potential health
effects of sludge exposure. Although Schwartzbrod ei al. (1987), present a
Iiterature review on the health effects of sludgekewage exposure, there is a
definite need for more epidemiological studies regarding sludge disposal. This
topic is addressed more fully in Section 2 . 5 .
1.1.4 Representative Pathogens and Indicator Organisms
Due to the limited data base on the pathogenic organisms in municipal
sludge and the lack of simple appropriate measurement methods,
"representative pathogens" are often tracked through waste treatment and
disposal pathways (Fradkin et ai., 1985). Past studies have utilized the
following representative species: SaIrnonella for enteric bacteria;
enteroviruses as an example of human enteric viruses; Entamorba hislolytîca
of Giardia lamblia for protozoans; Ascaris lumbricoides for helminths; and
Aspergillus fumigattrs for fungi (Fradkin et al . , 1985). However, when
reviewing test results it should be kept in mind that results of standard tests
for representative species are subject to variabiiity among different
laboratories (APHA el al., 1995). Gerba ( 1 9 8 3 ) as cited by Fradkin et al.
(1985) reported that quantitative detection of pathogens, especially viruses is
not highly precise and results should be compared on the basis of orders of
magnitude.
Another approach for testing the bacteriological quality of sludge is to
use indicator organisms. The following criteria are important in the selection
of an indicator organism: (1) the number of indicator organisms should be
greater than the number of pathogens; (2) the indicator organism should be
easy to detect; and (3 ) the indicator organism should not cause any disease.
Coliforms have been the traditional indicators of the presence of pathogenic
bacteria from fecal pollution and are probably sufficient in this role
(Funderburg and Sorber, 1985). Fecai streptococci (FS) are also a widely
used indicator of pathogenic bacteria from fecal pollution.
Coliforms can be tested for in terms of total coliforms (TC) and fecal
coliforms (FC). The total coliform group includes non-fecal bacteria which
can corne from such sources as soi1 and vegetation. Since there are no
pathogens in vegetation and soii, it is important to distinguish between total
coliforms and fecal coliforms. It follows that the total coliform group is a
more conservative indicator of pollution than the fecal coliform group.
Heterotrophic bacteria are sometimes considered to be an indicator
group, but their usefulness is quite limited, since it includes such a wide range
of different bacteria (Olivieri, 1982; as cited by Lohaza, 1985).
1.2 Objectives of This Study
It has been hypothesized that the bacterial leaching procedure could
destroy pathogens originally present in the sludge (Lohaza, 1985). The final
bio-leached sludge could then be spread on land and would impose less of a
health risk on humans and livestock than non-bioleached sludges.
A study by Blais et al, (1992) reported that the low pH environment
(pHC2.5 ) during the five day bioleaching process resulted in a considerable
reduction in bacterial indicators (Le. TC, FC and FS were reduced by 3 logs
( to below the detection limit of 1 o3 cfu/lOOmL)), for al1 sludges examined.
However, it was not evident that the study by Blais et al. (1992) used
appropriate media. Their study utilized direct plating of sludge samples ont0
selective media. This could have influenced the results since many of the
bacteria may b e injured by the acid environment and unable to grow when
directly plated ont0 the selective media. Such a result was reported by Roth
and Keenan ( 197 1 ) who stated that "unreliable results may occur if selective
media are used for the detection and enumeration of Enterobacteriaceae in acid
foods." Thus. i t is the objective of this present study to determine the effect
of first putting sludge samples into an enrichment media to strengthen the
injured bacteria and then to enumerate the total coliforms. fecal coliforms and
fecaI streptococci using selective media. In this way a more accurate
enumeration of al1 indicator bacteria should be obtained.
It was the conclusion of Lohaza (1985) that bacterial leaching of
anaerobically digested sludge did not reduce the number of total coliforms,
fecal coliforms, or fecal streptococci present. Interestingly, Lohaza (1985)
did utiiize initial enrichment media, then selective media (Le. the MPN
methods from Standard Methods) instead of direct plating onto selective
9
media. It should be mentioned that Lohaza's bioleaching process ( 1985)
involved T. ferrooxidans and ferrous sulfate as the substrate, whereas, Blais
el ai. (1992) employed sulphur as the substrate with adapted strains of
Thiobacillrrs isolated from sludge (which involved a lower pH environment
than in Lohaza's experiments).
Therefore, this present study will examine the fate of indicator
organisrns (TC, FC, FS, HPC) in the bioleaching process. The MPN procedure
will be compared with the direct piating procedure (Blais et al. 1992) in this
study for enumerating total coliforms. Also, considering that the indicator
organisms rnay be injured from acid exposure, alternative methods for
improved indicator organism enurneration will be investigated.
2.0 LITERATURE REVIEW
The monitoring of pathogens is important for any wastewater treatment
facility. The use of indicator organisms to predict the fate of pathogens
during sewage treatrnent is widely practiced and numerous studies have been
conducted in this area. This present literature review covers indicator
organisms in general, as well as an in depth review of four specific indicator
organisms (HPC, TC, FC, FS). Various rnethods available for indicator
organism enumeration are also presented. In addition, the effects of
environmental conditions on indicator organism and pathogen survival are
summarized. This literature review also covers past studies which have been
conducted on the fate of indicator organisms and pathogens during bacterial
leaching. Finally, the health aspects of on-land sludge reuse are presented.
2.1 Indicator Organisms
Although ernploying indicator microorganisms for assessing water and
wastewater quality is widely practised, there has been much debate in the
literature over how accurate they are and which indicators are best under given
circumstances. Unfortunately, unlike in wastewater treatment performance,
there are no widely accepted indicator organisms for measuring sludge
disinfection efficiency (Sorber, 1984). However, in the past, four basic groups
of microorganisms have served as measures of efficiency: ( 1 ) indicator
bacteria (Enierobocteria, fecal streptococci); (2) pathogenic bacteria; (3)
viruses; and (4) parasites (Sorber, 1984).
A comprehensive microbiological study of the suitability of potential
indicator organisms for assessing sludge disinfection was conducted by
Strauch (1989). The different disinfection processes involved included
pasteurization, aerobic thermophilic stabilization ( ATSD), ATSD followed by
mesophilic anaerobic digestion, treatment with slaked lime or quicklime and
composting i n windrows and in closed reactors. The microbiological tests
included total aerobic microorganism count, entero bacteriaciae, coliforms,
fecal streptococci, Salmonella, fRNA-p hages and the percentage of sludge
samples infected with parasite ova. Based on temperature and pH studies on
these potential indicator organisms Strauch (1 989) selected enterobacteriaciae,
Salmoneilae, fecal streptococci, coliforms and fRNA-phages as the most
suitable indicators. He also concluded that several microorganisms can
indicate the presence of other organisms, but the true extent of sludge
contamination can only be determined based on direct enumeration of the
relevant organisms.
2.1.1 Total Heterotrophic Bacteria
The aerobic heterotrophic bacterial communities, although not of direct
sanitary importance have been studied by some researchers. Since these
heterotrophs include such a wide range of different bacteria, the HPC1s
usefulness is quite limited as an indicator (Olivieri, 1982; as cited by Lohaza,
1985). For example, Legendre et al. (1984) monitored the heterotrophic
bacteria in sewage treatment lagoons in order to determine what role these
environmental bacteria play as a biological cornpartment of the ecosystem.
These authors point out that the pollution-indicator bacteria (total coliforms,
fecal coliforms and fecal streptococci) differ from the total heterotrophic
bacteria group in taxonomie complexity and adaptive potential. The pollution-
indicator bacteria represent only a fraction of the total heterotrophic bacteria
(Legendre et al., 1984). Legendre et al. (1984) suggest that in the newly
formed eutrophic aquatic ecosystem (the sewage treatment lagoon) the aerobic
heterotrophic bacterial community undergoes a regular strategy of graduai
occupation of ecological niches, characterized by a multiple species structure,
involving many different ecological functions (niches). The pollution-
indicator bacteria were originally grown in a very closed, stable environment
(digestive tract) to which they are well adapted but when they are transplanted
into a new open environment they will at best, survive (Legendre el al., 1984).
It was concluded by Strauch (1989) that the total aerobic microorganism
count cannot serve as a reliable indicator of the state of hygiene of sludge.
However, according to Murthy (1984) the total aerobic bacteria count (TABC)
is believed to provide as much information as any other microbiological index
for routine monitoring of foods.
I n studies on drinking water it has been shown that some species and
strains of Pseudomonas, Sarcina, Micrococcus, Flavobactertum, Proteus,
Bacillus, actinomycetes and yeasts have suppressed coliform detection
(Reithler and Seligmann, 1957; and Weaver and Boiter, 195 1 as cited by
LeChevallier and McFeters, 1985). LeChevallier and McFeters ( 1 985) showed
that HPC bacteria could reduce coliform densities in drinking water by more
than 3 logs within 8 days. Cornpetition for limiting organic carbon was
proposed as the cause of the observed effects. Yet, in sludge there is an
abundance of organic carbon, so t h e above results from drinking water studies
should not necessarily be expected in the sludge bioleaching process. In
addition, LeChevallier and McFeters (1985) reported that some HPC bacteria
were able to cause injury to the coliform population. AIso, Ahmed et al.
(1967) as cited by LeChevallier and McFeters (1985) report that pathogens
have been detected in the absence of detectable coliforms in drinking water
sampies with high HPC levels.
14
However, it is generally accepted that plate counts are inadequate for
counting al1 the naturally occurring aquatic bacteria which are viable (Buck,
1979; as cited by Fry and Zia, 1982). Fry and Zia (1982) studied the
viabilities of planktonic bacteria from nine freshwaters sources and one raw
sewage sample. They reported that viabilities determined from microcolony
formation in slide culture ( 1 8.4 - 48.7%) were higher than a conventional plate
count method (1.8 - 14.9%). The results of the HPC test are also influenced
by the inoculurn size, suitability of the culture medium, incubation temperature
and the incubation time.
A study by Mayo and Noike (1996) on the effects of temperature and pH
on the growth of heterotrophic bacteria (from waste stabilization ponds) in
Chhrella vuigaris-heterotrophic bacteria culture employed the pour plate
method as outlined in Standard Methods (APHA et al., 1985). Temperature
and pH were found to be two important environmental parameters governing
the activities and growth rates of heterotrophic bacteria in waste stabilization
ponds (Mayo and Noike, 1996). The pH of the medium in algal-bacterial
systems influences the biornass regulation, ion transport systern (Guffanti et
al., 1984; as cited by Mayo and Noike, 1996) and rnetabolic rate (Mayo and
Noike, 1994; as cited by Mayo and Noike, 1996). Mayo and Noike (1996)
concluded that an agar pH of 7.0 was optimal for enurneration of heterotrophic
bacteria. There was no significant difference in the number of cells capable
of forming colonies for incubation temperatures of 20 and 35°C. However.
they noted the lag time for colony formation was longer at 20°C. They
recommended t hat samples from algal-bacterial systems such as waste
stabilization ponds be incubated at 35°C for 72 hours. Mayo and Noike (1996)
also stated that, depending on the pH of the culture, 86-98% of cells capable
of forming colonies will be visible to the unaided eye after incubation at 35°C
for 72 hours.
Standard Methods (APHA et al., 1995) recommends an incubation
temperature of 35°C for 48 hours for enurneration of total heterotrophic
bacteria on tryptone glucose extract agar when the plate count technique is
used. However, other authors (Jen and Bell, 1982; and BrozeI and Cloette,
1992; as cited by Mayo and Noike, 1996) recommend a temperature of 30°C
for 72 hours. In a study by Lohaza (1985) on the fate of indicator organisms
during bacterial leaching the pour plate technique for HPC bacteria with an
incubation time of four days at room temperature (20-25°C) was employed.
However in a similar study by Blais et al. (1992) the spread plate technique for
total aerobic colonies with an incubation time of 48 hours at 35°C was
preferred.
2.1.2 Total Coliforms
Coliform bacteria consist of several genera of bacteria belonging to the
Enterobacteriaceae family (APHA et al., 1995). This group has been
historically defined based on the method of detection (lactose fermentation);
rather than on the tenets of systematic bacteriology (APHA et al., 1995).
Coliforrn bacteria are al1 Gram-negative rods, 2 to 5 pm long, about 0.5 pm
in diameter and they are classified as either fecal coliforms or non-fecal
coliforms (Lewis-Jones and Winkler, 1991 ). With the MPN fermentation
technique the coliform group is defined as al1 facultative anaerobic, Gram-
negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas
and acid formation within 48 hours at 35°C. Fecal coliforms, such as
Escherichia coli originate exclusively in the intestines of humans and warm-
blooded animals (Lewis-Jones and Winkler, 199 1). Non-fecal coliforms, such
as Klebsiella, Citrobacter and Enterobacter aerogenes, can be present in
unpolluted soi1 and vegetation as well as in the intestines of animals (Lewis-
Jones and Winkler, 1991). The total number of fecal and non-fecal coliforms
is referred to as total coliforms. Total coliforms are often monitored as a
criterion of bacteriological quality for water and treated sewage effluent
(Lewis-Jones and Winkler, 199 1). However, only the fecal coliforms are
definitive indicators of fecal pollution (Feachem et al., 1983). In wastewater,
17
total coliforms do not necessarily relate to either the occurrence or degree of
fecal pollution since many coliforms are nonfecal in origin and, especialiy in
hot climates, the total coliforms can multiply (Feachem et al., 1983). Over
90% of the total coliform bacteria in the fresh feces of warm-blooded animais
are Escherichia c d i , which are fecal coliforms (Lewis-Jones and Winkler,
1991).
Coliforms are useful as indicators since they are readily identifiable and,
in temperate climates, they can survive only a few hours or days outside their
host, so that their presence indicates the possibility of recent fecal
contamination (Lewis-Jones and Winkler, 199 1 ) . However, it is important to
distinguish the numbers of fecal coliforms and total coliforms in a sample since
it is possible to get coliforms from soil and vegetation and pathogens are not
usually present in soil and vegetation.
However, Lehmann et al. (1983) maintain that the coliform group of
bacteria are important with reference to fecal contamination and the potential
health hazards of sludged fields. They recommend the isolation and
enurneration of important members of the total coliform group in order to gain
useful insight from this information. Lehmann ef al. (1983) restrict total
coliforms to include only E. coli. , Citrobacter freundii and En~erobacter
aerogenes (in accordance with the 1 4 ' ~ edition of Standard Methods) and these
three organism counts are combined and called the total coliforms by Lehmann
et al. ( 1 983) . Based on this definition, Lehmann et al. ( 1983) evaluated the
health hazard of sludged fields and drew conclusions based on this parameter.
The ability of total coliforms to indicate pathogens. however. is
questionable. This is because pathogenic viruses, parasite ova or cysts can be
present i n water free of bacterial fecal indicators (Lewis-Jones and Winkler,
1991). Berg and Berman (1980) reported that total coliforms were poor
quantitative reflectors of the numbers of viruses detected in raw sludges and
in thermophilically digested anaerobic sludges. It was concluded that during
mesophilic and thermophilic digestion of anaerobic sludges, total coliforms
were destroyed more rapidly than viruses. However, total coliforms always
remained i n samples i n which viruses were no longer detected. I n a study by
Strauch (1989) on raw sludge and sludges disinfected by the following
processes: pasteurization; aerobic thermophilic stabilization (ATSD), ATSD
followed by mesophilic anaerobic digestion, treatment with slaked lime or
quicklime and composting i n windrows and in closed reactors, it was
concluded that there was no significant correlation between coliforms and
virus counts in the treated sludge samples. However, there did appear to be
a significant correlation between coliform and virus counts in raw sludge
samples (Strauch, 1989).
19
Funderburg and Sorber (1985) point out that coliforms (total and fecal)
are less than ideal as indicators of enteric viruses i n water and wastewater.
They state that coliforms are cellular, whiie viruses are not and because of
their acellular nature enteric viruses are less subject to environmental stress
than coliforms. Enteric viruses thus tend to survive longer in water
environments than bacteria. I t should also be noted that because viruses are
much smaller than coliforms and because of other morphological
characteristics, viruses behave as colloids in water while coliforms do not
(Funderberg and Sorber, 1 9 8 5 ) . As colloids, viruses have a surface charge and
adsorb to solids more readily than bacteria/coliforms (Funderberg and Sorber,
1985). I t has been hypothesized that viruses adsorbed to solids will be
sornewhat more protected from inactivation than unadsorbed viruses or
bacteria (Funderburg and Sorber, 1985). Also, the numbers of enteric viruses
in sewage depend o n factors such as the season of year, sanitary conditions
and population age; whereas the fecal coliform level will probably remain more
or less constant (Berg, 1969; as cited by Funderberg and Sorber, 1 9 8 5 ) .
Another study involving the relationship between total coliforms and
viruses was carried out by LaBelle et al. (1980). They reported that indicator
bacteria (TC and FC) were not observed to be reflective of the levels of
enteric viruses in marine waters. In this study statistical analysis showed no
20
significant correlation between viruses and total coliforrns in seawater or in
sediment (LaBelle et al., 1980).
Besides the problem of total coliforms not being 'absolute' indicators
of pathogens, difficulties can also arise with coliform enumeration when
coliforms are injured and bypass detection. in drinking water treatment
systems a number of chernical and physical factors can cause a form of
sublethal and reversible injury which results in the failure of coliforms to grow
on accepted media such as m-Endo (McFeters et al.. 1986). These factors i n
drinking water include; chlorine and other biocides, low concentrations of
metals, such as copper and zinc, temperature extremes and interactions with
other bacteria (McFeters et al., 1986). Examination of a water distribution
system water in New England, for example, revealed that 96.8% of the
confirmed coliforms were injured and were not enumerated as either typical or
atypical colonies on m-Endo agar LES (McFeters et al., 1986).
2.1.3 Fecal Coliforms
Fecal coliforms are differentiated from total coliforrns in practice by the
ability of fecal coliforms (mainly composed of E.coli and thermotolerant K.
Pneumoniae) to ferment lactose with the production of acid and gas within 24
t o 48 hours at a temperature 44.S°C (Feachem et al., 1983). According t o
Feachem et al. (1983) only the fecal coliforms (and especially E-col i ) give
definitive indication of fecal pollution. However. in a study by Rivera er al.
