Microbial Phosphorus Removal in Waste Stabilisation Pond Wastewater
Treatment Systems
Lydia A. Mbwele
Royal Institute of Technology
School Of Biotechnology
Stockholm 2006
© Lydia A. Mbwele
School of Biotechnology
Royal Institute of Technology
Albanova University Center
SE – 106 91 Stockholm
Sweden
Printed at Univesitetsservice US AB
Box 70014
100 44 Stockholm
Swden
ISBN 91 – 7178 – 280 – X
i
ii
To my late sister FLORA, rest in peace
iii
Lydia A Mbwele (2006): Microbial Phosphorus Removal In Waste Stabilisation
Ponds Wastewater Treatment Systems.
A Licentiate thesis from the School of Biotechnology, Royal Institute of Technology,
Stockholm, Sweden
ISBN 91-7178-280-X
ABSTRACT
Waste Stabilisation Ponds (WSPs) are characterised by low phosphorus (P) removal
capacity. Heterotrophic bacteria are principal microbial agents in WSPs in addition to
algae. As treatment proceeds in WSPs, algal growth increases and pH rises, this has
lead to believe that P removal is mainly through sedimentation as organic P algal
biomass and precipitation as inorganic P. In activated sludge treatment plants (AS),
microbial P removal has been improved and is termed as enhanced biological
phosphorus removal. There was a need to establish whether it was possible to enhance
P removal in WSPs. A performance assessment of pond system at the University of
Dare s Salaam (UDSM), Tanzania, has shown that 90% of the P removed was in the
primary pond (facultative) and the rest in the maturation pond (aerobic).
In these studies, a pure strain A. hydrophyla was isolated from an activated sludge
wastewater treatment plant in Sweden. This plant has a train that functions with
enhanced biological phosphorus removal. The strain was tested for P uptake in
minimal media supplemented with glucose, succinate or acetate, grown aerobically
and anaerobically/aerobically. This strain was able to take up P without having been
subjected to the anaerobic phase. It was observed that P uptake was enhanced after the
anaerobic phase with media supplemented with glucose, but not with succinate or
acetate. Phosphorus uptake repeatedly followed the bacterial growth pattern with
correlation coefficients of more than 95%. Therefore P removal has a direct
correlation with bacterial growth.
Two isolates Acinetobacter sp. (isolated from the primary facultative pond) and E
.coli (isolated from the maturation pond) were obtained from a tropical WSP
treatment system at the UDSM. They were subjected to aerobic P uptake experiment
similar to those of A.hydrophyla. The uptake per unit absorbance of bacterial growth
was found to be comparable to that of A.hydrophyla, isolated from AS.
These results showed that heterotrophic activity is important in WSPs. It is possible to
enhance P removal in these systems by designing the primary ponds for maximum
heterotrophic activity and probably enrichment.
Key Words: Wastewater treatment, waste stabilisation ponds, activated sludge,
biological phosphorus removal, tropical climate, A.hydrophylla, Acinetobacter sp,
E.coli, heterotrophic activity, polyphosphate, polyhydroxyalkoanates
iv
List of Papers
This thesis is based on the following original papers, which in the text will be referred
to by their roman numerals.
I
L. Mbwele, M. Rubindamayugi, A. Kivaisi and G. Dalhammar, (2003).
Performance of a small wastewater stabilization pond system in tropical
climate in Dare s Salaam, Tanzania. Water Sc. and Tech.Vol.48 No 11 –
12 pp 187-191
II
L.A.Mbwele, Gunaratna K. Rajarao and Gunnel Dalhammar. Isolation
of Phosphorus Removing Bacteria from a Waste Stabilisation Pond
System. Manuscript
III
L.Mbwele, G.K. Rajarao and Gunnel Dalhammar. Influence of
microbial growth and carbon source in phosphorus uptake in wastewater
treatment. Manuscript
Reprints are published with due permission from the respective journals concerned
v
ACRONYMS
A/O
Anoxic/Oxic
AS
Activated Sludge
BOD Biological Oxygen Demand
CH4
Methane
CO2
Carbon Dioxide
COD Chemical Oxygen Demand
DO
Dissolved Oxygen
EBPR Enhanced Biological Phosphorus Removal
FC
Feacal Coliforms
FISH Fluorescent In Situ Hybridization
H2S
Hydrogen Sulphide
N2
Nitrogen
NEMC
National Environment Management Council
O2
Oxygen
P
Phosphorus
PAO polyPhosphate accumulating organisms
PHA Polyhydroxyalkonoates
SS
Suspended Solids
TC
Total Coliforms
UCT
University of Capetown
UDSM
University of Dar es Salaam
VFA Volatile Fatty Acids
WHO World Health Organization
WQI Water Quality Index
WSPs Waste Stabilisation Ponds
WW
Wastewater
vi
CONTENTS
ABSTRACT
LIST OF PAPERS APPENDED
ACRONYMS
1. INTRODUCTION
1.1 Phosphorus removal in waste stabilization ponds
1.2 Improved phosphorus removal in other treatment systems
1.3 Scope of this thesis
2. TREAMENT PROCESSES
2.1 Types of Waste Stabilisation Ponds
2.1.1 Anaerobic Ponds
2.1.2 Facultative Ponds
2.1.3 Maturation/Tertially Ponds
2.2 Pathogen and Parasite Removal
2.3 Nutrient Removal
2.4 Wastewater Stabilisation Ponds in Dar es Salaam, Tanzania
3. Biology of Phosphorus removal
3.1 EBPR process configurations
4. Present Investigations
4.1 Stabilisation process, growth and activities of oxidation of pond
microorganisms
4.1.1 University of Dar es Salaam Stabilisation Ponds
4.2 Microbial Phosphorus removal in Waste Stabilisation Ponds
4.3Microbial Phosphorus Removal in Activated Sludge Treatment
Plants
5. Conclusions and Recommendations
ACKNOWLEDGEMENTS
REFERENCES
ORIGINAL PAPERS
vii
1. Introduction
Assurance of water quality has become an integral part of environmental quality
management in the world today. Wastewater treatment is one of the strategies for
water quality management. In Tanzania (East Africa), water quality management
began during the colonial era. Some oldest sewers were laid in Dar es Salaam
between 1948 and 1952; wastewater stabilization ponds began to be used in the 70´s
until now. Currently, Tanzania has 19 urban areas with local authority status, that is, 9
town councils, 6 municipalities, and 4 city councils. On top of that, 66 township
authorities are administered by district councils (the municipalities of Mwanza,
Mbeya, and Arusha acquired a city status since July 2005). The majority of the people
(about 80% of urban population and generally 90% of the people in the country) use
on site disposal (decentralized) systems mostly pit latrines. In urban areas 10% of
middle and upper class populations use septic tanks, 5% of the affluent class are
connected to the sewerage systems and 5% of the poorest have no proper sanitation at
all (Mwaikyekule, 1999; Rukiko, 1999; Uronu, 1999). Despite that Tanzania is
presently experiencing rapid population and economic growth especially in the urban
centres, the provision of services, including wastewater collection, treatment and
disposal has however not kept pace with these developments.
The level of water pollution is high, as is evident from findings on one of Dar es
Salaam’s rivers, the Msimbazi, known for its clean water during the colonial times
and currently the recipient of many industrial and domestic wastewaters. The coliform
count in the Msimbazi at entry into Dar es Salaam (at Kisarawe) is 75–100 per 100 ml
of water, relatively good-quality water. When the Msimbazi leaves Dar es Salaam (at
Salander bridge), the coliform count is between 2.5 x 105 and 4 x 105 per 100 ml of
water, indicating heavy contamination. This is more than 1 000 times the coliform
count considered safe to swimm in. The lower stretch of the Msimbazi is, therefore,
an open sewer. Causes of this pollution are varied, but they include the city’s
excessive reliance on on-site modes of sanitation and its tendency to discharge raw
domestic and industrial effluents into rivers and natural channels (Yhdegho 1992).
In the city of Dar es Salaam, wastewater ends up in the Indian Ocean directly and
indirectly, treated and untreated, from both point and non point sources. The point
sources include the effluents from waste stabilisation ponds and a sea outfall (Meghji
and Merinyo, 1989).
The demand for water and sanitation (including wastewater treatment) is increasing
with increased urbanization and population (figure 1). Aging and poorly maintained
plants as well as inadequate and unreliable water sources, unreliable power supply,
low tariffs and illegal connections characterise the existing supply (Kironde, 2000). In
general, inadequate human, institutional and financial resources adds to the problems
of water supply and wastewater treatment in Tanzania; that does not correspond with
an increase in population in urban areas (figure 1). Outbreaks of cholera, typhus,
malaria and diarrhoea are common especially during the rain season. So basically
water supply and sanitation should be handled in an integrated manner. Currently
there are emerging strategies and regulations (NEMC) to safeguard the environment
from pollution and therefore protection of public health and mitigation of
environmental pollution.
