PRESENCE OF PROTOZOA AND METAZOA IN ACTIVATED SLUDGE DURING FAVOURABLE AND UNFAVOURABLE CONDITIONS

PRESENCE OF PROTOZOA AND METAZOA IN ACTIVATED SLUDGE DURING FAVOURABLE AND UNFAVOURABLE CONDITIONS ABSTRACT 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. INTRODUCTION 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 (3). 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, freeswimming ciliates which often bloom to high numbers. This is an indication of the breakup 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 freeswimming 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).

Flagellates 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 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 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. METHOD 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 present. 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 wastewater. 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. RESULTS 35 30 Number of Protozoans and Metazoans 25 20 15 10 5 0 Ta Zoo SC Ne Spiro As Pa Va Ph Tr Ch Am An Eu Monas spp. Types of Protozoans and Metazoans Figure 1a) Number of the different types of Protozoans and Metazoans present in activated sludge under good operating conditions

35 30 25 Number of Protozoans and Metazoans 20 15 10 5 0 Ta Zoo SC Ne Spiro As Pa Va Ph Tr Ch Am An Eu Monas spp. Types of Protozoans and Metazoans Figure 1b) Number of the different types of Protozoans and Metazoans present in activated sludge under unfavourable operating conditions Key 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

DISCUSSION 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 (9). 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. REFERENCES 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 (1968). 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 (1970). 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, 2 nd edition, pub Lewis Publishers, United States of America (1993). 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