(1988). E-coli was isolated from brorneliad leaf surfaces in a rain forest in
Puerto Rico. The authors suggested that E-coli may be part of the
phyllosphere rnicrofiora and not simply a transient bacterium of this habitat.
The unexpected isolation of fecal coliforms from these sites weakens the
validity of using fecal coliforms as indicators of biological water quaiity in the
tropics (Rivera et al., 1983).
In addition, the absence of fecal coliforms is not proof that fecal
contamination has not occurred and in hot climates some sewage may not
contain fecal coliforms (Lewis-Jones and Winkler, 1991). The explanation for
this is that high ambient temperatures allow non-fecal coliforms to grow, but
not fecal coliforms and pathogens (Mara, 1978; as cited by Lewis-Jones and
Winkler, 199 1 ).
In a study by Hood et ai. (1983) it was reported that low fecal coliform
levels were reliable indicators of the absence of Salmonella spp., in the
analysis of freshly harvested and stored oysters from Florida. Although high
fecal coliforms were Iimited in predicting the presence of Salmone[la spp. in
this study. In addition, concentrations of E.colz correlated very strongly with
fecal coliform levels in both fresh and stored oysters and clams (Hood el al . ,
1983).
As was the case with total coliforms, fecal coliforms have also been
shown to be poor indicators of virus concentrations in raw sludges and
mesophilically and thermophilically digested anaerobic sludges (Berg and
Berman. 1980). Fecal coliforms were 7 to 8 times more sensitive to
destruction than the viruses to mesophiiic digestion and 9 to 10 times more
sensitive to thermophilic digestion (Berg and Berman, 1980).
LaLiberte and Grimes (1982) showed that E.coli has the abiIity to
survive for several days in aquatic sediment. Based on this, the authors
suggested that fecal coliforms in water may not always indicate recent fecal
contamination but rather that the resuspension of viable sediment-bound
bacteria rnay have occurred.
In a study by LaBelle et al. (1980) a positive correlation was
demonstrated between the number of viruses in estuarine sediments near Texas
and the number of fecal coiiforms in the same estuarine sediments. On the
other hand, no other physical, chernical, or biological characteristic of
seawater or sediment that was measured exhibited a statistically significant
relationship to virai numbers. Also, no correlation was observed between
bacterial indicators and viruses in the overlying waters (LaBelle et ai., 1980).
The environmental factors and indicators measured included total coIiforms,
fecal coliforms, Clostridia, pH, salinity and turbidity. These authors concluded
that indicator bacteria are not reflective of the concentration of enteric viruses in
marine waters (LaBelle et ai., 1980).
Another example of the use of fecal coliforms as indicators in wastewater
can be seen in the U.S. Environmental Protection Agency's (EPA) Clean Water
Act. In 1973 the EPA required that effluents from wastewater treatment plants
meet specific fecal coliform limitations (Sorber, 1984).
However, in one study on a bamyard and grazed pasture, inhabited by dairy
farm cattle in Alberta, Canada, fecal coliforms were not observed to be reliable
indicators of Salmonefia (Lehmann el al., 1983). In the barnyard the fecal
coliforms ranged from 1 -0 x 1 O' to r 2.4 x 1 O6 MPN/I 008 and yet no Salmonella
were found at any time. Similarly, in the grazed pasture fecal coliform counts in
MPN/100g ranged from c2.0 x 1 O) to 2.2 x 10' and again no Salmonella were
found at any time.
Feachem et al. (1983) daim that E.coli is an inappropriate indicator for the
quality of treated sludges. They suggest that for sludges from conventional
sewage treatment systerns, the viable eggs of Ascaris lumbricoides appear to be
the best pathogen indicator currently available. The U. S. Environmental
Protection Agency (EPA) regulations for Class A sludge application to land
require that limitations in one of the following areas be met: (1) fecal coliform
counts; (2) Salmonella counts; (3) enteric vimses and viable helminth ova counts;
or (4) treatment by an approved method (Outwater, 1994).
2.1.4 Fecal Streptococci
The fecal streptococci (or Group D streptococci) are a group of
rnorphologically similar bacteria (Gram-positive cocci, roughly spherical
bacteria about 1 pm in diameter and occurring in short chains) and are found
mostly, but not exclusively in the intestines of humans and other warm-
blooded animals (Feachem et al., 1983; Lewis-Jones and Winkler, 199 1 ). The
group includes species mainly associated with animals (Streptococcus bovis
and S.equinus), other species which occur in both man and other animals, as
well as two biotypes (S. faecalis var. liquefaciens and an atypical S. faecalis
that hydrolyzes starch) which occur in both polluted and unpolluted
environments (Feachem et al., 1983; Audicana et al., 1995; Knudtson and
Hartman, 1992). These last two strains are indistinguishable from the true
fecal streptococci under routine detection or counting procedures (Feachem
et al., 1983). For this reason fecal streptococci should not be considered as
indicators of the bacteriological quality of wastewater-irrigated crops, since
the two nonfecal biotypes may both be present as natural flora (Feachem et al-.
1983).
Thus, although fecal streptococci are not ideal indicators of fecal
contamination, they are easy to enurnerate, generally survive longer than fecal
coliforms and are less prone to regrowth than fecal coliforrns (Lewis-Jones and
Winkler, 1991). Thus, fecal streptococci rnay b e better indicators than fecal
coliforms for excreted bacterial pathogens (which have little regrowth
tendency) and also for viruses (which survive longer than fecal coliforms in
cool waters) (Feachem et ai., 1983). The ratio of fecal coliforms to fecal
streptococci was at one time utilized to differentiate between human and
animal fecal contamination, however this is now considered unreliable (APHA,
et a!., 1995)
Various investigators have examined whether fecal streptococci counts
reflect specific pathogen counts under various conditions. A study by Berg
and Berman (1980) showed that the rates at which fecal streptococci were
destroyed by mesophilic and thermophilic digestion of anaerobic sludges
approached those at which the viruses were destroyed. These authors suggest
that fecal streptococci could serve as useful indicators of virus reduction
during mesophilic and thermophilic digestion of anaerobic sludges. In another
study the bacteriological effects of hydraulically dredging polluted bottom
sediment in a navigation channel of the Upper Mississippi River known to be
heavily contaminated with metropolitan sewage effluent were examined
(Grimes, 1980). Although the fecal streptococci densities ranged from O to
4000 CFU/ 100mL depending o n the distance downstream, no salmonellae or
shigellae were recovered from either upstream or downstream water samples.
A microbiological investigation of the suitability of potential indicator
organisms for raw and treated sludges was conducted by Strauch (1989).
Based on temperature and pH studies on the indicator organisms Strauch chose
fecal streptococci as a potential indicator for further sludge disinfection
studies. However from these studies Strauch (1989) concluded that fecal
streptococci cannot be utilized as hygiene indicators since their susceptibility
to sludge treatment differs considerably from that of salmonellae. High counts
of fecal streptococci did not always correspond to high counts of Salmonella
and low fecal streptococci counts did not necessarily signify low Salmonella
counts (Strauch, 1989) . However, fecal streptococci did appear to give a
reliable indication of virus contamination, which agrees with the study by Berg
and Berman (1980) concerning mesophilic and thermophilic digestion of
anaerobic sludges.
Other researchers have compared the rates of fecal streptococci
reductions i n harsh environments with the rates of reduction of other
indicators. A study by Sayler et a l . (1975) observed prolonged survival of
fecal streptococci as compared to other indicator organisms such as total and
fecal coliforms i n most of the sediment samples taken in Upper Chesapeake
Bay, Maryland over a one year period. A study by Davies-Colley et a l . ( 1 994)
showed that inactivation of 90% of enterococci (a subset of the fecal
streptococci group) generally required 2 .3 times the solar radiation required
for 90% inactivation of feca1 coliforms.
A study by Hackney and Bissonnette (1978) sho-wed that S. faecalis (a
species of fecal streptococci) survived much longer than the coliforms tested
( E - c o l i and Enierobacter a r r o g r n e s ) when exposed to acid mine streams.
These results are presented in Figure 2.1. In addition, no significant injury
was incurred by S. jaecalzs , whereas the coliforms were sublethally injured
rapidly in the acid mine streams (Hackney and Bissonnette, 1978). Also, S.
faecalzs has also been shown to be more radiation resistant than the indicator
bacteriurn E-col i , Ktebsiella s p . , Enterobacter sp . , Salmonella typhimurium
and Proteus mirabil is (Yeager and O'Brien, 1983).
2.2 Enurneration of Indicator Organisms
2.2.1 The Fate of Pathogens during Sewage Treatment
Several unit processes combine to form a conventional sewage treatment
system. A flow diagrarn showing commonly utilized combinations of the
components in conventional sewage treatment systems is presented in Figure
2.2.
Obviously the numbers of pathogens in raw sewage will Vary, however,
an example of the possible pathogen concentrations in raw sewage is presented
Figure 2.1 - Cornparison of the recovery of E.coli (a); E. aerogenes (V); and S. faecaks ( O ) on nonselective TGEY medium after 24-hour exposure to an acid mine Stream. (from Hackney and Bissonnette, 1978)
Pretreatment I F
. .. P Primary sedimentation ! 9 Activated sludge (trickiing filters)
i 6 F Secondary sedimentation (humus tanks)
I
Sludge digesti
4 Sludge drying
6 Sludge disposai
9 (Tertiary treatment)
1 v Effluent discharge
Figure 2.2 - Components of a conventional sewage treatment system (Adapted fiom Feachem et al., 1983)
in Table 2.1 . Pretreatment by screening or cornminution will not affect any
change on the pathogen content of sewage (Feachem et al.. i 9 8 3 ) . During the
settling of suspended particles in primary settling tanks a proportion of the
pathogens will settle to the sludge layer either by direct sedirnentation or by
being adsorbed ont0 solids that are in the process of settling. Removal of
pathogens from the effluent is shown in Table 2.2. Similar performance can
be expected from secondary settling tanks.
The unit process between the primary and secondary sedimentation
tanks is often either trickling filters or activated sludge. Removal of various
pathogens by trickling filters is shown in Table 2.2. For the activated sludge
process in isolation the pathogen rernoval efficiencies are also shown in Table
2.2 .
From the removal efficiencies indicated in Table 2.2, wastewater sludge
would be expected to contain significant concentrations of pathogens.
However, not al1 sewage treatment plants are exactly like the one illustrated
in Figure 2.2 and the microbiological quality of the sludge will depend on the
extent and type of treatment that it has received.
A review of the literature reveals that indicator organism concentrations
from anaerobically digested sludge and raw sludge Vary significantly (Dudley
et al., 1980; Berg and Berman, 1980). The range of values obtained for
Table 2.1 - Possible output of selected pathogms in sewage in a develophg country (Adapted from Feachem et aL, 1983)
I Vinses Enteroviruses (a)
E. coli f i) Salmonella spp, Shxgellu spp. Vibrio cholerae
Helrninths Ascaris lumbricoides Hoohvomis (c) Schistosornu mmsoni Taenia saginara
I Trichuris trïchiuru 1 (a) - Inchdes polio-. echo-, and coxsackievinises @) - Pathogenic E coli - includes enterotoxigeriic, entaoinvasive, and enteropathogcnic E. coli (c) - Only Ancylostoma duodenale and Necator amencanus
Table 2.2 - Removal of pathogens by primary sedimentation tanks, trickling filters and activated sludge processes each acting in isoiation (Adapted from Feachem et al., 1983)
Vi ruses Bactena Protozoa Helmint hs
Reduction (log base 10 unit) Prirnary Sedimentation
O- 1 O- 1 0- 1 0-2
TrickIing Filters
0- 1 0-2 0-2 O- I
Activated Sludge
O- 1 0-2 0- 1 O- 1
anaerobically digested sludge and raw sludge are presented in Table 2.3.
2.2.2 Enurneration Methods
Before a discussion of the various methods available for determining
pollution indicator organism concentrations in sludge, the importance of
sample preparation methodology will be examined. A study by Dudley et al.
(1980) showed that the best recoveries of viable organisms (total aerobic
colonies, fecal coliforms, fecal streptococci) from sewage sludges (prirnary,
anaerobically digested and activated sludge) were obtained in samples
dispersed by vortex mixing with glass beads as opposed to sonication, or
blender homogenization. The indicator organisms surviving as cornpared to
those obtained by direct plating without any mixing, showed that vortex
mixing for two minutes yielded up to double t h e number of fecal coliforms
compared to the number from blender hornogenization for three minutes.
Vortex mixing always produced higher counts than direct plating without any
mixing, except for fecal streptococci in digested sludge, where counts were
87% of direct plating and in the aerobic count on EMB (eosin-methylene blue
agar) from digested sludge where counts were 88% of direct plating results
(Dudley et al., 1980). It was also shown that sonication at al1 wattage levels
was bactericidal (Dudley et al., 1980).
Table 2.3 - Range of indicator organism concentrations in anaerobically digested sludge and raw sludge (Adapted from Dudley et al., 1980; and Berg and Berman, 1980)
Total aerobic count Total coliforms Fecal coliforms Fecal Streptococci
(a) - Mixture of pnmary sludge (W3) and activated sludge (113)
Raw Sludge (a) CFWI O0 mL
- 1.5 x 10E8 - 1.2 YC 1OElO 5.0 x 10E6 - 8.7 x IOE8 1.3 x IOE6 - 5.6 x 10E7
Anaerobically Digested Sludge cm/ 1 OOmL
1.2 x 10E8 - 4.8 x 10E8 4.5 x 10E6- 1 . 6 ~ 10E8 4.8 x IOE5 - 1 . 1 x IOE7 6.9 x lOE4 - 3.6 x lOE6
35
In another study on sample preparation procedures by Doyle er al.
( 1984) on Arizona lake bottom sediments, it was reported that the distribution
of fecal coliforms as determined by the most-probable-number method, was not
significantly influenced by sedirnent settling for up to 16 minutes following the
dilution-mixing process o f the sample. These authors thus reported that
representative samples for MPN analysis could be obtained by sampling the
supernatant in the mixing vesse1 at any depth for up to 16 minutes after
mixing.
If the indicator organisms to be enurnerated are known to be injured
several things need to be considered concerning the method of analysis, i n
addition to the sample preparation procedures. Factors influencing the
recovery of injured microbial cells from dried foods for example include:
( 1 ) food type; (2) composition of the rehydration medium; (3) period of
rehydration; (4) temperature of rehydration; (5 ) sampleibroth ratio
(6)incubation environment (aerobic versus anaerobic); (7) pH (Andrews.
1986). Mossel and Ratto state that the members of t he family of
Enterobacteriaceae present in dried foods and feeds may carry metabolic
lesions that impair the physiology of the cells to the extent that they will not
proliferate in standard selective media. The cause(s) of such sublethal lesions
may be thermal stressing, sojourning in an environment of very low water
activity, oxygenation, or a combination of such factors (Webb. 1969; as cited
by Mossel and Ratto, 1970). Generally submitting stressed celis to a
"restoration treatment" prior to their exposure to selective media is
recommended with overnight incubation in lactose broth being a common
"restoration treatment" in the examination of dried food (Mossel and Ratto,
1970). Such a restoration treatment was shown to result in 57% higher counts
than when no resuscitation method was employed in analysis of dried foods for
Enterobacteriaceae (Mossel and Ratto, 1970).
Dudley e t al. (1980) enumerated total coliforms, fecal coliforms and
fecal streptococci in vortex-blended sludge samples by three different
methodologies: (1) completed multiple-tube-fermentation; (2) membrane
filtration; and (3) spread-plating directly ont0 appropriate selective media.
These authors concluded that for total coliforms and fecal streptococci direct
plating was the superior method, however the MPN method yielded higher
fecal coliform recoveries. However, the fecal streptococci recoveries obtained
by direct plating are not always superior to those obtained by the MPN
analysis. For example, in one of the samples tested fecal streptococci by
direct plating was 3.0 x 106 CFUllOOmL and by MPN analysis it was 14 x 106
MPN1100mL (this is one out of the three sample tests shown by the authors).
Perhaps more testing could have been done by these authors to confirm that
direct plating is superior t o MPN analysis for fecal streptococci testing in
sludges. It should be noted that Standard Methods (APHA et al.. 1995) only
allows MPN techniques for total and fecal coliforrn and fecal streptococci
enumeration in waters of other than drinking water quality.
Lin (1976) reported that the standard one-step M-FC broth-membrane-
filter procedure was significantly less effective than the MPN technique for
recovery of fecal coliforms from chlorinated sewage effluents. He proposed
a two-step membrane-filter method which was shown to b e comparable to the
MPN procedure. Considering the reported effects of chlorination on sewage
effluents, Qualls et al. (1984) decided to compare MPN and membrane
filtration recoveries of coliforms from UV-irradiated sewage effluents. It was
shown that for UV-disinfected wastewater effluents the standard one-step
membrane filtration method was comparable to the MPN technique recoveries
of total and fecal coliforrns.
Double and Bissonnette (1980) experimented with a two-step MF
procedure for the enumeration of total coliforrns from acid mine waters,
whereby bacterial cells were initially exposed to a rich nonselective medium
so that repair of injured cells could be obtained prior to application of the
standard selective medium. The resuscitation brot h consist ed of 409 peptone,
6g yeast extract, 30g lactose and IOOOmL distilled water (pH 7.4). The
recovery ratio of enriched versus direct MF techniques ranged from 1 . 1 to
74.4, however in most cases the MPN method produced the greatest coliform
densities compared to the MF direct and MF enriched approaches.
Other reports have stated that injured E.coli cells are sensitive to high
incubation temperatures and selective media (Feng and Hartman. 1982) - Also
the MPN method is susceptible to bacterial interference and false-negative
reactions (no gas production in the presence of coliforms) which can occur at
the presumptive, confirmed and completed stages of the MPN test (Feng and
Hartman, 1982). Other problems such as the synergistic gas production from
lactose by non-coliforms, cultivation of anaerogenic and non-lactose-
ferrnenting E.coli strains and the presence of lactose-fermenting noncoliforms
have al1 caused problems with the MPN analysis (Feng and Hartman, 1982).