8
Figure 1. Map of Dar es Salaam city, showing location of treatment cites and city
expansion
since
1947
(Briggs
and
Mwamfupe;
www.iio.org.uk/gallery/africa/zambia/DSM_urban.pdf)
WSPs have been plagued with many problems from design and operational related
ones, although they are effective for domestic, municipal and industrial wastewater
treatment. The dependency on weather conditions makes empirical design equations
derived elsewhere, inapplicable in other locations (Middlebrooks, 1987). Frequently,
the quality of the effluents does not consistently meet effluent quality requirements
for discharge or reuse because their performance vary with climatic conditions and
design.
9
Figure 2. (a) Primary Facultative Pond and (b) Secondary Facultative pond (note
bypass of untreated wastewater) to WSP treatment site in Mikocheni; this system
receives domestic and industrial wastewater
1.1 Phosphorus Removal in Waste Stabilisation Ponds
The nutrient removal processes taking place in the ponds include biological processes,
in which diversified groups of organisms are responsible i.e. bacteria, fungi and algae
(Hosetti and Frost. 1998). The origin of these microorganisms could be from the air,
soil, and animals living near the system and the wastewater itself.
Phosphorus removal is by adsorption to sediment, plant uptake and bacteria uptake
(Crites and Tchobanoglous, 1998; Bitton, 1999). Phosphorus removal has been
recorded up to 26% in arid climate in Marrakech, Morocco while in Catalonia, Spain
removal of up to 48% has been recorded. A first order plug flow predicts a 45% P
removal for a 90% BOD removal but this is mainly attributed to sediment adsorption
(Mara et al., 1992; Crites and Tchobanoglous, 1998; Bitton, 1999; Boussaid et al.,
2000; Garcia et al., 2000).
At low temperatures, nutrient removal of 50% - 70% is difficult to achieve. At higher
temperature, with well designed WSP series which include shallow maturation ponds,
better P removal can be achieved, but even then phosphorus is over 1 mg/l
respectively (Mara et al., 1996; Pearson et al., 1996). Generally, WSPs are
characterised by low phosphorus removal efficiencies.
1.2 Improved Phosphorus Removal in other Treatment Systems
Phosphorus removal has been improved in activated sludge treatment systems termed
as enhanced biological phosphorus removal (EBPR). EBPR has been considered to
depend on polyphosphate accumulating bacteria (PAOs) that are able to accumulate
polyphosphate, by storing more phosphorus than they need for growth, i.e. “luxury
uptake”. The ability to store the polyphosphate depends on the availability of volatile
fatty acids (VFAs), which are generated through the hydrolysis and fermentation by
non-polyphosphate accumulating organisms of the complex organic matter present in
the wastewater or introduced externally. The bacteria responsible for EBPR seem to
be diverse and to consist of several phylogenetic groups. (Kortstee, 1994;
Christensson 1997; Mino et al, 1998; Santos et al., 1999; Crocetti et al., 2000; Mino,
2000).
10
In activated sludge (AS), EBPR has been achieved by introducing an
anaerobic/anoxic phase as the initial stage to the conventional aerobic process to
enhance hydrolysis and fermentation; this has increased microbial P removal. Studies
on EBPR have shown that substrates for metabolic processes are VFAs and low
molecular organic carbon compounds. It is believed that VFAs are produced by nonPAOs, thereby creating a population structure comprising of PAOs responsible for P
removal and non-PAOs responsible for VFA production. However no difference in
microbial population have been observed between conventional and non-conventional
AS treatment plants, leading to a poor understanding of the microbial population of P
removing bacteria. In addition, the influence of different organic substrates other than
VFAs has not been fully investigated. Therefore, there is need to test the influence of
different carbon compounds and bacteria on P uptake, purely aerobic P uptake as
opposed to the anaerobic/aerobic sequencing in activated sludge.
The general treatment efficiency of wastewater stabilisation ponds in Tanzania is poor
(figure 1.2). In addition, nutrient removal needs to be addressed since discharge limits
will be getting more and more stringent.
The enhanced biological phosphorus removal (EBPR) biochemical models that have
been proposed for phosphorus removal cannot fully account for the total phosphorus
removal in wastewater treatment plants. The methods used to isolate phosphorusremoving bacteria seem to favour different genera, depending on the method and on
the enrichment culture. Thus the mechanism of microbial ecological selection for P
removing bacteria in EBPR processes is poorly understood. The microbial diversity
and biochemical processes responsible for phosphorus removal need to be studied, for
optimisation of the biology of wastewater treatment. It is necessary to characterise in
depth the microbial activity contributing to the global removal processes to
understand the EBPR mechanisms.
Understanding the processes responsible for phosphorus removal in wastewater
treatment is important for enhancement of biological phosphorus removal. Different
bacteria have been proposed to be candidates for EBPR. This suggests that P
removing bacteria may be phylogenetically and taxonomically diverse. This is
supported by the fact that pure cultures have not given any definitive and conclusive
information about the microbiology and biochemistry of EBPR, leading to a poor
understanding of the microbial ecological selection of P removing bacteria. The aim of
this study was to establish the capacity of microorganisms to remove phosphorus from waste
stabilisation ponds.
The main objectives of this study were to:a. Establish the treatment performance of wastewater stabilisation ponds in a
tropical climate and evaluate the level of P removal.
b. Establish the capacity for biological phosphorus uptake by pure cultures of
microorganisms isolated from wastewater stabilisation ponds treatment
To meet these objectives, the performance of a waste stabilisation pond system was
evaluated. Bacteria were isolated from the same stabilisation pond system and from
an activated sludge wastewater treatment system with enhanced biological
phosphorus removal, in Sweden, for laboratory investigation of their capacity to take
up phosphorus.
11
1.3 Scope of this thesis
This thesis is presented in 5 chapters. Chapter 1 is an introduction. Chapter 2 and 3
gives an overview of the background information on stabilisation ponds and enhanced
biological phosphorus removal. Chapter 4 describes (i) the investigation on
performance of wastewater stabilisation ponds in a tropical climate, Dar es Salaam,
Tanzania. (ii) The investigation carried out to compare P uptake by isolated strains
from a waste stabilisation pond system (same system) (iii)The investigation of
microbial phosphorus uptake, by a pure strain isolated from a biological activated
sludge treatment plant in Sweden. Chapter 5 gives some conclusions, followed by
acknowledgement..
12
2 Treatment Processes
The main components in municipal wastewater are organic matter, nutrients such as
nitrogen, and phosphorus, suspended solids, metals and pathogenic bacteria,
originating from kitchen food waste, human excreta, detergents and that originating
from industry. Discharge of organic matter in wastewater to recipient water sources
leads to a consumption of oxygen resulting in oxygen deficiency whereas nutrients,
like phosphorus and nitrogen result in eutrophication in aquatic ecosystems.
Phosphorus is the limiting nutrient in eutrophication in water systems, which makes
its removal from wastewater necessary before discharge. Nitrogen in the form of
nitrate is a cause of methemoglobinemia in infants and susceptible populations. In the
form of ammonia, nitrogen is toxic to fish, and exerts oxygen demand in receiving
waters by the nitrifiers (Bitton, 1999). Therefore wastewater treatment aims at
reducing the organic matter and nutrients as far as possible, remove suspended matter,
reduce pathogen content and toxic chemicals. The quality of the treated wastewater
may depend on the processes employed, volume and condition of the receiving water
and its ability to dilute the wastewater (Bitton, 1999).
Biological wastewater treatment is advantageous to chemical treatment because
energy consumption and sludge production is reduced, hence reduced cost. To be able
to maximise biological wastewater treatment, establishing the biological diversity and
processes responsible for wastewater treatment, especially nutrient removal and their
metabolic potential is important. The common biological treatment systems used for
domestic wastewater are activated sludge, ponds/lagoons and aerated stabilisation
ponds/lagoons. The aerated ponds/lagoons are deeper and more heavily loaded
organically than unaerated ones. The activated sludge and lagoons are
aerobic/anaerobic suspended growth processes. The less common treatment plants are
the ones with attached growth treatment processes, including the trickling filter,
rotating biological contactors, packed bed, Up Flow Anaerobic Sludge Blanket
reactors and constructed wetlands (Crites and Tchobanoglous, 1998; Bitton, 1999).
In Tanzania, it has been recognised that wastewater treatment by stabilisation ponds is
the preferred choice because they require minimum costs for both construction and
management and can be efficient when well maintained. It has been shown that WSPs
can meet the required discharge standards when properly designed and maintained.
