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                                  Kimoni Dhunpath

            eThekwini Municipality, Water and Sanitation, Scientific Services,
                             P.O.Box 1038, Durban, 4000
        Tel: 311 8457 Fax: (031) 311 8003   E-mail: Bacto_Lab@dmws.durban.gov.za


Activated sludge, a microbial community, consists of free, flocculated and filamentous
bacteria, protozoa, rotifers and a few other higher invertebrates. Protozoa and metazoa
are said to be an important factor in shaping the morphological and taxonomical
compositions of communities in the activated sludge process. The aim of this study was
therefore to determine which protozoa and/or metazoa prevailed during the favourable and
unfavourable conditions present. Activated sludge wet mounts and smears were
microscopically analysed under 100X magnification using dark field.             Each works
analysed was classified as a good or poor operating system. The different protozoa and
metazoa types were identified and enumerated. The results were graphically represented.
It was found that the Amoeba predominates during system start-up and the flagellates
along with the Amoeba are present when a toxic overload is experienced. The presence
of free-swimming ciliates was indicative of a good operating system. The attached ciliates
were found to be good bio-indicators of a toxic upset since ciliates leave their stalks when
exposed to this situation. Rotifers were found during favourable conditions and are said to
be present in stable activated sludge environments. This study showed that there is a
wide range of loadings over which each species may be found but a narrower range over
which they may be found in large numbers.


A number of reports demonstrate that grazing by protozoa is an important factor in shaping
the morphological and taxonomical compositions of communities in the activated sludge
process. These protozoa have an important role in maintaining a good balance in
biological ecosystems. They eliminate the excess bacteria, which stimulates their own
growth, as well as aids in flocculation. They also decrease turbidity and BOD of the
effluent by consuming the free bacteria. Microscopic observation of protozoa and other
higher life forms in activated sludge is a common and wide spread practice. The types of
organisms present can be related to plant performance and effluent quality. Moreover,
these organisms are useful for the assessment of toxicity. In literature there are many
contradictions concerning the effluent quality associated with particular organisms. These
arise partly from the fact that individual species can survive over a wide range of
environmental conditions and probably partly from poor identification of the species
present (2).

From a morphological point of view, activated sludge is a microbial community consisting
of free, flocculated and filamentous bacteria, protozoa, rotifers, nematodes, and a few
other invertebrates. Protozoa and other higher life forms are aerobic and bacteriovorous.
A few anaerobic flagellates and a number of saprophytic flagellates also occur. The
saprophytic flagellates use soluble organic matter for growth. Carnivorous protozoa, both
free ciliates and attached ciliates are known as suctorians. They feed on other protozoa.
Chlorophyll-bearing flagellates are incidentally observed and are derived from the aerobic
basin wall (1). It is known that the effluent quality of activated sludge plants changes from
day to day but studies have shown that it was unlikely that the protozoan population would
react quickly enough to reflect such variations unless a toxic discharge was being
considered (3).

Protozoa and other higher life forms constitute approximately 5% of the activated sludge
biomass. These organisms perform several important functions in activated sludge. The
most important of which is their removal of non-flocculated bacteria from wastewater
through their feeding activities, yielding a clarified effluent. Studies have shown species of
protozoa that excrete specific materials to cause flocculation of bacteria and suspended
solids in the wastewater. This suggests that protozoa play a role in effluent quality (4).
Protozoa by predation, indirectly increases bacterial activity by preventing bacteria from
reaching self-limiting numbers. Bacteria are thus kept in a state of prolonged youth and
their rate of assimilation of organic materials is greatly increased. They also contribute to
biomass flocculation through production of faecal pellets and mucus (8). Protozoa may
also function to break up large floc masses and encourage a more active biomass through
their motility. They perform beneficial roles in the wastewater system, including the
clarification of the secondary effluent and act as bio-indicators of the health of the sludge

Taxonomic classification of these organisms is based primarily on motility. The six basic
groups looked at are flagellates, amoebae, free-swimming ciliates, attached ciliates,
rotifers and a few other higher invertebrates. Protozoan populations can change rapidly in
activated sludge under circumstances of toxic upset. The presence of particular types of
protozoa indicates the chemical and physical performance of the works and the type of
influent being received. It can therefore be stated that food availability, freely dispersed
bacteria or turbidity is the primary determinant of which group will predominate (9).

One of the most valuable uses of the microscopic observation of these organisms is for
toxicity assessment. Protozoa, particularly the ciliates and rotifers, are generally the first
to be impacted by toxic materials and can serve as an in-situ bio-monitoring tests for
toxicants or other adverse stresses on the activated sludge process. The first noticeable
sign of toxicity or stress is usually the slowing or cessation of cilia movement in the ciliates.
Next, the predominant protozoan group shifts towards flagellates and small, free-
swimming ciliates which often ‘bloom’ to high numbers. This is an indication of the break-
up of the activated sludge floc and an overabundance of free bacteria, used up by these
organisms as a food source. In severe cases these protozoans die, which may lead to
foaming (5).