Various researchers have developed and proposed new methods for the
recovery of pollution-indicator organisms. For example Rose et al. (1975)
proposed an improved membrane filter (MF) method for fecal coliform analysis
of wastewater. Green et a!. (1977) proposed a two-temperature membrane
filter method for enumerating FC bacteria from chlorinated sewage effluents.
They reported that the average recovery of fecal coliforms by the standard MF
procedure was only 14% that of the MPN method, whereas with their new
technique recovery was increased to 68% of the MPN counts (Green et al.,
1977).
Recently the use of microbial enzyme profiles to detect indicator
bacteria has been developed as an alternative to MPN analysis (Park et al..
1995; Bitton er al., 1995; Feng and Hartman, 1982). Such methods involve
the enzymatic hydrolysis of chromogenic or fluorogenic substrates with
subsequent detection of the coloured or fluorescent products (Apte et al..
1995). A study by Feng and Hartman (1982) analyzed raw surface water and
wastewater effluents utilizing a fluorogenic assay. These authors reported
that their method was more efficient than the membrane filter - mFC broth
assay for the enurneration of fecal coliforms. However, although such
fluorgenic assays are permitted by Standard methods (APHA et al., 1995) for
testing water of drinking water quality, they are not permitted for testing other
waters. The MPN method is the only method permitted by Standard Methods
(APHA et al., 1995) for estimating coliform densities in water of other than
drinking water quality.
In addition to the incubation medium, the incubation time can also
influence the results obtained. Roth et al. (1994) examined the effect of
extended incubation of lauryl sulfate tryptose (LST) broth and brilliant green
bile (BGB) broth inoculated with a variety of food and water samples. It was
observed that 40% of the samples showed an increase in the presumptive MPN
40
when incubation was extended from 48 to 72 hours, however, only 5% showed
confirmed MPNs which exceeded the 95% confidence limits established for the
48 hour confirmed MPN. Extending the incubation of BGB tubes to 72 hours
resulted in less than 5% of the sarnples exhibiting increased MPNs, which
exceeded the 48 hours 95% confidence Iimits. The importance of incubating
LST tubes for at least 48 was also shown as 67% of the samples tested
exhibited an increase in the MPN as a result of continuing the incubation frorn
24 to 48 hours, however the extent of the increase was not stated. Some loss
in viability of coliforms was noted when LST tubes were incubated beyond 72
hours. Standard Methods (APHA et al., 1995) stipulates an initial 24 hours
incubation, which if the results are negative, is to be extended to 48 hours for
both LST (presumptive test) and BGB broth (confirmed test).
In addition to incubation time, incubation temperature can also influence
the recoveries of fecal coliforms. Standard Methods (APHA et al., 1995)
requires that in the MPN test for fecal coliforms the EC tubes should be
incubated in a water bath at 4 4 3 ° C for 24*2 hours. However, Weiss et al.
(1983) examined the recoveries of fecal coliforms and of E.coli from raw milk,
ground meat and raw sewage when incubation occurred at 44.5, 45.0 and
45S°C. For the sewage samples there was a trend of decreasing fecal coliform
counts with increasing temperature. At 44.5, 45.0 and 4 5 5 ° C the number of
positive EC tubes were 806, 758 and 679 respectively. This underlines the
importance of maintaining the EC tubes at 445°C as per Standard Methods
(APHA et al . . 1995). However given the nature of the equiprnent in many
laboratories it seerns unlikely that a constant 44S°C is always maintained.
2.3 Effects of Environmental Conditions on Indicator Organisms and
Pathogenic Bacteria
I t has been suggested tha t there are three subpopulations to be
considered when studying the survival of total bacterial populations in water:
( 1 ) those cells that can withstand the stresses of the aquatic environment and
can be detected utilizing selective media and standard laboratory procedures;
(2) those cells that cannot withstand t h e stresses of the aquatic environment
and subsequently become lethally injured and cannot be detected on any media;
(3 ) those cells that become physiologically debilitated. damaged, or injured
due to the stress of their aquatic environment and apparently become sensitive
to inhibitors in selective media and thus these injured cells can only be
detected on nonselective media. (Bissonnette et al., 1975; as cited by Hackney
and Bissonnette, 1978). It is the objective of this Section (Section 2.3) to
examine the reported effects in the literature that different environmental
conditions (pH, temperature, solids, oxygen availability) can have on bacterial
populations and their enumeration.
2.3.1 Effect of p H on Pathogens and Indicator Organisms
The majority of bacteria grow best within a pH range of 5 to 9 (Madigan
rr al., 19%). Only a few species can g r o w a t pH values o f grea ter than 1 O or
less than 2 (Madigan et al., 1996). Understandably then, it has been the
objective of numerous studies to determine the exact effect o f pH on
microorganism survival in various environments.
Hackney and Bissonnette (1978) for example, studied the survival of
indicator bacteria in acid mine water (AMW). They hypothesized that a toxic
microenvironment may result in AMW due to the high ion concentration in the
water, with the hydrogen ion probably being the most toxic ion in acid mine
water. The high hydrogen ion concentration can prevent cells from exchanging
ions and can inhibit growth even when a rich environment is provided
(Hackney and Bissonnette, 1978). Also, exposure to acid mine water, can
alter the interna1 pH of the ceil and thereby cause disruption of the protein and
nucleic acid structure (Hackney and Bissonnette, 1978). In t h e study by
Hackney and Bissonnette (1978) it was reported that the coliforms, (E-coli and
E-aerogenes) were very susceptible to injury in acid mine water and their
enumeration with selective media could lead to erroneous conclusions. In a
9-hour experiment with stream water at pH 3 and an average temperature of
17.2"C, less than 1% of the E.coli population and of the E-aerogenes
population survived (Figure 2.3). When enumerated after 3 hours o f exposure
the recovery ratio of non-selective to selective media was 40 000 for E-coli
and 28 000 for E-uerogenes. In another experiment, strearn water (pH 3 ,
2 4 A ° C ) was examined after 2.3 hours of contact time with the indicator
organisms. It was observed that 0.03% of the E.coli population survived
(non-selective medium), 70% of the E-aerogenes population survived (non-
selective medium) and 95% of the S-faecalis population survived (non-
selective medium) (Hackney and Bissonnette, 1978). Concerning the S.
faecalis population, a recovery ratio of only 1.2 for non-selective versus
selective media was observed. It was thus concluded that the Standard
Methods (APHA et al., 1971) selective media used to enumerate fecal
streptococci is acceptable for use in acid mine water. Also it should be noted
that the S-faecalis population was observed to be a much more conservative
indicator than the coliforms tested in these acid mine water experiments
(Figure 2.4).
A study by Wortman et al. (1986) investigated the morphological
alterations of E.coli that result from exposure to acid mine water (AMW).
These authors concluded that E-coli experienced considerable changes in
Figure 2.3 - Cornparison of the recovery of E.coli (O); and E. aerogenes (O) ; on nonselective (-) TGEY medium and selective (-) DLA agar during a 9-hour exposure to the environment of an acid mine strearn. (from Hackney and Bissonnette, 1978)
O 5 IO 15 20 25 Hours
p p p p p p p p p p p p - - - - - - - - - - - - - -
F p r e 2 . 4 - Cornparison of the recovery of E-coli (O); E. aerogenes (a); and S. faecalïs (O); on nonselective (-) and selective (-) mediums during a 24-hour exposure to the environment of an acid mine stream. (from Hackney and Bissonnette, 1978)
morphology when exposed to AMW. They reported that leakage of cytoplasrn
and lysis often resulted €rom exposure to AMW probably due to the bacterial
envelope being affected. Also, older bacteria were able to withstand injury
from AMW better than younger. smaller cells.
Although these studies clearly show that the external pH influences
indicator organism concentrations, no statistically significant relationship has
been proposed in the literature. Goya1 ei al. (1977) attempted to observe such
a relationship between pH and indicators (total coliforms, fecal coliforms.
Salmonellae) over a pH range of 7.4 to 8.7 (in canal waters), however no
statistically significant relationship could be found. A study by LaBelle el
aL(1980) examined the relationship between environmental factors and the
occurrence of enteric viruses in estuarine sediments. These authors reported
that the number of viruses only increased in a certain range of pH frorn about
7.8 to 8.4 and dropped at both higher and lower ends of the pH scale.
The type of acid and the exact pH may play important roles i n pathogen
inactivation. Roth and Keenan (1971) studied the effects of acetic. lactic.
malic, citric, tartaric, hydrochloric and sulfuric acids on E.coli. The sulfuric
acid had the lowest pH at 3.0 and almost always produced the highest death
rate. It was also observed that greater injury was inflicted by organic acids
than inorganic acids. One possible remedy (which they did not test) was
47
suggested, namely, the use of a highly buffered diluent. The suggestion of a
critical pH has been noted since 1941 when Cowles (1941). as cited by Roth
and Keenan (1 97 1) reported that when the pH of hydrochloric acid solutions
rose above p H 2 . 6 . a rapid loss in bactericidal power would occur.
According to Mayo and Noike (1996). pH is an important factor in the
activities and growth rates of heterotrophic bacteria. The pH of the medium
in algal-bacterial systems influences the biomass regdation, ion transport
system and metabolic rate (Mayo and Noike, 1996). In domestic wastewaters,
the pH is a function of the organic loading rate (Mayo and Noike, 1996). In
their study heterotrophic bacteria were grown in a chemostat and the pH was
controlled from 3 .O to 1 1.5. They enumerated the heterotrophic bacteria with
the pour plate method as described in Standard Mefhods and they reported
that an agar pH of 7.0 produced the most colonies regardless of the pH at
which the bacteria were initially grown. The authors suggested from those
results that the optimum pH for bacterial growth is probably near neutral. The
agar pH recommended by Standard Methods (APHA et al., 1995) is strictly
7.0k0.2.
El Hamouri et al . (1994) examined the ability of high-rate algal ponds
(HRAPs) to remove pathogens. In these systems the physico-chernical factors
that may affect fecal coliform die-off include; UV light penetration, dissolved
oxygen concentration and pH (El Hamouri el Q I . , 1994). With daily pH values
between 8.4 and 9 .4 in the HRAP these authors observed an average rate of
removal of 99.98 for FC and 99.89 for fecal streptococci counts per 1OOmL.
Curtis et al. ( 1992) concluded that humic substances, pH and dissolved
oxygen are important variables in the mechanism by which light damages
microorganisms in waste stabilization ponds (WSP). They reported that light-
mediated damage of fecal coliforms was highly sensitive to increased pH
values in a WSP with a pH range of 7 .5 to 9 . 5 . A rapid decline in fecal
coliforms was observed past pH 9.
Chung and Goepfert (1970) examined the growth of Salmonella at low
pH. Although the optimum pH for growth is thought to be between 6.5 and
7.5 they are also able to grow i n more acidic environments (Chung and
Goepfort. 1970). These authors inoculated 1-3 x 1 O' Salmonella cellslmL of
various acidified broths. They defined growth to mean an increase in ce11
numbers of at least 1 log over the initial load of organisms. Depending on the
acid, the minimum pH for growth of Salmonellae ranged from 4.05 to 5.50.
Also, it was shown that higher levels of inoculum (Le. 106 cells/mL) were more
Iikely to produce growth than Iow levels of inoculum (Le. 102 cells/mL). Also,
aeration was a factor in whether or not growth occurred. As well, Salmonellae
were most tolerant of low pH at 25-32°C. When the pH value would not
49
permit growth the rate of death was most rapid at the higher temperature. It
was noted by the authors however that the results were obtained under ideal
conditions with a nutritionally favourable medium which was free from naturai
inhibitors and competing microflora and the water activity (a,) was quite high.
They suggested that in foods. various parameters (e.g. OIR potential, water
activity, temperature) would act synergistically to prevent Salmoneilae
growth. In addition these authors were unable to "train" Salmonellae t o grow
at lower pH by repeated subculture in acidic environments.
Foster and Hall (1990) examined the adaptive acidification tolerance
response of Salmonella ~yphimurium. First, cells of Salmonel la ~yphirnurium
were grown at pH 7.6; then they were shifted to mild acid for one doubling as
an adaptive procedure. These adapted cells were 100 t o 1000 times more
resistant to a subsequent pH 3.3 environment than were unadapted cells shifted
directly from pH 7.6 t o 3 - 3 . This acidification tolerance response required
protein synthesis and the authors suggested that it appears to be a specific
defence mechanism against acid.
Henry et al. (1983) examined the factors affecting the survival of
Salmonel la and E-col i in anaerobically fermented pig waste. The authors
reported that Salmonella ~yphirnuriurn survived at pH 6.8 but not at 4.0 after
being incubated at 37°C for 24 hours in either fermented o r synthetic medium
50
containing volatile fatty acids (VFA). Also, E.coli could not be recovered
from a fermented medium after being incubated at 30°C for 24 hours with the
presence of VFA ( p H 4.0).
Rubin (1985). examined the protective effect of casein o n Salmonella
typhimurium in acid-milk. It was shown that at a pH of 3 3 5 , 4.2 and 4.5 the
die-off rate was 6.5. 13.0 and 40 rnin/log reduction of cells respectively in
milk with 1.42% lactic acid and was 4.0, 10.0 and 33.3 rnin/log reduction
respectively, i n whey with 1.42% lactic acid. These authors concluded that
the protective effect of casein toward Salmonella typhimurzum increased as
the pH increased.
2.3.2 Temperature
The effects of temperature on pathogenic organism survival have been
studied by various researchers (Goya1 et a l . , 1977; Mayo and Noike, 1996;
Chung and Goepfert, 1970). In a study of canal surface waters and canal
bottom sediments along the Texas Coast. no statistically significant
relationship was observed between water temperature and indicator organism
concentrations (total coliforms, fecal coliforrns and Salmonellae)(Goyal et ai.,
1977). Chung and Goepfert (1970) examined the effect of temperature on the
growth of Salmonellae at low pH values. The temperatures investigated
51
ranged from 16 to 43°C and the pH from 3 -9 to 4.6. It was observed that the
Salmonellae were most tolerant of low pH at 25-32°C and the minimum pH
that allowed growth increased at temperatures above and below this range
(Chung and Goepfert, 1970). Also, these authors noted that at the pH values
not allowing Salmonellae growth, the rate of death was most rapid at the
higher temperature.
Hackney and Bissonnette ( 1978) noted that most sanitary-indicator
organisms as well as enteric waterborne pathogens initially grow in the
intestines of man or warm-blooded animals which is a favourable environment
having a constant temperature. In their study on the recovery o f indicator
bacteria from acid mine streams it appears that the temperature o f the water
at the time of sampling affects the degree of injury to the indicator bacterium
population (E.colz). Injury in this experiment was defined based on those cells
that would grow in nonselective media yet would not g row in selective media.
Data from their work showed that a small (2 to 3°C) increase in temperature
significantly increased the amount of injury without a proportional increase in
death. A direct reiationship between temperature and the amount of injury to
E-coli was shown. However, Xfaecalis was more resistant to srnall increases
i n temperature and did not appear to incur much injury at any Stream
temperature tested although an increased death rate for S-faecalis was
observed at the warmer Stream temperatures.
Sinton et al. (1994) examined the inactivation of enterococci and fecal
coliforms from sewage and meatworks effluents in seawater mixtures. It was
observed that inactivation was generally slower at lower temperatures. It has
been suggested that increased inactivation at higher temperatures is due to
increased activity of predatory and lytic organisms (Gameson, 1984; as cited
by Sinton et al., 1994), and also to the detrimental effects of increased
metabolism at low nutrient levels (Gameson, 1988; as cited by Sinton et al.,
1994). In a sludge bioleaching process a lack of available nutrients is of
course, never a problem, unlike the seawater mixtures as described above.
El Hamouri et al. (1994) examined high-rate algal pond (HRAP)
performances in fecal coliform and helminth egg removals in Morocco. It was
observed that fecal coliform and fecal streptococci removal rates increased
from a -2.2 log unit removal rate in autumn and winter t o a -3.2 log unit
removal rate during summer conditions. However the seasons not only
involve temperature changes but also changes in light intensities which affect
indicator organism die-off in HRAPs. Also the light intensity and the
temperature influence the pH and the dissolved oxygen concentrations in
HRAPs, thus affecting the indicator organism's survival further. Therefore,
HRAPs and the bioleaching process cannot be compared directly.
2.3.3 T h e Role of Solids in Microorganism Survival
Numerous investigations have shown that solids protect indicator
bacteria and enteric pathogens from adverse environmental conditions (Marino
and Gannon, 199 1; LaBelle et ai., 1980; Sayler et al., 1975). An investigation
by Marino and Gannon (1991). for example. examined the survival of fecal
coliforms and fecal streptococci in storm drain sediment. I t was observed that
storm drain sediments function as reservoirs of large concentrations of fecal
coliforms and fecal streptococci during warm, dry weather periods of up to 6
days. Also, the fecal coliforms were able to multiply in drain sediment that
had a low organic content and reduced predator populations but not the fecal
streptococci.
LaBelle et ai. ( 1 980) investigated reiationships between bacterial
indicators and the occurrence of enteric viruses in estuarine sediments. I t was
observed that viruses were present in greater numbers on a volume basis in
sediment than in overlying seawater. Several times, viruses were isolated from
sediments when overlying seawaters met biological water quality standards for
recreational use.
Sayler et al. (1975) examined t h e distribution of fecal indicator
organisms in Upper Chesapeake Bay, Maryland, USA. The indicator
organisms studied included heterotrophic plate count, total coliforms, fecal
coliforms and fecal streptococci. Bacterial counts were not observed to be
directly correlated with the concentration of suspended sediment. However,
a large proportion of both HPC bacteria (53%) and fecal indicator organisms
(>80%) were associated with the suspended sediments.
Matson et al. (1978) exarnined the relationship betwcen heterotrophic
plate count bacteria. total coliforms, fecal coliforms and fecal streptococci in
river sediment. These authors noted that the fate of indicators which do attach
to sediment is regulated by their ability to metabolize benthic nutrients,
withstand predatory pressure and metaboiically compete with other microbes.