On the other hand, interrelationships that bond microscopic fauna and flora with the
chemistry of their circumstances can be manipulated to ensure effective treatment of
wastewater (Mara et al., 1992; Bitton, 1999; Crites and Tchobanoglous, 1998; Hosetti
and Frost, 1998; Boussaid et al., 2000; Garcia et al., 2000). However, the
conventional design of waste stabilisation ponds (WSP) do not consider some of the
ecological processes, which take place in the pond, instead the design parameters used
are organic matter and pathogen removal, nutrients have been disregarded (Mara et
al., 1992; Boussaid et al., 2000). Nutrients could be integrated as design parameters,
so that the desired level of nutrient removal can be achieved through uptake by the
living fauna and flora. WSPs have been plagued with many problems from design
and operational related ones, although they are effective for domestic, municipal and
industrial wastewater treatment. The quality of the effluents does not consistently
meet effluent quality requirements for discharge or reuse because their performance
vary with climatic conditions. If land availability and climatic conditions permit, then
WSPs provide cost effectiveness in investment, operation, maintenance and energy
cost (Escalante et al., 2000; Garcia et al., 2000; Pratap et al., 2000; Oakly et al.,
13
2000). Dependency on weather conditions may render empirical design equations
derived elsewhere inapplicable in other locations.
2.1 Types of Waste Stabilisation Ponds
Waste stabilisation ponds (WSPs) are shallow man made basins into which
wastewater flows through. After a retention time of many days, a well-treated effluent
is discharged (Mara et al., 1992). They are a good alternative in countries where land
and sunshine is plentiful. Saqqar and Pescod (1995) recorded BOD, COD and SS
removal efficiencies of 53%, 53% and 74% respectively and performances increased
with an increase in temperature. WSPs can withstand shock loads without significant
loss of efficiency in terms of BOD removal and bacterial pathogen removal, although
nutrient removal by maturation ponds may be reduced (Pearson et al., 1996). WSPs
have been classified according to the availability of oxygen for the stabilisation
process as anaerobic, facultative and aerobic (Mara et al., 1992; Crites and
Tchobanoglous, 1998; Hosetti and Frost, 1998; Bitton, 1999). There are three types of
ponds; anaerobic ponds, facultative ponds and maturation/oxidation ponds
2.1.1 Anaerobic ponds
These serve as a pre-treatment step for high BOD organic loading with high protein,
fat and suspended solids content, they more or less function like open septic tanks.
Complex organic materials, e.g. polysaccharides, fats etc
Hydrolytic bacteria
Low molecular weight products i.e. Monomers, sugars e.g. glucose, amino
acids, fatty acids etc
Fermentative acidogenic bacteria
Volatile fatty acids e.g. Organic acids, alcohols, ketones e.g. ethanol,
propionic and butyric acid etc
Acetogenic bacteria
Acetate, Carbon Dioxide and Hydrogen
Methanogens
Methane (CH4)
CO2, S2- H2
Figure 3. Metabolic microbial groups involved in anaerobic digestion of wastes
(Bitton, 1999; Pescod, 1996).
They are normally 2-5 m deep sometimes up to 9 m with long detention times of 20 –
50 days, (Bitton, 1999).
14
Where temperatures are higher than 20 0C, they have shorter retention times of 1 - 5
days and can achieve 60% to 75% BOD removal. A consortium of microorganisms,
mostly bacteria, is involved in the transformation of complex materials into simple
molecules.
Anaerobic biodegradation yields methane through methanogenesis, carbon dioxide
(CO2) and other gases such as hydrogen sulphide (H2S). Four major groups operate in
a synergistic relationship as summarised in Figure 3. The important stages are the
hydrolysis, fermentation, acetogenesis and methanogenesis. Anaerobic ponds require
temperatures above 10 0C, and so work very well in warm climate (Mara et al., 1992;
Crites and Tchobanoglous, 1998; Hosetti and Frost, 1998; Bitton, 1999).
2.1.2 Facultative Ponds
Facultative ponds can be primary or secondary. They are 1 – 3 m deep, designed for
low BOD loading of 100 – 400 kg /ha/day and a retention time of 5 – 50 days. The
biological wastewater treatment processes are carried out mainly by heterotrophic and
autotrophic bacteria, algae and zooplankton, in a mixture of anaerobic, aerobic and
facultative conditions. The biological activity in the facultative ponds can be
classified into photic, heterotrophic and anaerobic zones, figure 4 (Mara et a., 1992;
Crites and Tchobanoglous, 1998; Hosetti and Frost, 1998; Bitton, 1999).
i. The Photic Zone
This zone is aerobic, mainly dominated by algae, which are involved in nitrogen
fixation, nutrient uptake through photosynthesis and therefore produce oxygen for the
heterotrophic
bacteria.
Figure 4: Microbial activity in facultative ponds (Bitton, 1999)
Photosynthesis leads to an increase in pH, if alkalinity is low, and this favours
phosphorus precipitation as calcium phosphate, and ammonium ion may be lost as
15
ammonia (Bitton, 1999). Good wind mixing ensures a more uniform distribution of
BOD, DO, bacteria and algae and hence a better degree of waste stabilisation (Mara et
al, 1992).
The absence of wind mixing leads to stratification of the pond into an epilimnion, and
hypolimnion that are divided by a thermocline, and the algae tends to stratify into a
narrow band (about 20 cm thick) during the day and causes large fluctuations in
effluent BOD quality as the layer moves up and down through the top 50 cm, in
response to changes in the incident light intensity. Removal of hydrogen sulphide is
possible by photosynthetic sulphur bacteria that use H2S as electron donor instead of
H2O, which is important for reduction of odour (Mara et al., 1992; Bitton, 1999).
ii.
Heterotrophic activity
Heterotrophic bacteria are the principal microbial agents for degradation of organic
matter. The aerobic activity produces CO2 and micronutrients for algae growth and
utilises O2 produced by algae by wind mixing, as electron acceptor. The anaerobic
activity results in the production of gases such as methane (through methanogenesis),
hydrogen sulphide, carbon dioxide and nitrogen (through denitrification).
iii The anaerobic zone
The degradation in this zone is similar to that described in section 2.1.1
2.1.3. Maturation/Tertially Ponds
A series of maturation ponds receives effluent from facultative ponds, the size and
number is governed by the bacteriological quality of the final effluent (Mara et al,
1992). These are 1 – 2 m deep and has a significant role in pathogen and nutrient
removal but less significant in BOD removal (Bitton, 1999; Mara et al, 1992). Oxygen
is typically supplied by surface reaeration and shows no vertical biological and
physiological stratification (Mara et al, 1992).
2.2 Pathogen and Parasite Removal
Bacterial dye-off (pathogen removal) is dependent on long retention times, high pH
and DO generated by algal photosynthesis, necessary for photo-oxidative damage,
which in turn is promoted by high light intensity. Zooplankton predation is also
important. Some removal is by sedimentation in anaerobic ponds. A 100% removal
has been recorded in North East Brazil (Arridge et al., 1995). The protozoan cysts and
helminthes eggs are removed by sedimentation mainly in anaerobic and facultative
ponds. Removal of 100% was achieved in Kenyan WSP with overall retention time
ranging from 15 – 62 days (Grimason et al., 1996a; Grimason et al., 1996b; Bitton,
1999).
2.3 Nutrient Removal
The nutrient removal processes taking place in the ponds are purely biological
processes, in which diversified groups of organisms are responsible i.e. bacteria, fungi
and algae (Hosetti and Frost. 1998). The origin of these microorganisms could be
from the air, soil, and animals living near the system and the wastewater itself. In
waste stabilisation ponds, nitrogen removal is both assimilative and dissimilative, that
is by algal uptake, and through nitrification and leading to denitrification and
ammonification (Crites and Tchobanoglous, 1998; Bitton, 1999). Nitrogen removal in
wastewater stabilisation ponds is still not very well understood with respect to
16
denitrification, (the dissimilatory nitrate reduction), although Bitton, (1999) attributes
partial removal by this process. Otherwise the nitrogen cycle, ammonia volatilisation
and algae uptake are well known processes in stabilisation ponds. According to Mara
et al., (1992) there is little evidence for nitrification and denitrification (unless the
wastewater has high concentrations of nitrate). It is believed that the nitrifying
populations are very low in WSP, may be due to the absence of physical attachments.
On the other hand nitrogen removal by WSPs of 40 – 80% has been recorded (Silva et
al, 1995; Crites and Tchobanoglous, 1998; Bitton, 1999; Masudi, 1999).
Phosphorus occurs in natural waters and in wastewaters almost solely as phosphates.
These phosphates include organic phosphate, polyphosphate (particulate P) and
orthophosphate (inorganic P). Orthophosphates are readily utilized by aquatic
organisms. Some organisms may store excess phosphorus in the form of
polyphosphates. At the same time, some phosphate is continually lost in the sediments
where it is locked up in insoluble precipitates.
Phosphorus removal is by adsorption to sediment, plant uptake and bacteria uptake
(Crites and Tchobanoglous, 1998; Bitton, 1999). A first order plug flow predicts a
45% P removal for a 90% BOD removal but this is mainly attributed to sediment
adsorption (Mara et al., 1992; Crites and Tchobanoglous, 1998; Bitton, 1999;
Boussaid et al., 2000; Garcia et al., 2000).