Optimum activated sludge performance occurs with a balance among free-swimming and
attached ciliates and rotifers. An overabundance of flagellates, amoebae or free-
swimming ciliates is an indication of high organic loading while an overabundance of
attached ciliates, rotifers and other higher life forms indicates the opposite. Some plants
attempt to adjust process parameters based on the types of protozoans and other life
forms observed because sludge settling often deteriorates at these two extremes (1).
Many of these organisms feed on soluable organic matter and their presence can indicate
a high soluble BOD level. Flagellates are usually found in high numbers during recovery
from a toxic discharge to the treatment plant or at low DO levels. If Flagellates are present
as the dominant protozoan group, this could indicate an unstable wastewater environment
and a sludge that is in poor health (5).

Amoebae grow well on particulate organic matter and are able to tolerate low DO
environments. They are present in high numbers during start-up of a treatment plant that
has recovered from a toxic discharge (4).

Free-swimming ciliates
Ciliates usually occur under conditions of good floc formation and generally indicate good
activated sludge operation. Both Euplotes and Aspidisca are common in activated sludge
and their presence is desired as they indicate a well operating works. If the free-swimming
ciliates are the dominant protozoan group, this indicates that the bacterial population and
DO concentration are high. It also indicates a wastewater environment that is not yet
stabilised and a sludge that is intermediate in health (7).

Attached Ciliates
These protozoa are generally a sign of stable, healthy activated sludge operation. An
example of an attached ciliate is the Vorticella. If treatment conditions are bad, for
example, low DO levels or toxicity, Vorticella will leave their stalks. Therefore during
microscopic observation a bunch of empty stalks therefore indicates a poor condition.
Vorticella grow best in rapidly flowing water and seems to enhance nitrification (5).

Rotifers are the most abundant macro invertebrates found in the activated sludge process.
They are more complexed structures than protozoa. Most rotifers are motile and attached
to activated sludge flocs by a contractile ‘foot’. These organisms occur over a wide range
of mean cell retention time (MCRT). The presence of some species are indicative of a
high MCRT. Euchlanis an example of a rotifer is commonly found in activated sludge
when effluent quality is good. It requires a continual supply of DO. Their presence is
evidence to the fact that an aerobic environment has been sustained (9). Rotifers in
activated sludge therefore means a good, stable sludge with plenty of oxygen.

Higher Invertebrates
These include nematodes, tardigrades and Annelids. They are generally observed only in
higher MCRT systems. Tardigrades and Annelids appear to occur only in nitrifying
activated sludge systems, probably due to their susceptibility to ammonia toxicity (5).

This study was therefore undertaken to determine the activity of protozoans and
metazoans in the activated sludge system. The type of protozoans and metazoans
present in healthy and poor operating systems were identified and enumerated.


Collection of Samples
                                Activated sludge mixed liquor samples, from six different wastewater treatment works,
                                were collected.
                                Samples were microscopically analysed within 24 hours of collection.

                                Analysis of Samples
                                Only a few drops of sample was microscopically analysed .
                                Activated sludge wet mounts and smears were prepared in triplicate.
                                Wet mounts were analysed at 100× magnification (Dark field analysis) using a compound
                                microscope to determine the morphological characteristics of the protozoa and metazoa
                                The entire slide was observed and the protozoan and metazoan types were counted. All
                                protozoa and metazoa mentioned were previously identified in industrial and domestic
                                Each works analyzed was classified as a good operating system, i.e a variety of protozoa
                                and metazoa, good floc settling and a variety of different filamentous bacteria, and a poor
                                operating system.




Number of Protozoans and Metazoans





                                          Ta   Zo o   SC   Ne   S p iro     As        Pa        Va        Ph        Tr      Ch   Am   An   Eu   M onas
                                                                                                                                                 s pp.
                                                                          Typ e s o f P ro to zo a ns a nd M e ta zo a ns

                                Figure 1a) Number of the different types of Protozoans and Metazoans present in
                                activated sludge under good operating conditions


Number of Protozoans and Metazoans





                                               Ta    Zo o   SC     Ne   S p iro    As         Pa         Va          Ph          Tr        Ch     Am    An   Eu   M o na s
                                                                                  T yp e s o f P ro to zo a n s a n d M e ta zo a n s

                        Figure 1b) Number of the different types of Protozoans and Metazoans present in
                        activated sludge under unfavourable operating conditions


                                          Ta        Tardigrad                                                          Ph               Philodina sp.
                                          Zoo       Zoogloea sp.                                                       Tr               Trachelophyllum
                                          SC        Stalks ciliates                                                    Ch               Chilodonella
                                          Ne        Nematode                                                           Am               Ameoba
                                          Spiro Spirochetes                                                            An               Annelid
                                          As        Aspidicidae                                                        Eu               Euchlanis
                                          Pa        Paramecium
                                          Va        Vaginicola                                                         F                Fungi

With reference to the introduction it was found that the Amoeba predominates during
system start-up, from a toxic overload. It is believed that the amoeba has the ability to
ingest flocculated bacteria thereby creating a clarified effluent. From Fig 1a and 1b it can
be seen that a small number of amoeba was present during unfavourable conditions only.
This could have occurred because amoeba is able to tolerate low dissolved oxygen (DO)
environments. Due to their unique characteristics and the type of works analysed, their
low numbers were expected during this study.