In their study the concentrations of indicator organisms in the sediments were
always greater than in the overlying water. Mean sediment/water ratios for the
indicator bacteria ranged from 55 CFU/cmZ sediment/CFU/cm3, for fecal
streptococci, to 6700 CFu/cm2 sedimentlCFUlcm3 water, for HPC bacteria.
Protection by solids (>7 pm) has also been identified as a factor which
deceases the disinfecting action of chlorine in drinking water (Berman et al.,
1988).
In another investigation the recovery rate of Salmonellae from Stream
bottom sediments versus surface water was examined (Hendricks, 197 1). In
the analysis of river samples approximately 90% of the Salmonella recovered
were present in bottom sediments as opposed to the overiying surface water.
55
This phenornenon could be explained by the adsorption o f organisms to
suspended sand particles and subsequent sedimentation. Once o n the river
bottom these organisms could grow if suitable nutrients were present.
Wellings ei al. (1976) investigated the association of viruses and solids
in wastewater and sludge. Influent, effluent and chlorinated effluent samples
showed that from 16 to 100% of the total viruses enumerated were associated
with solids. In 50 % of the influent samples tested, four- t o fivefold more
viruses were present in the solids portion compared t o t h e supernatant. These
au thors emphasized the necessity fo r virus enurneration techniques being
applied to solids and not just liquids. Lund (1973) a s cited by Wellings e l al.
(1976) suggested that quantification o f viruses in wastewater and sludge is
invalid if solids are removed prior t o testing.
LaLiberte (1982) examined the survival o f E-coli in lake bottom
sediment in Minnesota, USA. It was observed that E.coli had the ability to
survive in aquatic sediment in situ for several days. Also, Goyal e l al. (1 977)
studied the occurrence o f bacterial indicators (TC and FC) and pathogens
(Salmoneiiae) in canals along the Texas Coast. No statistically significant
relationship was observed between the organism concentrations and the
suspended solids content of the water. However, al1 of the rnicroorganisms
studied were present in greater numbers in sediments than in t h e overlying
water, frequently by a factor of several logs.
A study by Bitton et al. (1972) showed that clay minerals afforded
significant protection to Klebsiella aerogenes during exposure to UV
irradiation. It has aiso been observed that wastewater bacteria occur
preferentially on smail-size particies (smaller than 20 pm) and particles of such
sizes are little affected by primary settling (Aubert and Aubert, 1977; as cited
by Legendre et al., 1984).
2.3.4 Metals
Lemmer et al. (1994) examined the population density and enzyme
activities of heterotrophic bacteria exposed to heavy metals in sewer biofilrns
and activated sludge. The authors cornpared the heterotrophic activity of
bacterial sewer biofilm biocenosis for biofilms exposed to domestic
wastewater (DW) and for biofilms exposed to trade wastewater (TW). The
TW biofilm biocenosis contained chromiurn concentrations in the range of
1660-2460 mg/kg dry weight and nickel was in the range of 840-1250 mg/kg
dry weight. The concentrations of these heavy metals in the DW biofilm
biocenosis were about two orders of magnitude lower. It was observed that
highly developed eukaryotic organisms such as slirne molds and a variety of
proto- and metazoa were abundant in the DW-biocenosis. However, in the
TW-biocenosis, they occurred only in low numbers or else were lacking. Yet
in the TW-biocenosis high activities of bacteria were observed similar to the
D W-biocenosis. Thus, the authors concluded that eukaryotic organisms, i n
contrast to bacteria, appear to be significantly more susceptible to pollution
by heavy metals.
Hackney and Bissonnette (1978) examined the recovery of indicator
organisms (E-coli. E-aerogenes and S. fuecalis) in two acid mine streams. The
environment of the "Scott's Run" (Fig. 2.4, p45) river section appeared to
cause more die-off than the "West Run" (Fig. 2.3, p44) river section. Yet b o t h
streams had an approximate pH of 3. Water in Scott's Run had an average
temperature 24.6"C, whereas the West Run had an average temperature of
17.2"C. Yet in the West Run the ternperature varied from 3" to 21°C. although
during any one experiment water temperature was always 17.2kZ°C. The
temperature variation was not reported for Scott's Run. However, the authors
did not think that temperature alone accounted for the greatly reduced
recoveries of E.coli and E.aerogenes in Scott's Run. Zinc, copper and
aluminum and sometimes arsenic and cadmium were present in high levels in
the acid mine drainage (AMD) and the concentrations of such ions may have
differed between the two streams causing greater organism die-off in Scott's
Run (Hackney and Bissonnette, 1978). Also, since Scott's Run had a higher
concentration of ferric ion than the West Run ( the amount was not quantified),
the authors suspected that organisms were more inhibited in Scott's Run.
2.3.5 Availability of Oxygen
Although dissolved oxygen would probably not be a major factor in the
survival of indicator organisms in the bacterial leaching process under normal
circumstances, various studies have reported that dissolved oxygen can have
a small influence on indicator organisms in certain environments. In a study
by Chung and Goepfert (1970), for example, the growth of Salmonella at low
pH was investigated. The effect of aeration was evident at pH 4.2, where
growth could be initiated at a low level of inoculum (102 cells/mL) only when
the culture was shaken, as shown in Table 2.4. However, i t was concluded by
the authors that the effect of aeration was rninor, since t h e difference in the
limiting pH value introduced by aeration effects was less than 0.1 pH unit.
Also, it was noted that the results in Table 2.4 describe conditions for t h e
initiation of growth and not the continuation of growth.
El Hamouri et a l . (1994) investigated high-rate algal pond (HRAP)
performances in fecal coliform and helminth egg removals. It was shown that
as the pH of the HRAP water rose from 8.4 (early morning, 8 a.m.) to around
9.2 (later afternoon, 4 p-m.) , the dissolved oxygen (DO) rose from 2.5 mg/L
Table 2.4 - Effecs of oxygen, pH and inocduni level on growth of Salmonella senjenberg at 30°C (Results fiom Chung and Goepfert, 1970)
Ino culum (cells/mL) Aerated Static
60
to 25 mg/L respectively. Both the pH and DO depend on light intensity and
temperature. The late afternoon oxygen-rich environment may have resulted
in the formation of singlet oxygen and/or superoxide molecule due to
excessive Iight capture by the algal chlorophyll. These two molecules are very
reactive forms of oxygen and they have been reported to provoke irreversible
damage to photosynthetic apparatus and to microorganism DNA (El Hamouri
cr al., 1994). Thus, to achieve optimal FC removal in HRAPs the authors
concluded that the retention time must be extended from 3 days in the hot
season to 6 days in the cold season. In the bioleaching procesç high light
intensities are not present, so the highly reactive forms of oxygen mentioned
above are unlikely to form. The photooxidative impact of sunlight has also
been reported by Sinton et al. (1994) in a study of enterococci and FC
inactivation from sewage and meatworks effluents in seawater. They stress the
importance of the DO concentration in the photooxidative impact of sunlight
on FC in the seawater-effluent mixtures. Once again however, the sludge
bioleaching process would probably not allow enough light penetration into
the sludge mixture for a photooxidative effect to occur.
An investigation into the effect of pH, oxygen and humic substances on
the ability of sunlight to damage fecal coliforms in waste stabilization pond
water was performed by Curtis et al. (1992). It was observed that the ability
of light to damage fecal coliforms was highly sensitive to and totaily
dependent on, oxygen. However, oxygen (up to 8 mg/L) in the absence of
light did not affect the FC levels (data shown in Table 2.5). Aiso, it was
reported t hat no FC removal was observed under anaerobic conditions.
I t should aiso be noted that i n the bacterial leaching process, the
availability of oxygen is essential for the bacterial oxidation of the added
elemental sulphur by the adapted strains of Thiobacillus (Blais et al., 1992).
2.3.6 Microbial Interaction
The native microflora in sludge can also affect indicator organisms and
pathogen survival, due to such things as competition and antagonism
(including predation). In a study by Marino and Gannon (1991) the effects of
interspecific competition, antagonism and predation on the survival of FC and
FS in storm drain sediment were investigated. It was reported that in the
untreated, control sample (predators, competitors and antagonists present) the
FC and FS concentration were both 10) 1100mL, whereas in the autoclaved.
seeded sample (predators, competitors and antagonists eliminated) the FC
were at 1 0 ~ / 1 0 0 m ~ and the FS were at 10' /100m~. It was concluded that
native microfloral competition/antagonism (including bacterial predation) and
protozoan predation are significant biotic factors with respect to FC and FS
Table 2.5 - Surviv.1 of FC in aerated pond water in the Iight and the dark (Results fiom Curtis et al., 1 992)
(a) Mean concentration
+
Exposure rime was 255 rnh; radiant energy received = 4.52 MJkquare meter
Illumination
Dark
Light
FC count (% of initial)
104.7
2.1
Dissolved Oxygen (nt g/ L)(a)
8.8
8.5
Mean pH
8.9
8.9
survival. Also the authors stated that competition/antagonism exerts an effect
on fecal bacterial survival which is two to three times greater than predation
effects under simulated conditions. Concerning the abiotic factors
(temperature, conductivity, pH) it was concluded that biotic factors were more
important that abiotic factors for bacterial survival (under the test conditions).
King et al. (1988) examined the survival of coliforms and bacterial
pathogens within protozoa during chlorination of drinking water. Bacteria
were first ingested by laboratory strains of Acanthamoeba castellanii and
Tetrahymena pyriformis (both protozoa). Chlorine was applied and it was
shown rhat the protozoa survived and grew under leveis of free chlorine
residuals that killed free-living bacteria. It was shown that bacteria could be
cultured from within chlorine treated protozoans well past the tirne required
for 99% inactivation of free-living cells. Also, al1 bacterial pathogens were
>50-fold more resistant to free chlorine when injested b T.pyriformis. Thus,
the bacterium-protozoan association can increase bacterial resistance to free
chlorine, thus Ieading to the persistence of bacteria in chlorine-treated
drinking water. It is conceivable that a simiiar mechanism could occur in the
sludge bioleaching process.
2.4 Past Studies Concerning the Fate of Indicator Organisms and
Pathogens During Bacterial Leaching
The bioleaching process itself is still in the early stages of development
and understandably the fate of pathogens during bacterial leaching has only
been the object of a few studies. Past investigations on the fate of pathogens
during bacterial leaching include: Lohaza ( 1985) ; Smith ( 199 1); Blais el al.
(1992); and Seth (1997). The results of these studies are not directly
comparable however, since the first two studies were done on bioleaching
processes where T. ferrooxidans and ferrous sulfate was the substrate. The
last two studies were performed with sulphur as the substrate in the
bioleaching process as described in Section 1.1.2 of this report.
In the study by Lohaza (1985) it was found that bacterial leaching of
anaerobically digested sludge under acidic conditions (pH 3.5 to 4.2) for up
to 15 days did not reduce the numbers of total coliforms. fecal coliforms or
fecal streptococci present. It was concluded that the survival of indicator
organisms was unaffected by the T. ferrooxidans inoculation and the source of
the indicator organisrns. In addition, it was noted that the growth of fecal
streptococci and fecal coliforms was inhibited in reactors with high
concentrations of suspended solids in the sludge. A concentration above 10
g/L of TSS was associated with a significant reduction in these indicator
organisms. Lohaza also carried out settling tests and concluded that total
heterotrophs, total coliforms, fecal coliforms and fecal st -eptococci in
anaerobically digested sludge were uniformly distributed in the mixed liquor
and that they remained at the same concentration after settling took place.
Finally, it was noted that since fecal streptococci show little tendency to grow
during leaching they may be better indicators than total or fecal coliforms.
In a study by Smith (1 99 1) it was observed that Salmonella iyphirnurium
in anaerobically digested sludge were inactivated completely after 7 hours of
bacterial leaching (at a pH of 4, aeration rate of lOOmL of air/L sludge/min.
at 20-3 0°C). Alt hough, the total coliforms persisted during the leaching
period of ten days, the total coliform population in al1 experiments decreased
substantially from the initial counts. It was also shown that suspended solids
had no observable effect on the survival of Salmonella typhimurium during
bacterial leaching. However at 1.5% suspended solids (SS), total coliforms
decreased from - 107/g sludge at time zero to - 102/g sludge after 25 hours of
leaching. At 0.9% SS, total coliforms decreased from - 107/g to - 10°lg sludge
after 4-5 days of leaching, and at 4.3% SS, coliforms decreased from - 106/g
to - 102/g sludge after 10 days of leaching. It was thus concluded by Smith
( 199 1 ) that the total coliform group was an inadequate indicator of the
presence of Salmonella ~yphzmurzum during bacterial leaching. Further, it was
also suggested that suspended solids played a significant role in the survival
of total coliforms during leaching, as the sludges with lower suspended solids
(Le. 0.9% vs. 4.3% SS) experienced significantly faster and more pronounced
coliform inactivation. However, the faster and more pronounced inactivation
may also be due to the faster pH drop in the systerns with lower suspended
solids. In the systems with 0.9% S S , pH dropped to 2.75 in 2-3 days bu t the
systems with 4.2% SS took 7-8 days to reach pH 2.75. Perhaps suspended
solids can absorb acid, thus buffering the system. Similar results concerning
solids were also observed by Lohaza (1985) who concluded that significant
reductions in fecal coliforms and fecal streptococci only occurred below 1.0%
SS. Perhaps the solids provide a physical barrier between the bacteria and the
acid environment as suggested by Smith (1 99 1) andlor perhaps the solids serve
as a source of food for the bacteria (Lohaza, 1985). Several researchers have
suggested that suspended solids serve to absorb acid in sludge (Smith, 1991;
Blais, 1992; Lohaza, 1985). Wong (1984) observed a reduction (15-20%) in
the suspended solids content of the sludge as a result of the lower pH. It was
attributed to the conversion of SS to soluble solids with some being lost in the
form of hydrogen sulfide and carbon dioxide (Wong, 1984 as cited by Lohaza,
1985). Lohaza (1985) also observed a 50% reduction in SS after a 15 day
leaching period under standard bacterial Ieaching conditions.
Blais et al. (1992) investigated reductions of indicator organisms in the
bioleaching process with sulphur as substrate as described in Section 1.1-2 of
this thesis. However, whereas Lohaza (1985) and Smith (1991) chose the
multiple tube MPN procedure, Blais et al. (1992) employed direct spread
plating onto selective media. This is a harsher environment than in the
multiple tube MPN technique which first involves inoculation into a general
enrichment media, to help strengthen the acid-injured cells from the sludge
before inoculation into selective media. Thus, it is postulated that the direct
plating of acid-injured cells directly ont0 selective media as done by Blais et
al. (1992) does not revive a significant proportion of the acid-injured cells,
which could be recovered if the multiple tube MPN technique were employed.
Blais et al. (1992) tested for total aerobic colonies (Le. total
heterotrophs), total and fecal coliforms and fecai streptococci. They observed
that with a pH c2.5 in the bioleaching reactors a considerable reduction in
bacterial indicators (3 log or under the detection limit of 10' CFU/lOOmL)
occurred for al1 sludges examined over a 5 day period. They examined
numerous digested and undigested sludges, with detailed results presented for
sludges with 0.684% TS and 1.74% TS. The rapid pH drop was due to the
heavy inoculum (5%) and high sulfur addition. These authors reported only
68
a slight decrease in total aerobic microorganisms after a 4 day leaching period.
However, the initially diverse microflora was replaced by 2 types of dominant
colonies (yeast and fungi).
2.5 Health Aspects of On-Land Sludge Reuse
Considering the widespread application of sewage sludges to
agricultural lands al1 over the world and the great variety of pathogenic
microorganisms that can be present in wastewater sludges, there is
considerable concern over the possibility of disease transmission to humans.
There are various possible pathways for the movement of sludge pathogens
from the disposa1 site to the exposure site as shown in Figure 2 . 5 .
Microorganisms present in wastewater and sludges and the potential health
effects are shown in Table 2.6.
In the discussion of the problems surrounding on-land sludge reuse the
biological hazards are consistently mentioned (Bruce and Davis, 1989; Coker
and Matthews, 1983; Davis, 1987; Feliciano, 1982; WPCF, 1989). The words
hasard and rzsk are often used interchangeably in such discussions. However,
it is best to define a health hazard as the "potentiai for adverse health effects"
and a health risk as "a distinct possibility for infections to occur" (Block et
a l . , 1986). Thus, the use of sewage sludge in agriculture poses a health
Table 2.6 - Bacteria, viruses, protozoa and helminths in wastewater and sludges ( h m Fradkin et al., 1985)
Salmonella ( 1 700 typts) Shigelia (4 specits)
Entcropatiiogcnic: Esclicrichia col; Yersinia enrerocolifica Canrpytobacter jejuni Vibrio cholerae Leptospira spp.
Enteroviruses: PoIiovirus (3 types) khovirus (3 2 types)
Coxsickievirus B (6 types)
New enterovimes (5 types)
Hcpatitis Type A (Enterovinis 72) Gastroenteritis virus (Nowak type agents) Rouvirus (4 types) (Reoviridae family) Rcovinis (3 types) A d e n o h (>4 1 types) ParvoWus (3 types)
Giardia lamblia Balunridium coli
Ascaris lum bricoides (Roundworm) A ticyclostoma duodenale (HooLwom) Necalor aniericanus (HooCc7vorm) Taenia saginata
Typhoid, pxatyphoid. and saImoncIlosis Bacillary dyscntcq
Gastrocn tcn t 1s Gastroentcnt is Gastroentcri t is Cholera Weil's d i scsc
Paraiysis, meningi tis, fcvcr Meningitis, respiratory discase, rash, diarrtiea, fevcr Kerpangma. respiratory discase, menhgitis, fcvcr Myocarditis, congzmtal h m anomalies, rash, fever, mcningtis, respiratory Jiseasc, plcurodynia Meningitis, enccp iialitis, rq i ra tory diseasc, acutc haernorrliagc ;onjunctivitis, fevcr [nfectious hepatltis
Epîdeniic vomiting and diarrhea, fever
Epidernic vomiting and diarrhea chiefly 3 f children Not clearly establishcd Xespiratory disease, cye dect ions 9ssociated wîth respiratory d i s in :hildren, but euology not clearly h o w n
9moebic dysentery , livcr abscess, mlonic uiceration Diarrhea, malabsorption bfild diarrhea, coIonic ulmation
Qscanasis
bernia
bernia
rami asis
hazard to humans and animals, however, whether or not there is a significant
health risk depends on such factors as the nature of the pathogen. its actual
numbers, sludee disposa1 practices, land use and other geographical.
climatologicai and demographic factors (Block et al., 1986). Other authors
(Mara and Cairncross, 1989) differentiate between a potenriai risk (Le. a
hazard) and an actual risk. These authors state that the following all must
occur for there to be an actlraf risk to health: ( 1 ) Either an infective dose of
pathogens reaches the field, or the pathogen multiplies in the field to form an
infective dose; (2) The infective dose reaches a human host; (3) The host
becornes infected; and (4) The infection causes disease or further transmission.