2.4 Factors affecting the Stabilisation Process, growth and activities
in the ponds
Natural purification of wastewater by stabilisation ponds begins immediately after
WW enters a pond. Settleable solids, suspended solids and colloidal particles either
settles to the bottom of the pond by gravity or may be precipitated by action of soluble
salts due to the rise in pH. Soluble materials are oxidised by bacteria. Settled organic
matter is converted to inert residue and soluble substances diffuse into the bulk of the
water above, where further decomposition is carried out by bacteria. Degradation of
dead bacterial biomass with release of ammonia promotes algae growth.
The stabilisation processes in WSPs is dependent on various factors. These include
types of ponds (anaerobic, facultative or maturation). The type of ponds is generally
determined by pond depth. In deeper ponds, light cannot penetrate the water because
of the excessive water depth; algae photosynthesis is therefore inhibited, rendering the
microbiological activity to be more heterotrophic than photosynthetic. Reduction of
pathogenic organisms may be minimized as a result of inadequate exposure to direct
sunlight. On the other hand maturation ponds are shallow (0.5 -1 m) and are good in
destruction of faecal bacteria.
Detention time is another important factor. Algae require sufficient time to grow and
multiply through binary fission. Each pond cell (compartment) should provide a
minimum retention time to avoid premature cell washout (Arceivala, 1986). Detention
time will also influence the achievement of the desired level of coliform and organic
matter removal. Sufficiently long detention times (dependent on type of pond) should
17
be provided to allow parasites (faecal and helminths eggs) to die off and sink to the
bottom and to allow sufficient nutrient removal and organic matter transformations.
Equation
λ = 60.3 (1.099)T
λ = 20T - 120
λ = 20T - 60
λ = 350(1.107 – 0.002T)T25
Valid for T > 100 C
λ = 100 for T < 100 C
λ = 10T for 10<T>200 C
λ = 50(1.072)T for T > 200
C
Source
McGarry and Pescod (1970)
Mara (1974)
Arthur (1983)
Mara (1987)
λ for 260 C
700
400
460
370
Mara and Pearson (1987). Based on
meeting recommendations and
assumption
that
degradation 305
doubles with every 100 C rise in
temperature
Table 1: Predicted organic loading rates (kg/ha/day), for Dar es Salaam by different
loading rate equations Mayo, (1995).
Allowable organic loading rate equations for the design of facultative ponds are
temperature dependent. Depending on the equation, the permissible organic loading
rates (λ) may vary from 305 to 700 kg/ha/d for the same design temperature for Dar
es Salaam. For example, the minimum mean monthly water temperature in Dar es
Salaam is 260 C and the predicted loading rates are shown in table 1 (Mayo, 1995).
Another important factor that influences pond performance is the design equations
which are temperature dependent and assumes that BOD reduction follows first order
kinetics and ponds are completely mixed. Temperature influences the organism yield
coefficient, die-off rates, availability of nutrients and dominant species, all of which
are influencing the organic matter decomposition (Friedman and Shroeder, 1972).
Temperature does not only influence the degradation of soluble matter in the
supernatant, but also the settled sludge that is most often not considered during the
design (Mayo, 1995). At temperatures below 170 C fermentation may be neglible, and
above 230 C it is so intense that sludge may float to the surface (Marais, 1974).
Light intensity has a great influence on photosynthesis, which in turn can influence
the stabilisation process. As the concentration of algae increases, light penetration
decreases so that the phytoplankton becomes self-shading, and thereby restricting
photosynthesis in the lower layers.
2.4 Wastewater Stabilisation Ponds in Dar es Salaam, Tanzania
In Dar es Salaam, the biggest city in the country, less than 5% of the population (of
about 3 million people) is connected to the sewer system, grouped into 11 systems
and supported by 17 pumping stations. This system covers the central areas, some
industrial areas, and a few outlying residential areas. Effluent is discharged into
oxidation ponds and local watercourses and directly into the ocean. The central areas
discharge directly into a sea outfall, where the end of the pipe lies in less than 2 m of
water. Some of the oxidation ponds no longer operate as expected as a result of a lack
of maintenance and overloading, and sometimes raw sewage is discharges into the
surface drainage system. About 80% of the 2.2 million people living outside the
central areas have access to on-site facilities — 70% of these use pit latrines; 30%,
18
septic tanks (DCC). Rough calculations suggest that about 94% of the industries in
Dar es Salaam are connected to a piped-water sewerage system (Chaggu, 2004).
About 6% are connected to septic tanks and soakaways. The major mode of treatment
is through oxidization/stabilization ponds, and the treated wastewater is then usually
discharged into rivers and ultimately into the ocean. Some of these WSPs receive
municipal, industrial and/or septage.
Figure 5: (a)At inlet to the UDSM WSPs, silt and sludge has accumulated over the
years and grass is growing at the entrance to the primary facultative pond (b)Influent
to the Mabibo/Ubungo WSPs (mostly receiving industrial WW), they no longer
function. Wastewater flows in a stream to the Msimbazi river.
Various studies suggest (Haskoning and M-Konsult 1989) that industrial wastewater
is not pre-treated before it is discharged into oxidization ponds.
In the city of Dar es Salaam, wastewater ends up in the Indian Ocean directly and
indirectly, treated and untreated, from both point and non point sources. The point
sources include the effluents from waste stabilisation ponds (Meghji and Merinyo,
1989). The present stabilisation ponds were designed for removal of organic matter
(BOD) and pathogens, while removal of nutrients and other toxic pollutants was not
considered, (Mara et al., 1992). A non-exhaustive treatment efficiency assessment
made in 1988 concluded that the low performance was due to operational constraints
(Meghji and Merinyo, 1990). From BOD removal results in WSPs in Dar es Salaam,
Mayo (1996) reported deviations of performance and concluded to be a result of
assumptions made during design.
There are 7 WSP sites in Dar es Salaam. Each site handles wastewater from a
population of about 2000 – 6000 people. Two of these receive domestic wastewater,
industrial wastewater and sludge from septic tanks, while the rest receive domestic
wastewater and sludge from septic tanks except for the WSPs at the University of Dar
es Salaam. Septic tank sludge is known to contain very high concentrations of COD,
BOD, nutrients, metals and total solids; this affects the performance of the
stabilisation ponds. These WSPs do not function well due to adoption of design
formulas that were not meant for hot climates (Mara et al, 1992).
19
3. Biology of Phosphorus Removal
Microorganisms play two roles in biological phosphorus removal: microorganismmediated chemical precipitation and microorganism-mediated assimilation and
accumulation. At low pH in oxic conditions, microbial activity solubilizes phosphate
compounds and as the pH increases, phosphates precipitate and are incorporated in
the sludge. Phosphorus precipitation can also be induced by: the increase in phosphate
concentration resulting from phosphorus release, from the polyphosphate pool under
anaerobic conditions and denitrification as the denitrification process produces
alkalinity (Bitton, 1999).
However, the common theory today is that biological phosphorus removal mediated
by microoganisms uptake is carried out by polyphosphate accumulating bacteria
(PAO) in the anaerobic-aerobic process (Ubakata and Takii, 1994; Petersen et al,
1998; Bond et al., 1999; Santos et al, 1999; Mino, 2000). This process exposes the
microorganisms to alternate carbon rich anaerobic environment and carbon poor
aerobic environments and thereby inducing the metabolic characteristics of PAO. The
substrate for the anaerobic metabolic process is the volatile fatty acids produced by
the fermentative bacteria from sugars and low molecular organic compounds, which
are converted to and stored as polyhydroxyalkoanates (PHA) and the subsequent
hydrolysis of polyphosphate and its release.
Intracellular accumulation of polyphosphate P takes place aerobically and energy is
produced by the oxidation of the intracellular stored organic products. PAO maintain
the redox balance in the anaerobic uptake of various organic substrates by storing
PHA (Christensson, 1997; Crites and Tchobanoglous, 1998; Petersen et al, 1998;
Sudiana et al., 1999; Mino, 2000). The models emphasise that alternating anaerobic
and aerobic conditions is necessary to induce EBPR and the fermentative bacteria.
Quinone profiling showed similar patterns for conventional EBPR processes
indicating that introducing the anaerobic stage into a fully aerobic conventional
process does not significantly shift the population. Similarly no significant difference
in respiratory quinone profiling and microbial structure as measured by FISH was
observed in glucose and acetate fed P rich and poor sludges (Sudiana et al., 1999;
Mino, 2000).
Different bacteria have been proposed to be candidates for enhanced biological
phosphorus removal. These include Acinetobacter, Lampropedia, Microlunatus
phosphovorus, Rastonia (Alcaligens), Rhodocyclus spp, Pseudomonus spp and
Aeromonus spp, Propionibacter pelophilus, Klebsiella oxytoca, Aeromonus
hydrophyla, Agrobacterium tumefaciens, Aquaspirillum dispar, some gram-positive
bacteria and high G+C content bacteria, like Actinobacteria (Bitton, 1999; Bond et
al., 1999; Merzouki et al., 1999; Nielsen et al., 1999; Santos et al., 1999; Crocetti et
al., 2000; Kortstee et al., 2000; Liu et al., 2000; Mino, 2000). This suggests that P
removing bacteria are phylogenetically and taxonomically diverse. Appeldoorn et al.,
(1994) reported that 80% of all aerobic bacteria were able to accumulate polyP.