The Monas spp. falls under the group flagellates. The flagellates similar to the amoeba
are dominant during recovery from a toxic discharge. This protozoa was present in higher
numbers during the unfavourable conditions. This result coincides with literature that
states that its dominance indicates unstable conditions at the works and produces a
sludge that is of poor health.

Spirochetes, Aspidiscidae, Paramecium, Philodina and Chilodonella all fall under the
group free-swimming ciliates. Spirochetes were the only protozoa that were found under
unfavourable conditions. They predominate under conditions of low DO levels. The
presence of free-swimming ciliates is indicative of a good operating system. This is
evident when comparing Fig 1a and 1b.

The attached ciliates are said to grow best under rapidly flowing water and seems to
enhance nitrification. It is generally found at the works with an effluent BOD range 0-
20mg/L i.e. good quality effluent. From Fig 1a and 1b it can be seen that the stalked
ciliates were much higher during favourable conditions. It was stated in the introduction
that the ciliates leave the stalks during system upset. This phenomenon was observed
during this study. It can therefore be stated that stalked ciliates are very sensitive to any
changes in the operating of the system. Some stalked ciliates like the Opercularia spp. for
example are still present when the effluent is of inferior quality. This protozoa is useful as a
bio-indicator because studies have shown that its numbers increased when the activated
sludge was of bad quality. Opercularia can endure certain environmental conditions better
than other ciliates and is able to survive low DO concentrations (4). During wastewater
sludge analysis it is therefore important to look out for their presence as one of the criteria
in determining whether the wastewater system is operating well or not.

The rotifers were present in larger numbers during favourable operating conditions. They
are not found in very large numbers because they are able to consume up to 12 000
cells/per day thereby reducing BOD. Moreover, they stimulate microfloral activity and
decomposition, enhancing oxygen penetration and recycle mineral nutrients. They also
secrete mucous which aids in floc formation. Those that live in the slime on the walls of
the aeration basin control slime growth and therefore prevents an anaerobic environment
from forming. Rotifers have been found in very stable activated sludge environments.
They are strict aerobes and are said to be more sensitive to toxic conditions than bacteria

During this study all three protozoa belonging to the group higher invertebrates were found
in larger numbers during unsatisfactory conditions. They are the nematodes, tardigrades
and annelids. According to literature nematodes are found in systems where the mean
cell retention time is high i.e. the rate at which the cell is able to break down the organic
matter present. Although their numbers were higher during unfavourable conditions, there
was a small difference in number between favourable and unfavourable conditions. With
regards to the introduction tardigrades and annelids occur only in nitrifying activated
sludge systems, probably due to their susceptibility to ammonia toxicity. Nematodes have
a lower maximum growth rate and generally develop only in long MCRT systems. A lack
of nematode activity can therefore be one of the indicators of a toxic condition that maybe
developing in the treatment process.

Fungi is also a constituent of the activated sludge composition. They metabolise organic
compounds and can successfully compete with bacteria in a mixed culture. A group of
fungi is also capable of oxidising ammonia to nitrite and some can then convert the nitrite
to nitrate. The most common sewage fungus is the Zoogloea sp. With reference to Fig 1a
and 1b the Zoogloea sp. were more prominent during unfavourable conditions. Fungi is
said to grow abundantly under specific conditions of low pH and toxicity.

The results from this study shows that the number and diversity of ciliates change with
time. Studies have shown that this change occurs due to the quality of the influent and the
operating conditions that prevail (2). The work described in this study has shown that
there is a fairly wide range of loadings over which each species may be found but a much
narrower range over which it may be found in large numbers.


Activated sludge microorganisms : http//www.engitech.com/asm.htm

Curds C.R., Cockburn A. and Vandyke J.M. An experimental study of the role of the
ciliated protozoa in the activated sludge process, Water Pollution Control, 67(3), p312-329

Curds C.R. and Cockburn A. Protozoa in biological sewage treatment processes II -
Protozoa as indicators in the activated sludge process. Water Research. 4, p237-249

Esteban G., Tellez C and Bautista L.M. Dynamics of ciliated protozoa communities in
activated sludge process. Water Research. 25, p967-972(1991).

Jenkins D., Richard M.G. and Daigger G.T. Manual on the causes and control of activated
sludge bulking and foaming, 2nd edition, pub Lewis Publishers, United States of America

Motta M.da., Pons M.N., VivierH., Amaral A.L., Ferreira E.C., Roche N and Mota M. The
study of protozoa population in wastewater treatment plants by image analysis. Brazilian
Journal of Chemical Engineering, 18(1), p1-8 (2001).

Sudo R and Aiba S. Growth rate of Aspidiscidae isolated from activated sludge. Water
Research, 6, p137-144 (1972).

The microbiology of activated sludge: http//www.tvt-bio.com/micro2.html

Wastewater organisms: http//www.jcw.org/html/EmatchGame.html

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