The risk will remain a potenfial risk (Le. a hazard) if only ( 1 ) , or ( 1 ) and (21,
or ( l ) , (2) and ( 3 ) occur, but not (4). Further to this, even if an actual risk
is involved i t will be of public health importance only if it causes a
"measurable excess incidence or prevalence of disease or intensity of
infection" (Mara and Cairncross, 1989). It is the role of epidemiology to
verify such occurrences. Epidemiological studies are meant to show the
actual, as opposed t o the potential health risks. The hazards of on-land
sludge reuse can thus be split into three parts: (1) The presence of pathogens
on the field; (2) Pathogen survival; and (3) Pathogen transmission.
Concerning (1), al1 pathogens in the sludge may reach the fieid. However,
72
different treatment technologies will remove different pathogens to varying
degrees. For siudge the only treatments that will yield a totally pathogen-free
product are batch thermophilic digestion, therrnophilic composting, or drying
for at least 1 year (Feachem et al.. 1983). If such treatments are not employed
then pathogens will reach the field and part (2) (pathogen survival) becomes
quite important. Numerous studies have been performed o n t h e survival of
sludge pathogens i n soil and other environments (Ibiebele and Inyang, 1986;
Edmonds. 1976; Schwartzbrod et al., 1987; Liu, 1982). Policy-makers must
take into account the survival times of sludge pathogens in soil and on crop
surfaces. An extensive literature review on the subject was performed by
Feachem et al. ( 1 9 8 3 ) . The survival times of selected excreted pathogens in
soil and on crop surfaces at 20-30°C are presented in Table 2.7.
From Table 2.7 it is apparent that pathogen survival on crop surfaces is
much shorter than in soil, since the pathogens are less protected from the
harsh effects of sunlight and desiccation (Mara and Cairncross. 1989). The
data in Table 2.7 is relevant where effluent, sludge, compost or other fecal
products are being applied to land. However. the survival of pathogens in
sludge alone (that has not been applied to land) is slightly different and is
presented in Table 2.8. It is important to note that the ranges of survival
times in Tables 2.7 and 2.8 are due to both strain variation, differing climatic
Table 2.7 - Survival tirnes of various excreted pathogens in soi1 and on crop surfaces at 20-30 'C (Adapted fiom Feachem et ai., 1983)
Pathogen
Viruses Enterovimses (a)
Bacteria Fecd coliforrns Salmonella spp. Vibrio cholerae
Pro tozoa Et~tamoe bn histo!vtica cysts
Helrninths Ascaris Iirn~brkoides eggs Hookworm larvae Taenia sagirlata eggs Trichuris trichirua eggs
Survival Time da s i <IO0 but usually <20
<70 but usually <20 <70 but usudly <20 <20 but usudly < 10
c20 but usually c 1 O -
Many months c90 but usually <30 Many months Many months
On Crops
<60 but usuaily < 1 5
<3 0 but usually < 15 <30 but usually <I 5
<5 but usually <2
< 10 but usuaily Q
<60 but usually e 0 <30 but usually <10 <60 but usuaily <30 6 0 but usually 0 0
a ) - Includes poliovims, echovirus and coxsackievims.
1 aole LU - survrvat rimes or vanous excrerea parnogens 1x1 siuugc: a L
20-30 OC (Adapted from Feachem et al., 1 983)
Pathogen
Viruses Enteroviruses (a)
Bacteria Fecal coliforrns Salmonella spp. shrgeiiu spp. Vibrio cholerae
?rotozoa E ~ m o e b a histolylica cyst s
ielminths Ascaris izmtbricoides eggs
SurvivaI Tirne (days)
< 100 but usually <20
€90 but usually -30 <60 but usually <30 <30 but usually < 10 <30 but usually <5
1 3 0 but usually c 1 5
Many months
(a) - lncludes poliovims, echovirus and coxsackievirus.
75
factors as well as different analyticai techniques (Mara and Cairncross, 1989).
Some of the factors affecting the survival of enteric bacteria in soil are
presented in Table 2.9. Lehmann et al. ( 1983) have also provided a thorough
review of the literature on the survival of sludge pathogens in soil.
However, as noted by Schwartzbrod er al. (1 987) the literature is very
scarce on the potential health effects of sludge exposure. Several papers
however have addressed the topic of risk assessment of on-land sludge
disposa1 (Hays, 1977; Fradkin et al., 1989; Fradkin et al., 1985) . Fradkin et
al. (1985) present a unique evaluation on the feasibility for performing a risk
assessment on pathogens. According to these authors a risk assessrnent
involves evaluating available information on representative species of microbes
and their potential health effects and then modelling their fate, persistence and
transport. The output is information on the potential for human health
impacts.
First, the likelihood of exposure associated with each of the pathways in
Figure 2.5 (p69) must be combined with the infectious doses (ID) of pathogens
in order to do a risk assessment on pathogens. A matrix of qualitative estimates
has been prepared by Fradkin et al. (1985). as is presented in Table 2.10.
It is known that bacteria and viruses are more likely than helminths and
protozoa to rnove along the exposure pathways and eventually corne in contact
Table 2.9 - Factors affecting the survivai time of entenc bacteria in soil (from Feachem et al., 1983)
Soi1 Factor
Antasonism from soil microflora
Moisture content
I~oisture-holding capacity
Organic matter
PH
Sunlight
Temperature
Effect on Bacterial Sumival
Increased survival time in sterile soi1
Greater survivd time in moist soiIs and during times of high rainfall
Survival time is less in sandy soils than in soils with greater water-holding capacity
Increased survival and possible regrowth when sufficient amounts of organic matter are present
Shorter suMval time in acid soils (pH 3-5) than in alkafine soiIs
Shorter survival time at soil surface
Longer s u ~ v a l at iow temperatures; onger survival in winter than in sumrner
Table 2.1 O - Likelihood of exposurc frorn pothogens to humons os related to the nurnber of organisms potentislly prescnt in each pathway and thc iiifcclious dosc (adnpicd îrom Fradkin ct al., 1985)
Number of organismsl infectious dose
water
1 Bacteria Viruses Helminhs Protozoa
Ground- water
I I I **** = Many organismsllow infectious dose *** = Many organismshigh infcctious dosc ** = Few organismsllow infectious dose * = Few organisms/high infectious dose - = Presence unlikclyhigh or low infectious dose
Pathway
Direct contact
*** ****
**** (a)
Sediments
(a) The **** score is for land based disposa1 sites; for ocenn disposal the score is **
wi th humans (Fradkin et al. , 1985) . However, minimum infectious doses
(MID) (Le . the dose required to infect 50% of the population) for bacteria are
between 10' to I O 6 whereas, a single viral un i t may initiate a n infection
(Fradkin et al., 1985) . Thus, as shown in Table 2.10 risk assessment efforts
should be directed towards viruses due to the large numbers of viruses in
sludge, their relative ease of mobility and their low infectious dose (Fradkin
el al., 1985). This appears contrary to the statement of Blais er al. ( 1 992).
stating that epidemiological studies show that pathogenic risk from utilizing
sludge as fertilizer is due to the presence of pathogenic bacteria and helminthic
Worms.
According to Block et al. (1986) risk assessment of sludge use in
agriculture has been a matter of much debate and so far there is no consensus
on desirable preventive hygienic measures. A good reference on this subject
has been prepared by Block et al. (1986) which is a collection of 19 research
papers and is the product of 39 scientists from 12 countries. These scientists
split the topic of the epidemiology of the agricultural use of sludge into four
areas: ( 1 ) Bacteria; (2) Parasites; (3) Viruses; and (4) Occupational hazards.
Concerning bacteria, the scientists decided to restrict the discussions to
salmonellae, since sludge is apparently not a factor in the spread of other
bacterial diseases. They concluded that sludge has been involved in the
79
transmission of salmonellosis to dairy cattle and through the sale of infected
unpasteurized milk, to humans. However if national guidelines on the
agricultural use of sludge had been followed, the disease outbreaks rnay not
have occurred. They decided that no new epidemiological studies regarding
salmonellosis were required due to the great amounts of knowledge already
accumulated.
Concerning parasites (protozoa and helminths) it was concluded that
sewage sludge which is subjected to disinfection procedures such as
pasteurization or composting under properly controlled conditions does not
present a risk to human or animal health when spread on land. However, if
sludge is not adequately disinfected some parasites will remain and can present
a potential risk to public and animal health. The scientists noted that
information on the types and concentrations of parasites in sewage sludge is
limited and the analytical methods for doing such determinations are
sometimes flawed. Also the sources of parasites in sludge (humans vs.
animais) require further study. It was accepted that sludge spread on land can
act as a vector for Ascaris spp. and Taenia saginaia. However, the role of
sludge in the epidemiology of Giardia, Sarcocystzc and Cryptosporidium (and
possibly Entamoeba and Toxoplasma) is less clear and requires further
investigation. It was also noted that stabilization processes may or may not
80
inactivate parasitic ova and therefore application of stabilized sludge to
grassland may not always be safe from a parasitological standpoint.
Concerning viruses the group of scientists noted that available detection
methods are not quantitative and that no more than 10 per cent of viruses
present will be detected. For many viruses no methods for routine detection
purposes are available and for other viruses there are virtually no known
methods. Such difficulties exist for hepatitis A virus, rotavirus of human
origin, coronaviruses and Norwalk agent. This is highly problematic since
these viruses are probably most significant from an epidemiological standpoint.
The group could only identify two published reports of sewage sludge being
a vector in the spread of virus infections. The scientists also noted that many
levels of treatment are inadequate for producing a virus-free material. This
is true for digested, composted and lime-treated sludges. Thus, a potential
risk exists in connection with land application of such sludges. Concerning the
potential risks of viruses and on-land sludge application, setting specific
standards is as difficult for sludges as it is for water, according to the
scientists. In epidemiological studies it has been difficult to separate the
specific case of virus spread from sludge and virus spread from other sources.
Prospective epidemiological studies would have immense costs and very
substantial practical difficulties. Instead of epidemiological studies the
81
scientists recommend studying the nature and survival of the various viruses
and to produce guidelines accordingly i n order to prevent major routes of
spread, if possible.
Concerning the occupational hazards for those that work with sewage
sludge (i. e . compost plantworkers, sewage treatment plantworkers, farm
families etc.), few cases have been reported in the Iiterature. Perhaps the best
study available today is an Ohio farm family study sponsored by t h e EPA from
198 1 to 1983 as presented by Ottolenghi and Hamparian ( 1 987) . According
to Block et a l . ( 1 986 ) , evaluation of existing data does not indicate the level
of risk, but such data does not warrant substantial changes to present
European Community sludge treatment and handling practices d u e to
occupational hazards.
Various other regulatory organizations have made statements on the
potential health effects of sludge reuse on-land. A World Health Organization
(WHO) working group in 1981 gave particular attention to four pathogens
present in sewage sludges, Taenia saginatai. Salmonella. Sarcocysris and
hepatitis A (as cited by Lewis-Jones and Winkler, 1991). Those of most
concern in the UK are Salmonella. eggs of the beef tapeworm, Taenia
saginata and its larval stage in cattle, Cysticercus bovis. potato cyst
nematodes, globodera spp. and a range of viruses (Lewis-Jones and Winkler,
82
1991). In a review of sludge disposa1 to land in 1981, the UK Department of
the Environment and the National Water CounciI stated that only Taenia
saginata (a helminth) was definitely being disseminated through the disposal
of sewage sludge, bu t ova of other parasites Taenia solir<m. Ascaris and
Trichuris were a cause for concern (as cited by Lewis-Jones and Winkler,
199 1). According to Feachem et al . (1983), although sewage sludge has an
alarming variety of different pathogens, in most cases it contains an
insignificant number of them and will have a negligible effect in transmitting
the diseases when it is used in agriculture. Modifying this da im of low risk.
Feacham er a l . ( 1 983) have added three exceptions o f human or veterinary
importance: beef tapeworm infection, salmonellosis and tuberculosis. The UK
North-West Water Authority in their 1983 code of practice stated that the two
types of organisms providing a major disease risk through the land application
of sewage sludge are the Salmonella group bacteria and the beef tapeworm
Taenia saginata (as cited by Lewis-Jones and Winkler, 1991). In the UK
Department of the Environment 1989 Code of Practice, the organisms
considered to be of most concern are Salmonella, the eggs of the beef
tapeworm Taenia saginala, potato cyst nematodes and a number of viruses (as
cited by Lewis-Jones and Winkler, 199 1).
Wastewater quality guidelines and standards are often in terms of
83
maximum permissible concentrations of total andlor fecal coliform bacteria.
Fecal coliforms can be used in wastewater as reasonably good indicators of
bacterial pathogens, but they are less effective as indicators of excreted
viruses and are of very limited use for protozoa and helminths for which no
reliable indicators exist (Mara and Cairncross, 1989).
Standards or guidelines for wastewater quality for crop irrigation
generally specify maximum indicator organism concentrations (such as fecal
coliforms) and minimum treatment requirements (primary, secondary or
tertiary) depending on the type of crop (consumable, non-consumable).
However, for sludges from conventional sewage treatment systems.
Feachem et al. ( 1983) state that the viable eggs of Ascaris lurnbricoides
appear to be the best pathogen indicator currently available. They suggest
that if ascariasis is endemic and there are no viable Ascaris eggs present in the
sludge, then other pathogens are absent as well, since Ascaris eggs are so
resistant. Feachem et al. (1983) also state that E.colz is an inappropriate
indicator for the quality of treated sludges. When it cornes to pathogen
removal from sludges intended for reuse in agriculture, major problems will
only be encountered where conventional sewage treatment plants are in use
(Feachem et al., 1983). Such plants produce both an effluent and a sludge that
have high concentrations of pathogens and require expensive additional
treatment before they can be recommended for unrestricted agricultural reuse
(Feachem et al., 1983). Achieving a strict quality standard for sludges (c 10
viable Ascaris eggs per 100 grams, for example), c m only be achieved by well-
managed thermophilic digestion or composting, or by retention times of > 1
year (Feachem et al . , 1983). Although a second-best choice would be
mesophilic digestion followed by several months on drying beds (Feachem e t
al. , 1983).
2.6 Summary
The present Iiterature review has covered numerous topics relating to
sludge disinfection. Unfortunately, unlike i n wastewater treatrnent
performance, there are no widely accepted indicator organisms for measuring
sludge disinfection efficiency (Sorber, 1984). Although, in the past, four basic
groups of microorganisms have served as measures of efficiency: ( 1 ) indicator
bacteria (Enterobac ter ia , fecal streptococci); ( 2 ) pathogenic bacteria; (3)
viruses; and (4) parasites (Sorber, 1984).
In the present literature review four indicator groups (TC, FC, FS, HPC)
and how well they predict the fate of pathogens have been examined. A thorough
search of the literature revealed only a few studies (Strauch, 1989; Berg and
Berman, 1980; Lehmann et al. 1983) have been concerned with the suitability
85
of indicator organisms for assessing sludge disinfection. Therefore, studies
involving the ability of indicator organisms to predict the fate of pathogens in
various environments, in addition to that of sludge, have been presented.
Concerning fecal coliforms, it was noted that t he EPA regulations
include limits on fecal coliforms for sludges applied to land. However, fecal
coliforms may be poor indicators of virus concentrations in raw sludges and
mesophilically and thermophilically digested anaerobic sludges (Berg and
Berrnan, 1980). In another study. however, fecal coliforms were shown to be
over-conservative indicators for the presence of Salmonella in environments
such as grazed Pasture lands (Lehmann et al., 1983).
The total coliform group consists of fecal and non-fecal coliforms and
is thus a more conservative indicator than the fecal coliform group. Lehmann
et al. (1983) maintain that the coliform group of bacteria are important with
reference to the potential health hazards of sludges fields. In addition,
Strauch ( 1989) did note a significant correlation between coliform and virus
counts in raw sludge samples, however total coliforms may not be correlated
with virus counts in treated sludge samples (Berg and Berman, 1980; Strauch,
1989) .
Fecal streptococci may be better indicators than fecal coliforms for
excreted bacterial pathogens and also for viruses (Feachem et al., 1983). Berg
86
and Berman (1980) showed that fecal streptococci could serve as useful
indicators of virus reduction during mesophilic and thermophilic digestion of
anaerobic sludges. Although Strauch (1989) also showed that fecal
streptococci were reliable indicators of virus contamination in treated sludges,
i t was also observed that fecal streptococci were not suitable indicators of
Sahoriella i n the same sludges. Concerning exposure to acid environrnents,
i t has been demonstrated that fecal streptococci in acid mine streams are
signifkanrly iess susceptible to injury than coliforms (Hackney and
Bissonnette, 1978).
The aerobic heterotrophic bacterial communities, although not of direct
sanitary importance have been studied by some researchers. Since these
hetertrophs include such a wide range of different bacteria, the HPC's
usefulness is limited as an indicator (Olivieri, 1982; as cited by Lohaza, 1985).
It was concluded by Strauch (1989) that the total aerobic rnicroorganism count
cannot serve as a reliable indicator of the state of hygiene of sludge.
However. according to Murthy (1984), the total aerobic bacteria count is
believed to provide as much information as any other microbiological index for
routine monitoring of foods.