The few pure cultures studied for P uptake have not given any definitive and
conclusive information about the microbiology and biochemistry of EBPR, leading to
a poor understanding of the microbial ecological selection of P removing bacteria.
Some strains of Acinetobacter isolated from an EBPR sludge were able to accumulate
polyP aerobically, but could not polymerise PHA in the anaerobic stage. Microlunatus
20
phosphovorus does not take up acetate and nor accumulate PHA anaerobically, only
2.7% of PAO detected were represented by these bacteria. Lampropedia spp, although
it accumulates poly P and stores PHA with subsequent release of P, it has a
morphology that is has a unique sheet like cell arrangement, which is not common in
EBPR processes (Mino, 2000). Hesselman et al., (1999) found Rhodocyclus
dominating by 81% in an EBPR community while Bond et al., (1995) found the same
Rhodocyclus occupying 14% of the total isolated clones from high phosphate removal
performance sludge. The difference in dominance raises more questions as far as
EBPR bacterial taxonomy is concerned.
Non-culture methods indicate a high genotypic and phylogenetic diversity of
microoganisms in EBPR. Fluorescent in situ hybridisation (FISH) technique with
group specific oligonucleotide probes targeting rRNA, showed an EBPR sludge
containing the alpha-, beta- and gamma- subclasses of proteobacteria, the cytophaga
group and Gram- positive bacteria with high G+C DNA contents (Mino, 2000).
An enzyme responsible for polyphosphate synthesis and build-up, polyphosphate
kinase (ppK) has been purified from diverse bacteria like Vibrio cholerae,
Pseudomonas aeruginosa, Acinetobacter sp., Escherichia coli, Klebsiella aerogenes,
showing a high degree of ppK conservation and polyP prominence in many organisms
(Kornberg, 1995; Barker and Dold, 1996; Christensson, 1997; Geiβdörfer et al., 1998;
Kornberg et al., 1999; Meinhold et al., 1999; Merzouki et al., 1999; Trelstad et al.,
1999).
Many studies have focused on sludge that had PHA and polyP accumulating
properties, i. e mainly on sludge that was stained or analysed for polyP and PHA
granules by use of acetate metabolism. Additional external carbon sources have been
used for enhanced biological phosphorus removal. Carbon sources that have
dominated in EBPR are the volatile fatty acids (VFAs), either generated internally or
externally, to be taken up by PAOs during the anaerobic retention time. The internal
VFA generation is supposedly by non-PAO from compounds that cannot be taken up
by PAOs (Mino et al, 1998).
The amount and form of carbon source play essential roles in the EBPR, which
applies for different VFAs as well. Different P uptake and release rates were observed
when acetic, propionic and butyric, acids were used as carbon source at different
concentrations, in which the best P uptake rates were obtained by Acinetobacter lwoffi
and Acinetobacter calcoaceticus with butyric acid as carbon source. M. phosphovorus
was able to ferment glucose to acetate, but could not grow under anaerobic
conditions. Carucci et al., (1999) observed that peptone, glucose and acetate can
undergo anaerobic P uptake through biochemical pathways other than the traditional
PAO like metabolism. Carbon source types, bacterial species, and different P
concentrations, may have an effect on composition and abundance of P removing
microbial populations (Christenssen, 1997; Rustrian et al., 1999; Santos et al., 1999;
Liu et al., 2000).
3.1 EBPR process configurations
There are three main configurations that are commonly used for EBPR. The
Phoredox, Phostrip and University of Cape Town processes (Christensson, 1997). The
Phoredox process uses a separate anaerobic reactor situated in the main stream ahead
21
of the aerated activated sludge basin. When the anaerobic and aerobic stages are
divided into a number of equally sized completely mixed compartments, then these
systems are referred to as A/O systems. The A/O systems can be operated at short
SRT and high organic loading, leading to high sludge production and phosphorus
removal rates, nitrification does not take place. Where as in the Phostrip process the
anaerobic reactor is situated in a side stream and is a combination of chemical and
biological methods for phosphorus removal. When combined nitrogen and
phosphorus removal are implemented, a range of different configurations are
available. They are all based on anaerobic, anoxic and aerobic compartments. One of
these configurations is the UCT process. In this process, sludge from the secondary
clarifier is recycled to the anoxic compartment and not to the anaerobic stage in order
to reduce the nitrate load on the latter. Instead mixed liquid is recirculated from the
end of the anoxic stage to the anaerobic basin to bring the PAOs to the anaerobic
reactor. At the end of the anoxic stage, the nitrate level is kept low by denitrification,
thus avoiding the recirculation of nitrate to the anaerobic reactor. In comparison with
the A/O process, a longer SRT (13-25 days) and a higher ratio of BOD to total
phosphorus in the fresh sewage are required for the UCT process (Sedlak, 1991)
Öresundverket, the municipal wastewater treatment plant in Helsingborg, Sweden is
operating on the UCT process configuration. The highly loaded AS process was
extended into an AS system for nitrogen and phosphorus removal, with a design
capacity of 3250 m3/h, 15000 kg/d BOD7, 2700 kg/d nitrogen and 490 kg/d
phosphorus. The plant comprises of four separate trains and one or two of these trains
have been operated with EBPR, while the others have been operated with chemical
phosphorus removal (Christensson, 1997).
22
4 Present investigations
In the present studies, pond performance was done by wastewater sampling once in
every two weeks, between 9 – 10.30 am, over a period of six months from February to
August 2001. Analytical procedures were carried out according to the Standard
Methods for the examination of Water and Wastewater (APHA/AWWA/WPCF,
1995). Parameters analysed were chlorophyll, dissolved oxygen (DO), pH, ammonia
nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, inorganic and total
phosphorus, BOD, conductivity, total dissolved solids (TDS), total and faecal
coliforms.
For the P uptake experiments (Paper II and III), strains A103 and D404 were isolated
from the WSPs at the University of Dar es Salaam, Tanzania. Strain L6 was isolated
from sludge obtained from an aerobic biological train, with reported enhanced
biological phosphorus removal, of an activated wastewater treatment plant at
Oresundsverket in Helsinborg, South of Sweden. The isolation was isolated by serial
dilution and plating on nutrient agar plates until pure cultures were obtained. Minimal
media containing glucose, succinate or acetate were designed. Growth tests were
conducted by measuring optical density (OD600). Colony forming units were
determined alongside absorbance (for L6). Calculation of uptake was done by
extrapolation from a regression on graphs for P uptake as a function of optical density
(and as a function of CFU for L6) so that phosphorus uptake was determined per unit
absorbance of biomass (and per cell for L6).
4.1 University of Dar es Salaam Stabilisation Ponds
The University of Dar es Salaam (UDSM) Stabilisation Ponds consists of seven
ponds, one primary facultative pond, which discharges to the first two facultative
ponds in parallel, then to another two facultative ponds also in parallel and finally to
two maturation ponds arranged in parallel (figure 6).
The effluent from the maturation ponds is discharged into a nearby stream. The
system was designed to serve 5000 people, but now serves more than 10,000. The
wastewater flow is about 840 m3/d (Mayo, 1995), mainly of domestic origin and from
the laboratories, workshops and a health centre.
The primary facultative pond has accumulated silt and sludge at the inlet, such that
grass is growing within that miniature island (figure 2.5a).
Table 2: Pond specifications (Kayombo et al, 2000)
PrFP
FP 1 & 2
MT
Pond depth (m)
HRT (days)
1.92
8.3
1.7
7.3
1.61
4.3
Water depth (m)
Pond area (m2)
1.54
4,326
1.2 and 1.02
2,914
0.54
2,108
On occasions, sludge is seen floating on the sides of the PrFP probably due to
fermentation arising from high temperatures (Marais, 1974). Otherwise this system
probably performs better than any of the systems in Dar es Salaam. These WSPs were
23
desludged 20 years ago, this is evidenced by shallow water columns measured in 2000
(Table 2), which is now lower than originally designed.
The second secondary facultative ponds were short circuited during construction; this
has an effect on the performance of these cells as indicated by the reduced total and
faecal coliform decay rate constants (table 3) and algal growth rate as indicated by
chlorophyll (figure 7). Sludge has accumulated much more in the secondary
facultative ponds and in the maturation ponds as indicated by the water depth (Table
2).