The fate of pathogens during sewage treatment was also examined in the
present fiterature review. It was noted that wastewater sludges contain high
concentrations of pathogens and ranges of indicator organism concentrations
in anaerobically digested sludge and raw sludge were presented (Table 3.3).
The topic of enumerating indicator organisms in sludges was also covered.
The importance of sample preparation methodology was demonstrated by
Dudley et al. (1980). These authors showed that the best recoveries of viable
organisrns (HPC. FC, FS) from sewage sludges (primary, anaerobically
digested and activated sludge) were obtained i n samples dispersed by vortex
mixing with glass beads as opposed to sonication, or blender homogenization.
Concerning enumeration methods Dudley et al. ( 1 980) concluded that for total
coliforms and fecal streptococci direct plating yielded the highest counts, but
the MPN method yielded higher fecal coliform recoveries in the analysis of
sludge samples. However a study in acid mine waters (pH=3) showed that the
coliforms, (E. colt and E. aerogenes) were very susceptible to injury in acid
mine water and their enumeration with selective media could lead to erroneous
conclusions (Hackney and Bissonnette, 1978).
Numerous studies have been conducted on the effects of environmentai
conditions (pH, temp, etc.) on indicator organism and pathogen survival in
various environments. Concerning pH, it is known that only a few species of
bacteria can grow at pH values of greater than 10 or less than 2 (Madigan et
al., 1996). Environments such as acid mine waters (pH=3) have been shown
88
to reduce indicator organism (E. colz, E. aerogenes and S-facalis)
concentrations significantly (Hackney and Bissonnette, 1978). Chung and
Goepfert also demonstrated that the minimum pH for growth of salmonellae
ranges from 4.05 to 5.50.
Concerning temperature effects Chung and Goepfert (1970) examined
the effect of temperature on the growth of salrnonellae at low p H values. I t
was observed that the salmonellae were most tolerant of low pH at 25-32°C
and the minimum pH that allowed growth increased at temperatures above and
below this range.
There are also numerous studies available reiating to the role of solids
in microorganism survival. An extensive number of investigations have shown
t hat solids protect indicator bacteria and enteric pathogens from adverse
environmental conditions (Marino and Gannon, 199 1; LaBelle et al., 1980;
Sayler et al., 1975).
The effects of microbial interaction, the availability of oxygen and the
presence of metals on indicator organism and pathogen survival were also
reviewed in this thesis. Such factors are not expected to be of great
importance in the bacterial leaching process compared to pH, temperature and
the presence of solids.
Past investigations on the fate of pathogens during bacterial leaching
89
include: Lohaza (1985); Smith (1991); Blais et al. (1992); and Seth (1997).
The results of these studies are not directly comparable however, since the
first two studies were done on bioleaching processes with T. ferrooxidans as
the inocula and ferrous sulfate the substrate. The Iast two studies had sulphur
as the substrate in the bioleaching process. In the study by Lohaza ( 1985) it
was found that bacterial leaching of anaerobically digested sludge under acidic
conditions (pH 3 . 5 to 4.2) for up to 15 days did not reduce the numbers of
total coliforrns, fecal coliforms o r fecal streptococci present. In the study by
Smith ( 1 99 1 ), it was observed that Salmonella ~yphzmirrit~m in anaerobically
digested sludge were inactivated completely after 7 hours of bacterial leaching
(pH 4), although, the total coliforms persisted during the leaching period of
ten days. Blais et al. (1992) tested fo r total aerobic colonies, total and fecal
coliforms and fecal streptococci during bacterial leaching. They observed that
with a p H C 2 . 5 in the bioleaching reac tors a considerable reduct ion in
bacter ial indica tors (3 log o r under t h e lower de tec t ion limit o f I O 3
cfu/100mL) occur red fo r al1 s ludges examined over a f ive d a y per iod .
However , Blais et al. (1992) employed direct spread p la t ing o n t 0
se lec t ive media f o r enumerat ing ind ica to r organisms. This i s a harsher
environment than in the multiple tube MPN technique which f i rs t involves
inoculation into a general enrichment media, t o heIp s trengthen t h e acid-
90
injured cells from the sludge before inoculation into selective media. Thus,
it is possible that the direct plating of acid-injured cells directly ont0 selective
media as done by Blais et al. (1992) does not revive a significant proportion
of the acid-injured cells. which could b e recovered if the multiple tube MPN
technique were employed. Thus whether or not the indicator organisms were
actually being reduced during bacterial leaching has been a subject of
controversy.
Another topic examined in the literature review was the health aspects
of on-land sludge reuse. The various possible pathways for the movernent of
sludge pathogens from the disposa1 site to the exposure site were covered, as
were the potential health effects. According to Block et al. (1986) risk
assessrnent of sludge use in agriculture has been a matter of much debate and
so far there is no consensus on desirable preventive hygienic measures.
Salmonellae, Ascuris spp., and Taenia saginata are pathogens of concern when
sludge is applied to land according to Block et al- (1986). Those of most
concern in the UK are salmonellae, eggs of the beef tapeworm Taenia saginara
and its larval stage in cattle Cysticercss bovis, potato cyst nematodes
Globodero spp. and a range of viruses (Lewis-Jones and Winkler, 199 1) .
3.0 MATERIALS AND METHODS
3.1 Experimental Setup
100 mL Batch Reacrors
Batch reactors consisted of 250 mL Erlenmyer flasks, containing 95 mL
of raw sludge (collected as described i n Section 3.2 and stored at 4°C before
use), and 5 mL of inoculum (pH<) ) , to which various amounts of sulphur
(between 1 and 3 g/L) were added. These batch reactors were agitated at 200
rpm at room temperature (20-25°C) utilizing a gyratory incubator shaker
apparatus (New Brunswick Scientific Co. Inc . , mode1 MS2). On the days for
microbiological analysis, lOmL of sample was removed from each batch
reactor.
1.5 L Batch Reactor
One batch reactor consisted of a 15 L glass reactor, which contained 1.5
L of raw sludge (collected as described in Section 3.2 and stored at 4OC before
use), and 60 mL of inoculum (pH<3) , to which sulphur (3 g/L) was added.
Aeration was provided through rubber latex tubing at a rate of 600 crn31minute
at room temperature (20-25°C). On the days for microbiological analysis,
approximately 50mL of sarnple was removed from the batch reactor.
140 L Contznuous Svstem
A continuous feed bacterial leaching pilot plant was installed at the
Main Wastewater Treatment Plant in Toronto. I t consisted of a number of
components. The raw sludge was contained in a 30 L capacity feed tank,
which was equipped with a Carframo Ltd. mixing device (rnodel RZDRI-64)-
rotating at 250 revolutions per minute (rpm) for mixing and aeration purposes.
The raw sludge was pumped from the feed tank to the solubilization tank in
flexible tubing (!hW inner diameter by 1/16" wall diameter). The flow from the
feed tank was regulated by a Piper (RPT) timer. A 200 L capacity plastic
container was used as the solubilization tank, although the working volume
was 140 L, due to the positioning of t h e outlet. A stirring device (Carframo
Ltd., rnodel RZR-1) was operated in the solubilization tank at 250 rpm. A
foam-breaking device was installed on the stirrer and over the liquid surface
to control foaming when necessary. Aeration was provided in the
solubilization tank via latex tubing positioned at the bottom of the tank. The
outflow from the solubilization tank was carried by flexible tubing to the
settling tank. The settling tank was of 4 L capacity, conical in shape and made
of plexi-glass. The underflow of the settling tank was pumped in flexible
tubing back to the solubilization tank. The outflow from the settling tank was
carried in flexible tubing to a discharge tank of 100 L capacity. Polymer of
93
the required dosage was pumped in flexible tubing by a Beachman Solution
Metering Pump (mode1 746) from a 500 mL beaker to the inlet of the settling
tank. The timing of the polymer pump was coordinated with the timing of the
raw sludge feed pump, to ensure that the fi ow of polymer was occurring
simultaneously when there was flow from the solubilization tank.
3.2 Source of Samples
The raw sludge samples were collected from Toronto's Main
Wastewater Treatment Plant in volumes of 100 mL or greater, shipped cold
and stored at 4°C at the University of Toronto laboratory for between 4 and
6 hours before analysis commenced (iisually no more than 5 hours). The
bioleached sludge samples (IOOmL) were collected from the top of the 140 L
solubilization tank of the pilot plant located at the Main Wastewater
Treatment Plant. They were shipped, stored and analysed in the same way as
the raw sludge samples.
3.3 Bacteriological Methods
Enumeration of bacteria included heterotrophic plate count, total
coliforms, fecal coliforms and fecal streptococci. Samples were prepared by
vortex mixing 5 mL of sludge at high speed for 2 minutes with 15 mL of sterile
94
phosphate-buffered saline (0.1 M, pH 7.2) containing approximately lg of
sterile 3-4 mm diameter glass beads in a 50 mL centrifuge tube. Sarnples were
diluted serially in sterile phosphate-buttered saline. Commercially available
dehydrated media (BDH Laboratories) was exclusively utilized in this study.
3.3-1 Resuscitation Broth
During the 100 ml-batch reactor studies and steady-state operation of
the 140 L bioleaching reactor, experiments with a pre-enrichment resuscitation
broth were studied. This resuscitation broth had been used by Mossel and
Ratto (1970), for resuscitating sublethally injured cells. The resuscitation
medium called "CAS0 tryptone soya broth" contained tryptic digest of casein,
17 g; papaic digest of soya protein, 3 g; glucose, 2 S g ; NaCl, 5 g; K,HPO,.
2Sg; distilled water, 1 000 mL. The broth was sterilized by autoclaving for
15 minutes at 12 1 OC and 1 . 1 kg/cm2 pressure. Resuscitation was effected by
incubating 5 mL of the sludge sample under investigation in 15 mL of this
broth for 2 hours at room temperature (20-25OC) in an upright 50 mL
centrifuge tube prior to analysis. Essentially it simply involved replacing the
15 mL of phosphate-buffered saline in the standard method with the recovery
broth and waiting for 2 hours before following the standard analysis technique.
Note that the vortex mixing did not occur until after the 2 hour incubation
period at room temperature.
3.3.2 Total Heterotrophic Count
Total counts of heterotrophic bacteria were analyzed by the pour plate
technique described in Standard Methods (APHA el al.. 1995), combined with
some minor modifications. The growth media was Standard II Nutrient Agar,
obtained in dehydrated forrn. Sufficient cyclohexirnide (BDH Laboratories)
was added to the nutrient agar (45°C) just prior to pouring the plates to give
a final concentration of 0.1% in the pour plates. Cycloheximide inhibits the
growth of fungi but can be tolerated by bacteria. A 1% stock solution of
cycloheximide was prepared in distilled water, autoclaved for 15 minutes at
121°C and 1 .1 kglcm' pressure and stored at 4°C until needed. About 3-4
dilutions of each sample were plated in duplicate. AI1 plates were incubated
at room temperature (20-25°C) and counted at 4 and 7 days.
3.3.3 Total Coliforms
Total coliforms counts were determined by two techniques:(l) The
presumptive and confirmed tests of the MPN technique (five-tube) as
described by Standard Methods (APHA et al., 1995); and (2) Direct plating
on m-Endo Agar LES as described by Blais el al. (1992).
96
I n the MPN technique, lauryl sulphate broth was used for the
presumptive tests and brilliant green bile broth (BGB) for t h e confirmed tests.
Each t e s t tube contained 10 mL o f media and one Durham fermentation tube
t o tes t fo r gas production. In al1 cases 1 mL of serially diluted sample was
delivered to each presumptive tes t tube. Presumptive tes t tubes were
incubated at 35°C. After 24*2 hours o f incubation, tubes showing the
production of gas were interpreted as positive and three loops o f media (or 1
mL from a sterile pipet te) were transfered from each positive tube into BGB
tubes t o be confirmed. Also, presumptive tubes showing gas production at the
end o f 48*3 hours were confirmed in BGB tubes in a similar manner. The
BGB tubes were incubated a t 35°C for 48*3 hours. BGB tubes showing gas
product ion at the end o f o f 24*2 hours o r 48*3 hours were interpreted as
positive for total colifoms. Total coliform counts were based on the BGB test
tube results and the MPN table in Standard Methods (APHA et al., 1995), for
determining total coliform densities.
In the direct plating technique, 0.1 mL of serially diluted sample was
spread with a steri le glass L-rod ove r each o f two replicate plates for each
dilution tested. Each plate contained approximately 1 1 - 12 mL o f m-Endo
Agar LES. The plates were incubated a t 35°C for 24 hours.
3.3-4 Fecai Coliforms
T h e fecal coliform MPN procedure described in Starzdard Methods
(APHA et al., 1995) was employed with EC broth as the confirmatory medium.
T h e same presumptive tubes described in Section 3 . 3 . 3 were exarnined after
24*2 hours of incubation. As before, tubes showing the production of gas
were interpreted as positive and three loops o f media (or 1 mL from a sterile
pipette) were transfered from each positive presumptive tube into EC tubes t o
be confirmed. Also, presumptive tubes showing gas production at the end o f
48*3 hour s were confirmed in EC tubes. The EC tubes were incubated in a
t empera tu re controlled water bath a t 44S°C for 24I2 hours. EC tubes
showing gas production at the end of this incubation period were interpreted
a s positive fo r fecal colifoms. Fecal coiiform counts were based on the EC
test tube results and the MPN table in Standard Methods (APHA et al., 1995),
for determining total coliform densities.
3.3.5 Fecal Streptococci
The multiple-tube technique, with five tubes per dilution, a s described
in Standard Methods (APHA et al., 1995) was employed for the enurneration
of fecal streptococci. For the presumptive test, each test tube contained a one
mL inoculation of diluted sample and ten mL of azide dextrose broth media.
The presumptive tubes were incubated at 35°C. Each tube was examined for
turbidity at the end of 24I2 hours. If no definite turbidity was observed, then
each tube was reincubated and observed again at the end of 48*3 hours. Al1
tubes showing turbidity after 24- or 48-hours of incubation were subjected to
the confirmed test. The confirmed test involved streaking a portion of growth
from each positive azide dextrose broth tube onto bile esculin azide agar. The
dishes were then inverted and incubated at 35°C for 24*2 hours. Brownish-
black colonies with brown halos confirmed the presence of fecal streptococci.
Fecal streptococci densities were estimated from the number of tubes i n each
dilution series that were positive on bile esculin azide agar. The most
probable number was found from the MPN table in Standard Methods (APHA
et al., 1995) .
3.4 Physical and Chernical Methods
Varoius parameters were measured in both the batch and the continuous
reactors. These parameters included; pH, temperature, ORP, dissolved
oxygen, hydraulic retention time in the reactor and several types of solids.
Detailed descriptions of the methods employed are given below.
3.4.1 p H
The pH was measured by a VWR Scientific (model # 801 5) pH meter for
al1 batch reactor experirnents. The pH of the raw sludge and the bioleached
sludge from the continuous pilot plant reactor was measured within 20 minutes
after collection by an Orion Research Digital Ion Anfyzer (model #601A) .
The pH in t h e solubilization tank of the pilot plant was also monitored with a
cordless Checker (mode1 #632) pH meter.
3.4.2 Dissolved Oxygen
The dissolved oxygen was monitored in the continuous system by a
portable Orion dissolved oxygen meter (model #80i), by placing the dissolved
oxygen probe into the solubilization tank of the pi lot plant.
3.4.3 Temperature
The temperature was monitored in the continuous system with a portable
Orion dissolved oxygen meter (model #80i) that also gave a reading for
temperature. The probe was inserted into t he solubilization tank to obtain
readings.
3.4.4 Solids
Total solids (TS), suspended solids (SS), total volatile solids (TVS) and
suspended volatile solids (SSVS) were determined. Total solids and total
volatile solids were deterrnined as per Standard Methods (APHA et al., 1995).
Suspended solids and suspended volatile solids were determined by
centrifuging 10 mL of sarnple (@3000rpm for 10 minutes) and recovering the
pellet for determination of S S and SSVS as per Standard Methods (APHA et
a , 1995) .
3.4.5 Oxidation Reduction PotentiaI
The ORP of the raw sludge and the bioleached sludge from the
continuous reactor were measured (wit hin 20 minutes after collection) by an
Orion Research Digital Ion Anlyzer (mode1 #60 1 A), which was attached to a
platinum redox electrode (Orion Research, Mode1 97-78-00) which allowed for
ORP deterrninations.
3.4.6 Sufphate
Sulphate was determined using the procedure in Standard Methods
(APHA et al., 1989) as modified by Rich (1993).
4.0 RESULTS AND DISCUSSION
4.1 Batch Reactors
A series of batch reactors was set-up and rnonitored over periods
ranging €rom 7 to 17 days. The initial microbial counts on day O for each of
the reactors containing 100 mL of sludge were based on the raw sludge
analysis on the same date as the reactor start-up date. The solids were also
measured in the raw sludge and the total solids in the batch reactors were
assumed to be constant. In fact, the batch reactor containing 1.5 L of sludge
was sampled on day 1 and day 8 and the total solids only changed by O. 1%.
It should be noted that in al1 the batch experiments containing 100 mL of
sludge t h e lowest detection lirnit of the MPN analysis is 80 MPN/100 mL.
4.1.1 Experiment 1
This experiment involved a batch reactor that contained 95 mL of raw
sludge, plus a 5 mL inoculum (pHC3.0). At tirne zero, 1 g of sulphur/L was
added to the reactor. The total solids in the raw sludge at time zero were also
measured to be 2.25% and were assumed to be constant throughout the
leaching period in the batch reactor. It was analysed for total coliforms and
fecal coliforms versus tirne via the MPN technique. The results are displayed
in Figure 4.1. As the pH decreased a corresponding decrease in the
10 Time (Days)
1 O Timc (Days)
Presumptivc (TCRC) - TC - FC i
Figure 4.1 - Effect of bacterial leaching on presumptive, total and fecal coliform counts - Experiment 1
103
presumptive, total and fecal coliform counts can be observed. At day 12 the
fecal coliforrns could no longer b e detected since they went below the
detection limit of 80 MPN/100 mL. The pH stabilized in this reactor around
pH 2.7 beyond day 7. The total coliforms seemed to stabilize around 1000
MPN/100 mL from around day 12 until at least day 17. The presumptive
counts are also shown in Figure 4.1. Near the beginning of the graph (days O-
10) the presumptive counts were good indicators of the total coliform counts.
However, after day 10 t h e number of so-called false positives increased. That
is to say the percentage of tubes that were positive in the presumptive media
which could not be confirmed in the BGB media increased beyond day 10
compared to days O through 10. This is probably due to acid injury of the
total coliform cells. The graph indicates a >6 log reduction in fecal coliforms
in 12 days. It also indicates approximately a 5 log reduction in total coliforms
in about 12 days.
4.1.2 Experiment 2
This experiment involved a batch reactor that contained 95 mL of raw
sludge and a 5 mL inoculum. At time zero, 3 g of sulphurlL was added to the
reactor. The total solids in the raw sludge at time zero were measured to be
2.24% and were assumed to be constant in the batch reactor throughout the
leaching period. Total and fecal coliforms and fecal streptococci versus time
were determined using the MPN technique. The results are displayed in
Figures 4 . 2 and 4.3. As the pH decreased a corresponding decrease in t h e
presumptive, total and fecai coliform counts and fecal streptococci counts can
also be observed. By day 10 the fecal coliforms could no longer be detected
since they went below the detection limit of 80 MPNllOO mL. The pH
stabilized in this reactor around pH 1.75 beyond day 8. Near the beginning of
the graph (days 0-5) the presurnptive counts were good indicators of the total
coliform counts. However, at day 10 the number of so-called false positives
increased. This is probably due to acid injury of t h e total coliform cells. The
graph indicates a >5 log reduction in fecal coliforms in 10 days. It also
indicates approximately a >S log reduction in total coliforms in 10 days.
Figure 4.3 indicates a 3 log reduction in fecal streptococci counts after 10
days of bacterial leaching.
4.1.3 Experiment 3
This experiment involved a batch reactor that contained 95 rnL of raw
sludge and a 5 mL inoculum. At time zero, 1.5 g of sulphurlL was added to
the reactor. The total solids in the raw sludge at time zero were also measured
to be 2.25% and were assumed to be constant in the batch reactor throughout
107
the Ieaching period. Total coliforms and fecal coliforms from the MPN
technique are shown versus time in Figure 4.4. As the pH decreased a
corresponding decrease in the presumptive, total and fecal coliform counts can
be observed. At day 7 the fecal coliforms could no longer be detected since
they went below the detection limit of 80 MPN/100 mL. The pH stabilized in
this reactor around pH 1.9 beyond day 10. However, the pH reached a low of
1.81 by day 7. The Figure 4.4 indicates a >6 log reduction in fecal coliforms
in 7 days. It also indicates approximately a >4 log reduction in total coliforms
in 7 days.
4.1.4 Experiment 4
This experirnent involved a batch reactor that contained 95 mL of raw
sludge and a 5 mL inoculum. At time zero, I g of sulphur/L was added to the
reactor. The total solids in the raw sludge at time zero were also measured to
be 2.76% and were assumed to be constant in the batch reactor throughout the
leaching period. Results for total coliforms and fecal coliforms versus time
from the MPN technique are shown in Figure 4.5. As the pH decreased a
corresponding decrease in the presumptive, total and fecal coliform counts can
be observed. Although the same sulphur content was added as in Experiment
1 the pH profile is different. The pH in this reactor went down sharply to pH
Time (Days)
1
Presumptive TC lndicator Organism
Figure 4.5 - Effect of bacterial leaching on presumptive, total and fecal coliform counts - Experiment 4
2.1. The explanation for this is that the inoculum in Experiment 1 had been
on the gyratory shaker for almost a month before it was added t o the reactor
in Experiment 1 and probably had a low residual sulphur content compared to
the inoculum in Experiment 4 which came directly frorn the solubilization tank
of the continuous pilot system. B y day 8 the fecal coliforms could n o longer
be detected since they were below the detection limit of 80 MPN/IOO mL. A
>4 log reduction in fecal coliforms occurred in 8 days (Figure 4.5). Figure 4.5
aiso indicates a >5 log reduction in total coliforms after 8 days.
4.1.5 Experiment 5
This experiment involved a batch reactor that contained 1.5 L of raw
sludge and a 4% inoculurn. At time zero, 3 g of sulphur/L was added to the
reactor. The total solids in the raw sludge on day one were measured to be
2.10% and were assumed to be constant in the batch reactor throughout the
leaching period. This assurnption appears to hold true as the total solids in the
reactor were measured on day 8 and were determined to be 2.00%. The MPN
results for total coliforms and fecal coliforms versus time are depicted i n
Figure 4.6. Unfortunately the pH increased over the first 8 days of this
experiment for some unknown reason. Perhaps the inoculurn was improperly
prepared. In any case, as a result of the pH increase in the reactor, a
O 2 4 6 8 1 O 12 14 16 Time (Days)
6 8 Time (Days)
..- ma. .. . Presump tive (TCFC) TC I
Figure 4.6 - Effect of bacterial leaching on presumptive, total and fecal coliform counts - Experiment 5
corresponding increase in the presumptive, total and fecal coliform counts as
measured on day 7 can be observed. The pH in this reactor did finally reach
2.7 on day 1 5 . It should also be noted that the transfers in this experiment
were done with the standard 3 loop technique as opposed to transfer by a 1 mL
pipette. As well. certain alterations in the trmsfer conditions and the
sterilization procedures occurred and these are detailed in the Appendix
(Table A6). Presumptive counts are shown in Figure 4.6. Near the beginning
of the graph (days 0-7) the presumptive counts were good indicators of total
coliform counts. However, by day 15 the number of so-called false positives
increased greatly. This is probably due to acid injury of the total coliform
cells. In terms of reductions per 100 mL the graph indicates a > 3 log
reduction i n fecal coliforms after 15 days. It also indicates approximately a
>3 log reduction in total coliforms after 15 days. Compared to the other batch
reactors studied, t h e counts of indicator organisms i n this reactor (Figure 4.6)
on day zero are about 2 logs lower. Perhaps this is because no mixing of the
sludge sample was done before analysis in this particular experiment
(Experiment 5) .
4.2 Continuous Reactors
A continuous pilot plant for bacterial leaching was set-up at the Main
Wastewater Treatment Plant in Toronto during the summer of 1997. I t was
fed with raw sludge and thus it was necessary to determine the indicator
organism counts in the raw sludge as well as the bioleached sludge in order to
determine the fate of the indicator organisms. Raw sludge was sampled as
indicated in Figure 4 .7 which shows that the indicator organisms remained
relatively stable over time. From an average of the FC counts and the FS
counts for the first 4 sarnples a FC:FS ratio of 3.4 can be obtained. A value
of greater than 4 was at one time thought to indicate fecal pollution from
human sources, whereas a ratio of less than 0.7 was suggestive of
contamination by nonhuman sources, however it should be noted that these
limits are no longer considered reliable (APHA et al.. 1995) .
The pilot plant was operated under two different steady state conditions
during which indicator organisms were monitored. The complete data
describing the steady state parameters is shown in the Appendix (Table A10).
A summary table of the reductions in the indicator organisms for both steady
states is provided in Table 4.1. The counts shown for t h e raw sludge were
obtained by taking an average of two last two raw sludge samples ( November
7, 1997 and November 26, 1997). The counts shown for the leached sludge
Table 4.1 - Summary Table: Reductions in indicator organism counts during two different operating conditions of the coritinuous bactcrial leaching process
Steady Statc #1 1 Steady Statc #2 :T = 8.2 days
Reduction (%)
pH = 1.90 ; HRT = 10 days 1
Organism Counts
pH = L I S ; E
Organism Counts Reductian (%)
Organism Counts
@er g dry sludge)
lndicator Organism
HPC - 4 Day
7 Day l
Presumptive TCIFC
TC (Spread Plate)
FC
3.2E+08
1.1 E+08
are mean values of four samples each taken at each steady state.
As can be seen from Table 4.1. the fecal streptococci are always more
numerous than the fecal coliforms in the bioleached sludge at each steady
state. Also, total coliforms by the MPN method are always higher than by the
spread plate method. In general, the same reductions occurred in both steady
states in terms of total and fecal coliforms and fecal streptococci, considering
how high the initial counts in the raw sludge are. Although slightly larger
reductions occurred in total and fecal coliforms in the second steady state this
could easily be accounted for in terms o f reductions in the raw sludge itself.
Considering that bacterial counts in raw sludge are normally in the millions,
small changes of one thousand at steady state do not necessarily indicate a
change in the reduction efficiency of the bioleaching system. In fact fecal
streptococci counts are essentially the same at both steady states when one
considers the accuracy of the MPN method. Thus, the changes between the
two steady states in terms of pH and HRT do not seem to change the reduction
efficiency in terms of fecal coliforms, total coliforms, or fecal streptococci.
However, the HPC counts in the second steady state increased by about 1 log
compared t o the first steady state. No explanation can be provided for this
observation.
The occurrence of false positives at steady state is also shown by Table
4.1. With the raw sludge values there are n o false positives, however, at both
steady states the presumptive counts are consistently higher than the total
coliform counts. In fact, i t can be noted that the presumptive counts remain
virtuaily the same at both steady states. The explanation for the increase in
false positives is probably that acid injury of the total coliforms occurred, such
that they can recover in the general enrichment media of the presumptive test,
but they are not able to recover and produce positive results in the stressful
environment of the selective BGB media. Injury probably also occurred to the
HPC bacteria. I n observing the HPC plates for the raw sludge t h e colonies
appeared slightly larger than the colonies in the plates for the bioleached
sludge. The expianation for the srnaller colonies is probably acid injury from
the bioleaching process.
4.3 MPN vs. Spread Plate Method
An additional topic that was investigated during the microbial analysis
of the continuous pilot bioleaching system was the difference in the MPN
technique versus the spread plate method for the enurneration of total
coliforms. As shown in Table 4.2. there appears to be no difference in the
results for raw sludge between the MPN and the spread plate method. It is
possible that these results might Vary with the type of sludge being analyzed.
Table 4.2 - Cornparison of the MPN and the spread plate method for the enurneration of total coliforms in raw sludge
Date
14-Aug-97 04-Sep-97 30-Sep-97 07-NOV-97 26-NOV-97
MPN Method (MPN/g dry sludge)
6,39E+07 2.86Et08 1.24EtOS 1.89E+OB 3,48E+08
Spread Plate (CFUlg dry sludge)
8.3 1 E+07 1.25E-t-08 4,18E+08 1.81E-tO8 4.49EtO8
Dudley et al. (1980) concluded that the spread plate technique was superior
to the completed multiple-tube-fermentation and the membrane filtration
methods for the analysis of sludge samples. However, the sludge examined by
Dudley et al. (1980) is not clearly specified. At the outset of their paper these
authors state that primary, anaerobically digested and wasted secondary sludge
samples were collected for analysis. An examination of their results however,
reveals that the differences between the spread plate method and t h e MPN
technique are never greater than 0.2 log units. In fact, the results frorn Table
4.2 show that the spread plate count is slightly higher than the MPN.
Analysis of bioleached sludge, however, requires special attention, in
that the indicator organisms could be sublethally irnpaired. The MPN method
allows for resuscitation to occur in the general enrichment media of the
presumptive broth, whereas, the spread plate technique does not provide such
a "restoration treatment." It would thus be expected then that the bioleached
sludge would produce higher MPN counts than those in the spread plate
rnethod. This was the case in the present study where the MPN technique
produced higher counts for total coliforms at both steady states (Table 4.1).
At steady state # 1 the spread plates only produced between one to three
colonies per plate. At steady state #2 no colonies were observed on any of the
plates. However, except for sample #4 at steady state #1, the MPN technique
120
always produced positive tubes and higher counts than the spread plate
method. In addition. i t should b e noted that if spread plates contain fewer
than 30 colonies they are not really acceptable for counting under the
guidelines i n Standard Methods (APHA et al., 1995) . For this reason the
numbers shown have a "<" syrnbol shown at the left indicating that the counts
are less than the actual nurnber shown and they are less by an unknown
amount. Therefore it can be argued that the spread plate is an unacceptable
method for counting total coliforms in bioleached sludge when the numbers are
in the <IO00 CFU/100mL range, which is the limit of the spread plate method.
4.4 Experirnents Designed i o Improve Microbial Counting
4.4-1 Initial Trials
Various trials, designed to improve the microbial counting procedure
were conducted prior t o gathering the results shown in Sections 4.1, 4.2 and
4.3 (except 4.1.5). If the harsh acidic environment of the bioleaching process
sub-lethally impaired some of the indicator organisms they would not recover
i n the standard tests and thus go undetected. These sub-lethally impaired
organisms could recover when the sludge is applied to land, thus increasing the
health risks to humans and livestock. To recover such sub-lethally impaired
organisms during testing, a nurnber of rernedies were tried. These included
121
increased inoculum, extended incubation, double strength presurnptive media
and a new pre-enrichment technique.
4.4.1.1 Pipette Transfer Technique
Seth (1997) observed that transferring a culture with a sterile 1 mL
pipette instead of the standard 3 loop transfer in the MPN analysis was
superior in terms of recovering total coliforms. However, the fecal coliform
counts did not increase significantly above the values obtained with the three
loop transfer. His explanation for the increase in total coliform counts was
that the higher volume of inoculum (1 mL) for the confirmed tests "allowed a
higher probability of capturing the coliforms which recovered during the
presumptive test." Concerning the lack of fecal coliforms counts, Seth ( 1997)
stated that the "growth conditions for the enurneration of fecal coliforms were
too stressful even for the growth of coliforms which recovered during the
presumptive test."
The objective of this initial trial was to confirm the explanation
provided by Seth (1997). A batch reactor was set-up containing 3 g suIphur/L
and 100 mL of sludge ( i x . the reactor from Section 4.1.2). Transfers were
made on the last day of leaching (day 10) with both the 3 loop technique and
a sterile 1 mL pipette. It should be noted that the batch reactor in 4.1.2 was
122
analyzed o n the other days of leaching only with the 1 mL pipette (i t was
assumed to be the best method before more rigourous testing was performed).
As shown in Figure 4 .8 the 1 rnL pipette transfer did increase the total
coliform counts by about 1 log unit. During this experiment the recovery
broth was also being evaluated and it too showed similar irnproved results.
The pH at the time of sampling was 1.74 and the HRT was 10 days. This is
quite a low pH value and as rnight be expected lethal to the fecal coiiforms
which could not be recovered with any method. A more detailed investigation
of the I mL transfer technique was also done in a later experiment and the
results are shown in Section 4.4.2.
4.4.1.2 Recovery Broth/Double Strength BrothlExtended Incubation
The topic of recovering sub-lethally impaired coliform cells has received
quite a bit of attention in the food industry. Mossel and Ratto (1970)
investigated a pre-enrichment treatment to recover such sublethally impaired
cells of Enterobacteriaceae in dried foods. They stated that "restoration of
gram-negative rod-shaped bacteria may be complete within the order of a few
hours at temperatures in the range of 20 to 30°C, provided that the right
external conditions are chosen." In their study they tried incubation in shallow
layers of Tryptone soya peptone broth for 1 to 6 hr a t room temperature. One
t t : : : : : : I
to two hours incubation proved to be the best time period according to these
authors.
To try and recover some of t h e sub-lethally impaired cells from the
continuous leaching process, an experiment was conducted with the same
(Tryptic Soy) broth of Mossel and Ratto (1970), on this leached sludge. The
exact methodology is described in Section 3.3.1. Also it was hypothesized
that a double strength presumptive broth instead of the standard single
strength presumptive broth could increase indicator organism counts since the
increased nutrient level of the broth might help in the recovery of sub-lethally
impaired indicator organisms. The results from this initial trial are shown in
Figure 4.9. Although the continuous pilot system was not quite at steady
state. pH had been about 3 ( I 0 . 3 ) for 12 days. As can b e seen in Figure 4.9
total coliforms are around 1000 MPN/lOOmL by a11 three methods. However,
the fecal coliforms by the standard method (single strength presumptive broth)
are <80 MPN/100 mL. Considering that over 90% of the total coliform
bacteria in t he fresh feces of warm-blooded animais are Escherichia c d i ,
which are fecal coliforms, it seems improbable that the fecal coliform count in
this particular sludge (Figure 4.9) could be <80 MPN/100 mL. A close-up of
the fecal coliform counts in Figure 4.9 is shown in Figure 4.10. I t indicates
that with the recovery broth the fecal coliform count is increased by over 1 log
Figure 4.9 - Initial Trial : Single strength presumptive broth versus double strengtli presuinptive broth versus recovery broth in a sample analysis from the continuous bacterial leaching process (pH = 2.95, HRT = 10 days)
Single Strength Double Strength ' Recovery Brotli Method of Analysis
Figure 4.10 - Enlarged view of Figure 4.9 (fecal coliform results only)
unit compared to the standard analysis technique. The double strength broth
also increased the counts over the standard analysis technique but these results
do not seem significant enough to warrant further study on the double strength
broth. It was very unusual that the fecal coliform tubes that came from the
single strength presumptive tubes showed significant growth but did not
produce any gas. The fecal coliform tubes that came from the recovery broth
technique showed significant growth and they produced significant arnounts
of gas in the Durham fermentation tubes. Two possible explanations for the
occurrence of growth without gas production are: ( 1 ) non-lactose fermenting
bacteria were able to grow at 44S°C and produce growth and (2) the fecal
coliforms were acid injured such that they were unable to produce gas. These
results seemed so significant that the presumptive tubes were a11 left in the
incubator for a total of 5 days and numerous transfers were taken from the
presumptive tubes on days 2 through 5 to try and confirm the results and to
test an earlier hypothesis that extended incubation in presurnptive media may
allow for irnproved recovery of injured cells, and thus increase the fecal
coliform counts. There were various concerns that the EC tubes rnay have
been left at room temperature too long before they were incubated at 44S°C
and thus the extended incubation experiment helped to confirm that the results
shown in Figures 4.9 and 4.10 were reproducible. These data are presented in the
Appendix (Table A13). These results seemed so significant at the time that
immediately batch reactors were set u p o n the gyratory shaker to further
investigate the recovery broth.