(a)
Outlet
Outlet
3
2
1
3
2
1
PrFP – Primary Facultative Ponds
1. 1st Secondary Facultative Ponds
PrFP
2. 2nd Secondary Facultative Ponds
3. Maturation Ponds
(b)
Inlet
Figure 6: (a) The WSP system at the University of Dar es Salaam, (b) Schematic
Diagram of the Pond Systems at UDSM (Mayo, 1995)
24
There is a healthy algal growth from the first secondary facultative ponds to the
maturation ponds. This is shown by the increase in DO, chlorophyll and pH (Paper I).
Fish are thriving in the maturation ponds. The overall organic matter and nutrient
removal (Paper I), was not sufficient. BOD removal was 27%. However there is an
increase in nitrite and nitrate indicating nitrification taking place in the ponds. Picot et
al., (2005) reported nitrification in Mèze (France) stabilisation ponds. Total nitrogen
remained constant. But ortho-P removal was significant in the PrFP (figure 8) than in
the other cells (except in the MT pond), in this step alone the removal was about 18%.
Since there was hardly any algal growth in the PrFP, this significant removal is
attributed to the heterotrophic activity; the pH in this cell was neutral. Ouazzani et al,
(1997) reported significant P removal in the primary pond.
160
6
5
4
3
a
Total Colifrom
Feacal Coliform
b
Inf
140
Chlorophyll (mg/l)
Log10 (TC & FC)
7
120
Chlorophyll
100
80
60
40
c
20
PrFP FP1 FP2 MT
Inf PrFP FP1 FP2 MT
Pond Series
Pond Series
Ortho-P (mg/l)
Figure 7. (a) Short circuiting in the second facultative ponds at the UDSM ponds (b)
and (c) effect of short circuiting on performance (coliform removal and algae growth
in second facultative ponds) in the respective ponds.
7
6,8
6,6
6,4
6,2
6
5,8
5,6
5,4
5,2
5
Inf
PrFP
FP1
FP2
MT
Pond Series
Figure 8: Soluble phosphorus removal trend in the UDSM pond series.
The P removal was once more significant in the maturation pond, probably due to
precipitation as the pH in these ponds was an average of 9.21. Picot et al., (2005)
reported that most of P removal took place in polishing (maturation) ponds. This trend
25
indicates that algae play a minor roll in the removal of phosphorus, as there is hardly
any reduction in the secondary cells. The overall ortho-P removal was about 20%.
However, in the present system, there is a significant nutrient recycling due to lack of
desludging.
Total and faecal coliform removal was by 4 log units in each case (6 to 2 for FC and 7
to 3 for TC), the final numbers were 653 per 100 mls for FC and 7700 per 100 mls for
TC. Brissaud et al., (2005) reported 4 log units removal in Mèze (France) stabilisation
ponds. Caution is raised in the interpretation of data with respect to FC and TC. For
example in this study, percentage removal for FC and TC are 99.98% and 99.96%
respectively. For TC, although the percentage removal rate is very impressive, the
number is still very high. Considering the WHO standards with respect to faecal
coliforms for treated wastewater reuse, the effluent from the UDSM ponds qualify for
unrestricted irrigation for agricultural use, i.e. category B effluent. (WHO, 1989;
WHO, 2001). This standard states that category B treated wastewater should contain
not more than 1000 per 100 mls of faecal coliforms, although it is silent about total
coliforms. The removal rate for coliforms was affected by the short circuiting in the
second facultative ponds as indicated by the decrease in the decay rate constants
(table 4.3)
Pond cell
Total Coliforms Faecal Coliforms
PrFP
1.24
1.23
FP1
2.70
2.73
FP2
0.29
0.50
MT
0.59
1.57
Table.3: Calculated decay rate constants KT-day
Despite good pathogen removal in these ponds, generally the system is not
performing well in nutrient and organic matter removal. This may be attributed to
nutrient and carbon recycling that results from the accumulated sludge, overloading
could also be one of the reasons, since they were designed to serve 5000 people. Now
they are serving more than 10,000 people. The short circuiting in the second
facultative pond contributes to the low performance as well (figure 4.2 and table 4.3).
The overall organic matter removal was by 45% COD and 27% BOD. For COD, 10%
and 19% was reduced in the primary facultative pond and first secondary facultative
ponds respectively. Equally for BOD, there was removal of 10% in the primary
facultative pond and 10% in the first secondary facultative pond respectively. From
the results of this investigation it is noted that organic matter reduction takes place
mainly in the primary ponds and immediate following ponds. Picot et al., 2005
reported higher percentage removal of organic matter in the primary anaerobic pond.
The percentage removal decreased as the ponds got shallower as in this study.
Therefore, organic matter reduction is carried out mainly by bacteria, implying that in
primary or facultative ponds heterotrophic activity is much more pronounced than
photosynthesis. This is understandable because algae do not need complex organic
matter for photosynthesis. Algae can utilise carbon dioxide. Therefore anaerobic
conditions have to be optimised to maximise on heterotrophic activity. On the other
hand polishing ponds/maturation ponds have to be optimised to maximise pathogen
removal, by having them as shallow as practically possible.
26
It is important to optimise depth of the anaerobic or facultative ponds that receive
influent wastewater. The allowable organic loading rate therefore, may be decided
upon depending as to what extent the anaerobic/aerobic conditions to be developed
are desired in the ponds to produce the desired organic matter and nutrient reduction.
This should be determined hand in hand with pond depth for maximum desirable
anaerobic/aerobic layers that are bound to develop (e.g. depth of heterotrophic layer
that is desired to develop). Boussaid et al, (2000) varied organic matter loading, pond
depth and retention time for a one cell pond in Marakech, Morroco where the average
low temperature is 100 C. They concluded that a pond depth with a water column of
2.3 m, retention time of 9.5 days and organic matter loading as high as 780 – 1000 kg
BOD/ha.day was good enough to remove nematodes and faecal coliforms to achieve a
WHO class B effluent quality suitable for unrestricted irrigation. The pond area was
5000 m2. Pearson et al., (2005) reported higher removal rate constants for faecal
coliform in shallow ponds than in deeper ponds. Mara et al., (2001) proposes a
maximum loading rate of 350g BOD/m3.day at 250 C, 1 day retention time at 2.5 m
deep primary anaerobic pond.
The UDSM stabilisation pond performance is limited, the reasons being lack of
desludging which needs to be done at least every five years. Desludging may enhance
organic matter removal and therefore give more life to the pond system. Sludge
accumulation causes problems for the functioning of the pond system. The reduction
of effective volume (hence hydraulic retention time) affects performance (Nameche et
al., 1997; Mbwele et al., 2003); also feedback to the water column augments the
nutrient and organic load. Sludge can be a source of odour and resuspended solids
entering the water column and effluent. Sludge activity has no significant detrimental
effect on algae growth.
WSPs can perform well if properly designed in tropical climate. One good example
available is that from Ghana in Akuse, where mean temperature is 27.5o C (Hodgson,
2000). In his study, Hodgson observed P removal of 94%. But the respective pond
system is having a low organic loading and too long retention time. The raw
wastewater has a maximum BOD of 100mg/l. Assuming the effluent discharge rate is
the same as the influent flow rate, then the organic loading rate is 57kg/day to an
overall pond area of 15000 m2. The overall retention time is 40 days.
The average water temperature during the study time at UDSM was 27o C. According
to predicted design equations for organic loading, the Mara and Pearson (1987)
equation should be suitable (Table 4.1). This equation predicts an organic loading of
326 kg/ha.day. Mara et al., (2001) suggests a maximum loading rate of 350 kg/ha.day,
at 25◦ C. Therefore the predicted organic loading rate is within the acceptable range
for this temperature.
At a temperature of 25o C, Mara et al., (2001) obtained a good BOD removal from a 1
day retention time, 2.5 m deep anaerobic pond and a 3 day retention time, 1 m deep
facultative pond that complied with EU effluent requirements of 25 mg/l filtered
BOD, 50 m SS. Probably, at these temperatures retention times should not be so long.
Therefore the depth of the primary facultative pond at the UDSM should be adequate
for good stabilisation of the wastewater entering the ponds, without sludge
accumulation.
27
In the present study, ortho–P was significantly reduced in the primary facultative
pond. This corresponded with an equally significant reduction in organic matter (COD
and BOD) in the same pond, since phosphorus removal goes hand in hand with
organic matter removal. Therefore P reduction can be improved by optimising
bacterial activity in the primary ponds. The average minimum temperature of 260 C in
Dar es Salaam for example would allow having a primary facultative pond that is
deep as possible (2.5 - 3 m) to allow better stratification into anaerobic (enhance
hydrolysis and fermentation), heterotrophic activity (enhance P uptake) and minimise
on the photic zones of the pond (for facultative ponds) to minimise photosynthesis.
This will maximise P removal by heterotrophic activity in the primary pond. This is
possible because bio-P bacteria have been isolated from the UDSM ponds (Paper III).
The subsequent cells should become shallower along the train, having maturation
ponds as shallow as it is practically possible, to maximise on pathogen removal.