4.4.2 Recovery Broth - Detailed Study
The recovery broth was investigated in four batch experiments and one
continuous experiment. The recovery broth was afso experimented with on a
raw sludge sample. As shown in Figure 4.11 the recovery broth was observed
to have no influence on the HPC, TC, FC or FS counts in raw sludge compared
to the standard method of analysis.
In three of the batch reactors studied, the recovery broth did not appear
to influence the fecal coliform results at ail. However, in one of the batch
reactors, the recovery broth may have affected the fecal coliform counts. The
results from this reactor are shown in Figure 4.12. As shown in Figure 4.12
there was a 2 log increase in fecal coliform counts on day 12 with the recovery
broth technique compared to the standard method. Admittedly t h e previous
data points on day 10 do look spurious, however these were the results
obtained. Numerous EC tubes on day 10 showed growth, however. not al1 the
tubes showing growth produced gas. If al1 the tubes showing growth on day
10 had produced gas then the data points for day 10 could easily be around
Presuinptive (FS)
@ TC (spread plate) O 8 Cr TC 8 3 Presumptive (TC/ FC) c(
HPC (7 day)
HPC (4 day)
[I:] Single Strength Presumptive Broth Recovery Bioth
Figure 4.1 1 - Cornparison of the recovery broth technique and the standard metliod in the analysis o f a raw sludge sample for indicator organisrns
10 Time (Days)
- - A- - Standard hielhacl + Recovey Broih 2
Figure 4.12 - Comparison of the recovery brot h technique with the standard method for enurnerat ing fecal colifornis duriiig bacterial leaching in a batch reactor
1.00E+4. Nevertheless, a significant difference in the fecal coliform counts
obtained by the two methods was observed on day 12. The results of this
graph lead to the formulation of a hypothesis to explain the recovery broth
effect. The recovery effect in Figure 4.12 occurs right around the region
where fecal coliforms can be detected by the standard method (day 10) and the
point where fecal coliforms cannot be detected by the standard method (day
12). Thus the recovery effect may occur right at this "threshold of injury" to
the coliform cells. The recovery broth may possibly make fecal coliform cells
healthier at al1 levels of injury but only at the "threshold of injury" do the fecai
coliform cells recover just slightly more than in the standard method such that
they can produce gas in the EC tubes and the fecal coliforms from the standard
method cannot. Thus the recovery broth effect may only be noticeable over
a narrow range of injury which explains why it was so difficult to observe in
the earlier batch systems. The explanation for why the effect only occurs on
fecal coliforms and not the total coliforms may well be related to the fact that
fecal coliforms have a much more stressful incubation temperature (44.SoC) as
compared to the incubation temperature for the total coliforms (35°C). It
should be noted however, that there are not enough data points on Figure 4.12
to rigourously confirm that the recovery broth increased fecal coliform counts
in any way.
In addition to the batch systerns, 3 samples were taken at steady state
# 2 to try and observe the recovery broth effect. The results shown in the
Appendix (Table A M ) , show no difference in fecal coliform counts with both
methods, since al! samples had counts of <80 MPNI100rnL. It can be
postulated that the fecal coliforms in steady state #2 were too far beyond the
"threshold of injury" and the recovery broth couid not increase the fecal
coliform count. However, an extremely important observation was made in
that the amount of gas production in the presumptive test tubes was
consistently 3 to 4 times greater with the recovery broth as compared to when
the standard method was used. This was a consistent observation for a11 three
samples analysed. However, the greatly increased gas production did not lead
to an increase in total coliform or fecal coliform counts. Thus the recovery
broth may have made the cells healthier at this particular level of injury
(based on the presumption that healthier cells can produce more gas) but the
counts of total and fecat coliforms were not increased over the standard
method.
In order to observe the overall effect of the recovery broth at many
different levels of injury, a total was taken of al1 the positive presumptive,
BGB and EC tubes that were involved in parallel testing with the recovery
broth and the standard method. The results are shown in Table 4.3. in
Table 4 .3 - Cornparison of the standard method with the recovery broth technique in terms of the total number of positive presumptive, BGB and EC test tubes produced by each method
Standard Method
Recovery Broth
-
Number of Positive Presumptive Tubes
Number of Positive BGB Tubes
Number of positive EC Tubes
comparing the number of positive presurnptive tubes by both methods there is
seen to be no difference. With the BGB tubes, a minimal difference in
positives is also seen. However, there is a 25% difference in the total number
of positive EC tubes. This difference in positive EC tubes is only due to two
samples, however. Specifically, these two samples are the results on day 12
of Figure 4.12 and the result frorn the initial trial with the recovery broth
shown i n Figure 4.9. If these two observations are removed, then the total
number of EC tubes would be 54 and 5 1 respectively for the standard method
and the recovery broth. These data show that the recovery broth cannot
increase total or fecal coliform counts compared to the standard method at
most levels of injury. However, the data may lead one to hypothesize about
the recovery broth being able to increase fecal coliform counts over a narrow
range of injury around the "threshold injury level" as discussed earlier. In
general, it can be concluded that the experiments designed to study the
recovery broth were inconclusive.
4.4.3 1 mL Pipette Transfer - Detailed Study
I n order t o further verify the effect of the sterile 1 mL pipette
transfer, a number of consecutive samples from a batch reactor were taken
and analysed by both the standard 3 Ioop transfer and the 1 mL pipette transfer.
135
The batch reactor from Experiment 1 was chosen and the results are shown in
Figure 4.13. The samples were tested by both methods on days 10, 14 and 17.
There were, for all practical purposes, no increases in fecal coliform counts
on any of the sampling dates. On day 10 the level of injury to the total
coliform population is not significant enough for a difference to-be observed
between the 1 mL pipette transfer and the 3 loop technique. However, on days
14 and 17 there is clearly an increase in total coliform counts with the 1 mL
pipette transfer, as compared to the standard 3 loop transfer. A possible
explanation for this is that as the HRT increases, the injury to the total
coliform population also increases and the 1 mL pipette transfer increases the
chances of healthy cells being transferred from the presurnptive tubes into the
BGB tubes, thus increasing the chances of BGB tubes going positive.
5 10 Time (Days)
1 O Time (Days)
. - 9- - 3 loop TC __a__ 3 loop FC - 1 mL pipette TC -ie, 1 mL pipette FC
J
Figure 4.13 - Cornparison of the 3 loop and 1 mL pipette transfer in a batch reactor
CONCLUSIONS
The main conclusions arising from this investigation into the fate o f
indicator organisrns in raw during biological solubilization of metals, are listed
as foilows:
1. The mean indicator organisms counts in raw sludge from Toronto's
Main Wastewater Treatment Plant analyzed between 0811411997 and
1 11261 1997 were: (1 ) 2 4 x 10' CFU/g dry sludge for HPC bacteria (counted
after 7 days incubation); (2) 20x10' MPNlg dry sludge for TC; (3) 2 5 x 1 0 '
CFU/g dry sludge for TC; (4) 6 3 x 1 0 6 MPN/g dry sludge for FC; and (5)
4 3 x 1 O6 MPN1g dry sludge for FS.
3 - The maximum observed reductions in indicator organisms (per gram of
dry sludge) during biologicaI solubilization of metals in batch reactors during
laboratory experiments were: (1) > 5 log reductions in TC after 8 days (final
pH = 2.37); (2) > 6 log reductions in FC after 7 days (final pH = 1.80); and
( 3 ) 3 log reductions in FS after 10 days (final pH = 1.74).
3. Observed reductions in indicator organisms (per gram of dry sludge) in
the continuous pilot plant reactor during steady state # 1 (pH= 1.90/HRT= 1 Od)
were: ( 1 ) 98% reduction in HPC; (2) 5 log reduction in TC; (3 ) 5 log
reduction in FC; and (4) > 3 log reductions in FS.
4 . Observed reductions in indicator organisms (per gram of dry shdge) in
t h e continuous piiot plant reactor during steady state #2 (pH=2.15/HRT=8.2d)
were: ( 1 ) 87% reduction in HPC bacteria; (2) > 5 log reduction in TC;
( 3 ) > 6 log reduction in FC; and (4) > 3 log reduction in FS.
5. The spread plate method yielded equivalent results to the MPN method
for the analysis of total coliforms in the raw sludge examined.
6. Investigation into a new recovery broth method as described in this
thesis has proved inconclusive.
7. The sterile I mL pipette transfer technique as described in this thesis,
yielded higher MPN total coliform counts than the standard 3 loop transfer,
in the analysis of bioleached sludge.
RECOMMENDATIONS
The fate of indicator organisms (HPC, TC, FC and FS) during the
bacterial leaching process is now quite clear. However, various unknowns
should be investigated before t he bioleached sludge is deemed "safe" for land
application, from a pathogenic point of view:
1. Parasitic egg destruction was not investigated directly in this thesis and
should be a priority for further research. In particular, the acid resistance of
Ascaris eggs is a concern for the bioleaching process.
2. The bioleached sludge will need to be neutralized before it is applied to
land and thus it would be interesting to test the neutralized bioleached sludge
to determine the effect on indicator organisms (HPC, TC, FC, FS).
3 . The true effectiveness of the "recovery broth" investigated in this thesis
is unknown. If a continuous bioleaching reactor is in operation and fecal
coliform counts are not consistent with anticipated fecal coliform counts based
on total coliform counts, then investigative testing with the recovery broth
technique is recommended, if time and money permit.
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APPENDIX
Table Al : Experiment 1 - Fate of total and fecal coliforms during leaching (al1 counts in MPN1100mL)
24 hrs.
2.00E+OS
.6.40E+07
2,00E+06
3.60E+04
4.40Et03
1,20E+03
1.20Et03
- 48 hrs. 24 hrs.
~t ive Recoveq
. I
I
r Broth 48 hrs.
8.8OEtO8
6.40E+07
6.40Et06
2.OOE+O4
2.OOE+O4
1,20E+04
9.60E+O3
Total C Single Strength
48 hrs,
2,80E+08
>6.40E+07
3.60E+06
3.6OE+O4
2.00Et03
4.40E+03
1.20E+03
iiforms Recovery Broth
48 hrs,
8.80E+08
6.40E+07
6.40Et06
2.00E+04
1.20Et04
1,36E+03
5.20Et-02
Fecal C Single Strength
liforms Recovery Broth
Table A2: Experiment 2 - Fate of total and fecal coliforms during leaching (al1 counts in MPN1100mL)
Single 24 hrs.
mngth 48 hrs.
Recove 24 hrs. 48 hrs,
Total Coliforms Single Strength
48 hrs. Recovery Broth
48 hrs.
Fecal Coliforms Single Strength Recovery Broth
Table A3: Experiment 2 - Fate of fecal streptococci during leaching (al1 counts in MPN/I OOmL)
Presumptive Fecal Streptococci I Single !
24 hrs. irengt h
48 hrs. Recove
24 hrs.
2 . O O E + O 6
1 Broth 48 hrs.
2 . O O E + O 7
Confirmed Fec Single Strength
48 hrs.
Streptococci Recovery Broth
48 hrs.
Table A4: Experiment 3 - Fate of total and fecal coliforms during leaching (al1 counts in MPNll OOmL)
Total Coliforms Fecal Coliforms rength
48 hrs. Recove
24 hrs. r Broth
48 hrs. Single Strength
48 hrs, Recovery Broth
48 hrs. Single Strength Recovery Broth
24 hrs.
Table AS: Expriment 4 - Fate of total and fecal coliforms during leaching (al1 counts in MPN1100mL)
Single i
24 hrs.
Presumptive :rength 1 Recoveq
I 48 hrs. 24 hrs. Broth
48 hrs.
Total Coliforms Single Strenfi Recovery Broth
48 hrs. 48 hrs.
Fecal Coliforms Single Strength ( Recovery Broth
1
Table A6: Experiment 5 - Fate of total and fecal coliforms during leaching (al] counts in MPN/f OOmL)
Presurnptive
r
Totat Coliforms Fecal Coliforms
Note: Day
D ~ Y L
O 1 3 4 5 7 8 9 1 O 1 1 13 14 15
Day 7 - Presumptive tubes incubated for 48hrs before transfers occurred Day 15 - Presumptive tubes incubated for 48hrs before transfers occurred (Dilution water was not sterilized in this experiment)
1
Table A7: Main Treatment Plant Raw SIudge Parameters, (al1 bacterial counts in MPNtg dry sludge, or CFUtg dry sludge)
Date pH 1 Prisumpive TC 4 Dey 7 Day TCFC
TC' (Spreud Plate)
SSVS (%)
1.90 1.57 1.43
1.53 1.49 1.92
TVS (%)
2.03 1.64 1.46
1.55 1.57 2.03
DI0 cm&>
SS
(%)
2.92 2.50 2.24
2.19 2,18 2.58
Temp. (deg. C)
c c 0 - c m m m m m m *
Table A10 : Performance characteristics of the continuous pilot plant reactor
pH ORP (mv)
DO Temp. Sulphate TS SS TVS SSVS Comments (mgn) (deg. C) ( m m (%) (%) (%) (%)
1
Started Continuous Feediny on Aug./I 2/97; 14 Yday, 2.5 g S/L
5.40 1
4.50 1380 1710
6.50 23,2 2030 1.44 1,06 0,83 0.72 4.50 1890
1910 2030
22.9 1 .58 , 1.16 0.93 0.78
1
1800
Table A 10 : Performance characteristics of the continuous pilot plant reactor (continued)
Comment s ssvs (%)
Sulphur mixing problems. blender introduced
I J
1.71
TVS (%)
Sulphur increased to 4 g/L
TS (%)
1.6
1.9
1.8 2.3
Date
01-Sep-97 02-Sep-97 03-Sep-97 04-Sep-97 05-Sep-97 06-Sep-97 07-Sep-97
--
08-Sep-97 09-Sep-97 10-Sep-97 11-Sep-97 12-Sep-97 13-Sep-97 14-Sep-97 15-Sep-97 16-Sep-97 17-Sep-97 18-Sep-97 19-Sep-97 20-Sep-97 21-Sep-97
SS (%)
3.9
ORP (mV)
333
485
450 450
Day
21 22 23 24 25 26 27 28 29 30 31 32 33
, 34 35 36 37 38 39 40 41
DO ( m m
4.50
pH
3.00
3.60 3.10 3.00 2.85 2.85 2.90 2.90 2.85 2.85 2.85 2.85
3.10
Temp. W. C)
Sulphate (mg/L)
1680 2240 2300 2650 2230
2300 2570 2240
Table A 1 O : Performance characteristics of the continuous pilot plant reactor (continued)
SSVS (%)
1.08 1.06 1.05 1 . 1
Comments TVS (%)
1.56 1 .55 1.55 1.59
DO ( m a )
4.65
5.61 6.00 4.80 6.40
TS (%)
2.35
2.43 2.42 2.43 2.51
ORP (mv)
525
590 585 590 585
Date
13-0ct-97 14-Ott-97 15-0ct-97 16-Oct-97 17-0ct-97 18-Oct-97 19-Oct-97 20-0ct-97 21-Oct-97 22-Ott-97 23-Oct-97 24-Oct-97 25-0ct-97 26-Oct-97 27-Oct-97 28-Oct-97 29-Oct-97 30-Oct-97 31-Oct-97 0 1-NOV-97 02-NOV-97
SS (%)
1.38
1.47 1.46 1.44
, 1.49
Temp. m g . C)
18.5
15.5 17.1 17.3 17.7
Day
63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83
Sulphate ( m a )
5620
pH
1.92
1.90 1.90 1.94 1.90
1
Table A10 : Performance characteristics of the continuous pilot plant reactor (continued)
ORP DO Temp. Sulphate TS SS (mV) ( m a ) (deg. C) ( m m (%) (%)
TVS SSVS (%) (%)
Comments
Table Al 1 : Initial trial: 3 loop versus 1 mL pipette transfer (al1 counts in MPNIl OOmL) (from Experiment 2, tested on day 10, pH= 1.74)
Analy sis
Standard Method
Recovery Brot h
Bacterial Indicat or
Transfer Method 1 mL pipette 3 loup
Table A 12: Initial trial: Singie/Double strength presumptive broWRemveq broth (kom pilot plant, 08/26/97, pH=2.95) ( d l counts in MPN/I OOmL)
hdicator Single 1 Double Recovery
Presurnptive (TC/FC) 1
Presumptive (FS) 1
Standard method with single strength presumptive broth Standard rneihod with double strength presumptive broth Standard metfiod with recovery broth
Table A 13: Initial trial: Extended incubation; Single strength presumpûve brothRecovery broth (FC results ody) (from pilot plant, 08/26/97, pH=2.95) (al1 counts in MPNf 100rnL)
Recovery Broth
FC
4.40EM3 3.20EM2 4.40E+03 1.30E+03
1
Days in , Presurnptive Medium
before transfer to EC tubes
2* 3**
q*** 5****
* - 3 loop tramfer (EC tubes lefi for 1 hr at roorn temperature d e r irinoculation) ** - 3 loop transfer (wata temperatun at 46.5 degrees celsius) *** - sterile Iml pipette transfers **** - sterile Iml pipelte transfers
Singie S trength FC
4 0 4 0
1.60EI02 <80
Table A1 4: Single strength presumptive media versus recovery broth: raw sludge analysis (al1 bacterial counts in MPN/1 OOmL, or CFU/l OOmL) (Three loop transfer used exclusively)
Method of Analysis
Standard method
Recovery Broth
J
4 Day
î.4OE+ 10
2.02E+10
Presumptive (TC &FC) 7 Day
3.34E+ 10
2.38E+10
TC FC Presumptive (FS)
FS TC (Spread Plate)
Table A 1 5 : Detailed study - 3 1 loop versus mL pipette transfer (100 rnL batch reactor) (al1 bacterial counts in MPN/100 mL)
3 loop
II
-
ipette FC
Table AI6: Detaiied stuciy - Standard meihod versus Recovery broth (Samples h m pilot plant at steady sbte #2) (bacteriai counts in MPN/g chy sludge)
Sample Date
Method OC Analysis
- -
Standard Method
1 Recovery Broth
Standard Method
Recovery Broth
Standard Method
Recovery Broth
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