To upgrade the UDSM pond system, installation of screens is necessary for removal
of grit which has contributed to the accumulation of silt at the influent point. There is
no routine or periodical monitoring of influent and effluent quality. The short
circuiting adds to the hydraulic problems. Desludging of the ponds is very important.
Many of the ponds in Dar es Salaam for example have been rendered redundant (e.g.
Ubungo and Lugalo). Some are overloaded, for example the Vingunguti (Buguruni)
and Mikocheni ponds that receive industrial wastewater in addition to domestic
wastewater, a bypass has been created to allow untreated water to pass through. The
embarkment for the Kurasini ponds is broken and the last maturation pond has been a
grass field.
It is important for the authorities responsible for the treatment systems to have an
operator who will be responsible for operation and maintenance. There should be a
monitoring (evaluation) programme that is continuous or periodical. In Tanzania it
has been pointed out that for better WSP performance, at least minimal operator
training and funds for operation and maintenance should be made available (Mara,
1992)
On the other hand, considering the complexity of the ecology of the WSPs, it may be
important to develop a tool that would be simple and easy to apply for the purpose of
WSPs performance monitoring. This tool could be the water quality index. From this
study (paper 1), it has been observed that the algal photosynthesis has a significant
impact on pond performance. Therefore, suitable parameters (for spot assessment)
that may be used to develop the water quality index may be dissolved oxygen, pH,
chlorophyll and coliform bacteria. But this kind of WQI will be useful if the treated
water is for agricultural reuse. In this case, the nitrogen concentration may be
monitored since very high concentrations may be detrimental to some plants.
Nutrients, BOD and SS may need to be included in the WQI if the treated wastewater
is for direct discharge to the water sources.
28
4.2 Microbial Phosphorus removal in Waste Stabilisation Ponds
The Waste Stabilisation Pond system is one of the most effective and suitable method
of treatment of domestic, municipal, and industrial wastewater. However its use is
plagued with design, construction and operational related problems. The design of this
type of wastewater treatment systems has relied on the application of empirical or
semi-empirical models for many years. However, experience has shown that these
models are rarely, transferable and moreover they are based on only few parameters;
BOD, SS and pathogens.
Nutrient removal in wastewater treatment depends on the type and design of the
treatment process. In an activated sludge treatment plant, with wastewater containing
35mg/l of total nitrogen and 200mg/l of BOD (Hammer and Hammer, Jr. 1996),
nitrogen can be removed up to 25%; phosphorus removal can be approximately 20%.
With enhanced biological phosphorus removal, a modification of a conventional
biological process is made to favour removal of an increased amount of phosphorus
by bacteria (Chritensson, 1997). The traditional design of WSPs does not favour
nutrient removal to a great extent. There are no design equations for phosphorus
removal in WSP. Phosphorus removal in unaerated facultative WSPs has been
recorded up to 26% in arid climate in Marrakech, Morocco, while in Catalonia, Spain;
removal of up to 48% has been recorded (Boussaid et al., 2000; Garcia et al., 2000).
Mbwele et al., (2003) recorded a total soluble P removal of 19.52%. The removal of
phosphorus in WSPs rarely goes down to a 1mg/l level that is desirable. Hodgson
(2000) reported removal efficiencies of 92%, 48.8% and 94% for ammonia; nitrate
and phosphate respectively (mean concentrations in raw sewage were 18mg/l, 7.9mg/l
and 3.3mg/l of ammonia, nitrate and phosphorus). But this system seems to be under
loaded as it receives wastewater with BOD concentration of less than 100 mg/l (a
mean of 66mg/l), especially if temperature is taken into consideration. The mean low
temperature in Akuse, Ghana, is 250 C.
However, the nutrient removal processes taking place in the ponds include biological
processes, in which diversified groups of organisms are responsible i.e. bacteria, fungi
and algae (Hosetti and Frost. 1998). The origin of these microorganisms could be
from the air, soil, and animals living near the system and in the wastewater itself. In
waste stabilisation ponds, nitrogen removal is both assimilative and dissimilative, that
is by algal uptake, and through nitrification and leading to denitrification and
ammonification (Crites and Tchobanoglous, 1998; Bitton, 1999, Mbwele et al., 2003).
The efficiency of total phosphorus removal in WSP depends on how much leaves the
pond water column and enters the pond sediments. This occurs due to sedimentation
as organic P in the biomass and precipitation as inorganic P (principally as
hydroxyapatite at pH levels above 9.5), compared to the quantity that returns through
mineralization and resolubilization (Mara et al, 1992; Mara et al., 2004). The
phosphorus associated with the non-biodegradable fraction of the biomass cells
remains in the sediments. A first order plug flow predicts a 45% P removal for a 90%
BOD removal but this is mainly attributed to sediment adsorption (Mara et al., 1992;
Crites and Tchobanoglous, 1998; Bitton, 1999; Boussaid et al., 2000; Garcia et al.,
2000). Therefore it was believed that the best way of increasing phosphorus removal
29
in WSP is to increase the number of maturation ponds, so that progressively more and
more phosphorus becomes immobilized in the sediments.
4.2.1 Isolation of Phosphorus removing Bacteria from the UDSM
stabilisation ponds
From our studies (Paper II), we isolated and selected bacteria from a waste
stabilisation pond system in a tropical climate; that have a high capability to take up
phosphorus. These results indicate that it may be possible to enrich stabilisation ponds
so as to optimize phosphorus removal (nutrient removal) in waste stabilisation pond
systems. These strains were isolated from a primary facultative pond and a maturation
pond in a pond system. The values for phosphorus removal were found to be
comparable to those of a standard strain that was isolated from an activated sludge
treatment system with enhanced biological phosphorus removal (Paper III). These
results indicate that phosphorus (and in general nutrients) removal can be optimised in
waste stabilisation ponds. This can be made possible by maximising on heterotrophic
activity in the primary pond of the pond system, where most of the BOD is reduced.
BOD5 removal in primary facultative ponds is about 70% unfiltered or 90% filtered.
In secondary facultative ponds the removal is less, but the combined performance of
anaerobic and secondary facultative ponds generally approximates (or is slightly
better than) that achieved by primary facultative ponds
(http://stabilisationponds.sdsu.edu/). From the UDSM ponds performance evaluation
study, much of the phosphorus was removed in the primary facultative pond and in
the maturation pond (Paper 1). This implies that the phosphorus that is removed in the
primary facultative pond is associated with the BOD removal in these ponds (Mbwele
et al., 2003; Picot et al., 2005). Therefore in the primary facultative pond, BOD
removal is attributed to heterotrophic activity (and probably sedimentation) rather
than photosynthesis, since there was hardly any algal growth in the primary pond
(Paper I).
In the case of typical municipal sewage, it is generally accepted that a maximum
anaerobic pond loading of 300 g BOD5/m3/d at temperature higher than 200C will
prevent odour nuisance (Mara et al. 1992). However, results obtained from a more
recent study suggest that maximum design volumetric loadings may be increased to
350 g BOD5/m3/d at 25°C rather than restricting it to 300 g BOD5/m3/d (Pearson et
al.1996; Mara et al., 2001). Furthermore, Mara and Pearson (1986) propose a
maximum sulphate volumetric loading rate of 500 g SO4/m3 /d (equivalent to 170 g S/
m3/d) in order to avoid odor nuisance. At a pond temperature of 250 C, the effluent
from a 1-day retention time, 2.5 m deep anaerobic pond and a 3 day retention time, 1
m deep facultative pond produced an effluent of 25 mg/l BOD (Mara et al., 2001).
This suggests that long detention times may not be necessary in warm climate.
Enrichment of the primary ponds could be an additional strategy to improve nutrient
removal in anaerobic pond since the biological activity (heterotrophy) is carried out
mainly by bacteria rather than algae. Avelar et al., (2001) enriched laboratory
stabilisation ponds with activated sludge; this significantly increased the COD
removal rate. The high COD removal will in turn enhance the removal of phosphorus
by the bacterial growth, since P removal is associated with organic matter removal.
Therefore it is assumed that maximum organic loading rate (depending on
temperature and ww strength) and bacterial enrichment will maximise on bacterial
growth and thereby enhance phosphorus removal. Optimisation of phosphorus
30
removal in WSPs may seem impossible considering their size, but with proper
designs, the right organic loading, good management and operation, there is a good
possibility that phosphorus removal can be improved.
4.3. Isolation of Phosphorus Removing Bacteria from Activated
Sludge Treatment Plants
The EBPR process is reputed to be an economical and reliable option for the removal
of phosphorus from wastewater (Henze, 1996). However, full scale EBPR plants
show high variability in P removal efficiencies, and full control of the process can be
achieved only when the biological mechanisms involved are well understood.
As described earlier, the physiology of EBPR is divided into anaerobic and aerobic
phases for accumulation of substrate and phosphate uptake respectively. Selection of
bio-P organisms for removal of P from wastewater needs to give a selective advantage
by properly using their unique metabolism
(http://www.nhm.ac.uk/minerology/phos/loosdr.htm). Introducing an anaerobic period
at the first stage of a treatment process in which sludge and wastewater are mixed,
fermentation converts the organic carbon compounds into VFA. These are then taken
up by PAOs and stored inside their cells. In the presence of oxygen or nitrate, the
PAOs have substrate inside their cells, whereas other bacteria do not access to this
substrate and cannot grow.
The PAOs then can grow and become the dominant bacterium in the process. This is
a
b
the selection that is expected in the UCT activated process, where EBPR takes place.
a
31
Figure 9: Schematic description of (a) changes in concentration of phosphorus during
anaerobic and aerobic conditions in EBPR (b) uptake of carbon source during the
anaerobic phase and uptake of phosphorus during aerobic conditions in the EBPR
physiology by PAOs (http://www.iea.lth.se/sbr/lectures/L3_Dynamics.pdf).
The fermentation of organic matter to volatile fatty acids, such as acetate is ascribed
to non-PAO heterotrophes. But volatile fatty acids can be used by other heterotrophic
bacteria which may be more efficient than the PAOs. The disadvantage is that VFAs
become limiting and therefore contributes to low P removal in this way of selection.
Christenson (1997) in his study addressed this problem by using the primary clarifier
as a combined separation unit and fermentation tank at Öresundsverket wastewater
treatment plant. There was an increased amount of substrate for bio-P bacteria being
created through hydrolysis and fermentation of primary sludge, which led to an
increased P uptake. This was possible because the clarifiers at this plant are deep
enough. For better /higher achievement of satisfactory P removal, a more complete
hydrolysis and fermentation of sludge would be needed. This would require a separate
reactor instead of in the clarifier. However, investment costs have to be considered.
In the present study (Paper II), a pure strain was selected from among 174 isolates
from a train that has EBPR established at Öresundverket, Helsingborg, Sweden. The
strain was dominant in both the anaerobic and aerobic phase of the train. It was
selected for the study of P uptake in minimal media containing glucose, succinate or
acetate. Phosphorus was taken up anaerobically as well as aerobically when media
containing glucose was used. Santos et al., (1999) tested Microlunatus
phosphovorus for phosphorus uptake. M. phosphovorus was able to take up
phosphorus anaerobically and aerobically with glucose in their experiments.
Phosphorus was taken up in both types of media (containing glucose, succinate or
acetate) without going through the anaerobic phase. Carruci et al., (1999) also showed
that EBPR can be obtained with substrates other than VFAs with neither their preconversion to VFAs nor their storage as PHA. In the present study (Paper II), P
uptake was possible without the anaerobic phase with A.hydrophyla.
32
Figure 10. A.hydrophyla as observed under an Olympus BX 51 microscope with
bright field and photographed by an AnalySIS ColorView 2 digital camera and
analysis Docu 3.2 image processing software.
The possibility to obtain biological phosphorus removal in strictly aerobic conditions
has been investigated by Ghigliazza et al., (1999) as well. They carried out
experiments in a continuous stirred tank reactor. They showed that it was possible to
obtain phosphorus removal without the anaerobic phase. In other single batch reactor
anaerobic/aerobic experiments, Ghigliaza et al., (1998) have observed that the
anaerobic phase had no influence on Acinetobacter lwoffi phosphorus removal ability.
The strain showed the feasibility to obtain EBPR without the anaerobic phase and
phosphorus uptake that was beyond metabolic needs was observed with the same
strain.
In the present investigations (Paper II), the phosphorus uptake was found to
correspond directly with the growth of the microorganism (A.hydrophyla). The uptake
of phosphorus per cell varied with the carbon source that was present in the media.
Phosphorus uptake per cell was highest with media containing glucose, followed with
media containing succinate and finally with media containing acetate. In the case of
media containing acetate or succinate, there was no P uptake in the anaerobic phase. P
uptake was observed in the anaerobic phase when media supplemented with glucose
was used, since there was growth in media with glucose under anaerobic conditions. It
is well known that in EBPR under anaerobic conditions, there is no P uptake, instead
there is release. On the other hand, in this study it was observed that P uptake was
growth dependent. That is why P uptake was observed in media supplemented with
glucose.
This study has shown that the same bacteria can behave like a PAO and a non-PAO
by using glucose as carbon source. Other cultures have shown this property with
glucose (e.g. Microlunatus phosphovorus by Santos et al., 1999). This observation
indicates that facultative bacteria may be important in EBPR. The ability to
accumulate both polyP and PHA by the same strain is evidence that the same strain
can take up and accumulate carbon source and store them for use during poly-P
accumulation. The use of facultative bacteria for EBPR may be useful because it will
minimise competition for carbon source.
On the other hand uptake of phosphorus was achieved for aerobic cultures (i.e.
without initial anaerobic phase) due to the fact that P uptake was growth dependent. P
uptake did take place in the logarithmic phase during culturing but in the stationally
phase, there was no P uptake.
33
5. Conclusions and Recommendations
1. The performance of traditionally designed WSPs does not favour nutrient removal,
in this case phosphorus removal. However significant phosphorus removal (of the
phosphorus reduced) was observed in a primary facultative pond (neutral pH).
2. A strain isolated from an activated sludge treatment system, A.hydrophyla showed
that the anaerobic phase may not be very important for phosphorus removal in
wastewater treatment systems, since complete P uptake was possible when this strain
was tested for P uptake in minimal media supplemented with glucose, succinate or
acetate.
3. Phosphorus removal is dependent on bacterial growth.
4. Good phosphorus removing bacteria was selected from the Dar es Salaam ponds
and had comparable removing capacity to that isolated from an activated sludge
treatment system.
5. It is possible to enhance phosphorus removal in WSPs by maximising heterotrophic
activity in primary ponds of a WSP system. Good management and operation should
be given priority to maintain good performance.
6. More studies are needed to:(a) Desludge the UDSM ponds and evaluate their performance after
desludging
(b) To determine optimum organic loading rate of the UDSM ponds.
(c) Bacterial enrichment of primary ponds to evaluate more on the
importance of bacterial activity in phosphorus removal
7. Desludging (sludge removal) is very important for efficient functioning of WSP.
8. It is also recommended that some funding should be made available for regular
maintenance and operation of the WSPs.
34
6. ACKNOWLEDGEMENTS
I am very grateful to Prof. Gunnel Dalhammar for accepting me as her student and her
patience in guiding me throughout my studies. In addition to that, she (and her family)
provided continuous assistance and support, especially during the difficult times,
when she took care of me while in Sweden.
The tireless technical and scientific assistance from Dr. Guratna Kutuva Rajarao is
highly appreciated, not forgetting her moral support. Guna; thank you so very much.
My sincere thanks go to Dr. Rubindamayugi and Prof. Kivaisi, both of the University
of Dar es Salaam, Tanzania, for believing in me and accepting me to undertake these
studies. I am also indebted to the staff at Applied Microbiology Unit, University of
Dar es Salaam, for their tireless support and help. The Botany Department, University
of Dar es Salaam, especially the head of department, Dr. Magingo and to Charles
Kweyunga for assisting with the analyses.
My heart felt thanks go to Dr. Magnus Ngoile, the former Director General of the
National Environment Management Council of Tanzania, for letting me undergo this
training, and to NEMC as well, for granting me a study leave during all this period. I
am also thankful to my colleagues and friends who in one way or another contributed
to the success of my studies.
I am indebted to the entire staff at the Biotechnology Department, especially the
Environmental Microbiology group (Anna, Karin Sophia, Pele and others) and floor
two for their friendship and hospitality. Special thanks go to Kaj Kauko for his tireless
assistance with the microscope (photographing), file formatting of manuscripts and
thesis and his chats. Not forgetting Lena Gumelius (and her family), for being a friend
and who had their doors, open for me anytime. My sincere thanks go to Assoc.Prof.
Harry Brummer, for correcting the English language.
My stay in Stockholm would have been extremely difficult and probably impossible,
if it was not for my friend Grace and her husband Karl-Åke for their tireless help,
assistance and moral support, especially during the most difficult times that I faced
while living here. May God bless you always. I am equally indebted to my friends in
Stockholm; Agnes, Lyamchae and others not mentioned, who always supported me in
one way or another throughout my stay.
I am indebted to Dr. Ivar Virgin and Ms Benita Forsman from the Stockholm
Environmental Institute for their continuous commitment and support in the overall
management of the BIOEARN project. The financial support from Swedish
International Development Agency ( Sida-Sarec) is highly acknowledged
Last but least, I am deeply indebted to my family. My husband Assue, my sons
Ambakisye, Lusekelo and Lwitiko, my father Ambakisye Kaminyoghe, my mother
Margaret Bukuku, my sisters and brothers, for your blessings, encouragement and
support. Special thanks go to my sisters Sarah Ambakisye and Mary Msokwa and her
husband Dr. Zakayo Msokwa, for supporting me during the most difficult times.
35
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