Analysis of the possibility of using protozoa inhabiting activated sludge to evaluate the efficiency of wastewater treatment

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Wydział Biologii Instytut Nauk o Środowisku

ANALYSIS OF THE POSSIBILITY OF USING PROTOZOA INHABITING ACTIVATED SLUDGE TO EVALUATE THE

EFFICIENCY OF WASTEWATER TREATMENT

Mateusz Sobczyk

Rozprawa doktorska wykonana pod opieką dra hab. Janusza Fydy i dr hab. Agnieszki Pajdak-Stós w Zespole Ekosystemów Wodnych

w Instytucie Nauk o Środowisku

Kraków 2018

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PODZIĘKOWANIA

Serdecznie dziękuję dr hab. Agnieszce Pajdak-Stós i dr. hab. Januszowi Fydzie za wieloletnią opiekę promotorską i pomoc w odkrywaniu świata mikoorganizmów wodnych.

Dziękuję również Oli za pełnienie nieofcjanie funkcji trzeciego promotora, Edycie, Wioli i Asi za storzenie wzorcowego zespołu badawczego i owocną współpracę.

Dziękuję całemu zespołowi Biospektu: Piotrowi, Marcinowi, Ani i Kasi bez pomocy których nie powstałby rozdział VI, Panu Edwardowi za liczne wspólne delegacje po próbki osadu i ciekawe opowieści z życia wzięte, Pani Małgorzacie Pławeckiej za pomoc w zrozumieniu technologicznych aspektów procesu oczyszczania ścieków, Panu dr. hab. Krzysztofowi Wiąckowskiemu za liczne inspirujące rozmowy naukowe, Terezce i Kasi za pomoc w przygotowaniu i zrozumieniu Sekwencjonowania Nowej Generacji. Ulfowi za motywację i inspiracje naukowe, Łukaszowi za bratnią pomoc statystyczną, Karolinie za korektę językową oraz mojej Żonie Joannie za cierpliwość, wsparcie i ostateczną korektę.

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ABBREVIATIONS

AOB – Ammonia Oxidizing Bacteria AS – Activated Sludge

BNR – Biological Nutrient Removal process BOD5 – Biological Oxygen Demand

CAS – Conventional Activated Sludge system COD – Chemical Oxygen Demand

CV – Coefficient of Variation DO – Dissolved Oxygen

EBPR – Enhanced Biological Phosphorous Removal process EPS – Extracellular Polymeric Substances

FISH – Fluorescent in situ Hybridization GAOs – Glycogen Accumulating Organisms HRT – Hydraulic Retention Time

MCRT – Mean Cellular Residence Time ≈ SRT MLE – Modified Ludzack-Ettinger process MLSS – Mixed Liquor Suspended Solids

MLVSS – Mixed Liquor Volatile Suspended Solids NOB – Nitrite Oxidizing Bacteria

PAOs – Polyphosphate Accumulating Organisms PHA – Polyhydroxyalkanoate

PCA – Principal Component Analysis PCoA (PCO) – Principal Coordinate Analysis PE – People Equivalent

PRC – Principal Response Curves WWTP – Waste Water Treatment Plant RAS – Return Activated Sludge RDA – Redundancy Analysis SBI - Sludge Bitic Index SBR – Sequencing Batch Reactor SRT – Sludge Retention Time ≈ MCRT SVI – Sludge Volume Index

VFA – Vollatile Fatty Acid

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ABSTRACT

Protozoa are inherent elements of the food web of the semi-natural aquatic ecosystem, which is the active sludge. Over decades the role of protozoa in conventional wastewater treatment plants was well known and the subject of many studies. Nowadays, among the wastewater treatment plants (WWTPs) dominate facilities with biological nutrient removal (BNR). These WWTPs are adapted to efficient reduction of nitrogen and phosphorus compounds concentration. The conventional WWTPs differed in biological characteristics of activated sludge in comparison to activated sludge in new technologies such as advanced treatments for nutrient removal. This fact suggests that the results from previous studies should not be automatically transferred to microbial communities that purify sewage in a new type of treatment.

In this PhD thesis, the ecological role of protozoa in WWTP with BNR was investigated. It was also checked whether protozoa can be used as bio indicators in assessing the effectiveness of the BNR process. For this purpose, three experiments were carried out on a laboratory scale and annual monitoring of four full-scale treatment plants.

In laboratory experiments conducted in bioreactors, the increased density of crawling ciliates species Aspidisca cicada did not affect the number and efficiency of ammonia oxidizing bacteria (AOB), and in consequence efficiency of reduction rate of ammonia in sewage.

The influence of protozoa on the formation of activated sludge and on the degree of mineralization of nitrogen and phosphorus compounds in the "start-up" phase was not unambiguous, and the results depended on the experimental setup and scale. On a small scale and in a simple experimental setup, the bacterial community with presence of protozoa efficiently mineralized the compounds of carbon, nitrogen and phosphorus. In bioreactors simulating the sequencing batch reactor (SBR) process, differences in the mineralization of carbon, nitrogen and phosphorus compounds were not found between experimental groups of bacteria, protozoa and rotifers. Differences were observed in the mean sludge flocs size between different community of bacteria, protozoa and rotifers. The smallest flocs were found in bioreactors with the presence of microbial community consisted of flagellates, amoebas, ciliates and rotifers. This fact can be explained by the increased consumers pressure, mainly rotifers. In the experiment simulating the twenty-four-hour oxygen shortage in bioreactors, a lower rate of reduction of nitrogen and phosphorus compounds was observed in bioreactors treated oxygen shortage. On the other hand, no significant changes were noticed in the structure of the nitrifying bacteria, polyphosphate accumulating organisms, protozoa and metazoa community between experimental groups. In laboratory conditions, none of the protozoa and metazoa species could be treated as bio indicator for oxygen shortage condition. During the research in bioreactors a high variability in the densities and species community composition of microorganisms was observed within experimental groups. High variability

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6 within experimental groups, along with a small number of repetitions, probably limited the detection of significant differences between treatments.

The annual monitoring of protozoa and metazoa community in four full scale wastewater treatment plants showed that the microorganisms community composition in activated sludge depends on the individual characteristics of a given bioreactor and the year seasons. From the studied process parameters, the temperature best explained the variability among protozoan and metazoa community.

Based on the collected data on the microorganism group, process parameters and pollution reduction rate, it was not possible to clearly identify potential species useful for assessing the quality of treatment plants with biological nutrient removal.

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STRESZCZENIE

Pierwotniaki (Protozoa) są nieodłącznym elementem sieci troficznej półnaturalnego ekosystemu wodnego jakim jest osad czynny. Rola pierwotniaków w konwencjonalnych oczyszczalniach ścieków był przedmiotem wielu badań na przestrzeni dziesiątków lat i jest dość dobrze poznana. Obecnie wśród oczyszczalni ścieków dominują obiekty o podwyższonej skuteczności usuwania biogenów, przystosowane do wysokiej/wydajnej redukcji stężenia związków azotu i fosforu.

Osady pracujące w oczyszczalniach zaprojektowanych do usuwania biogenów różnią się od osadów z konwencjonalnych oczyszczalni składem gatunkowym bakterii jak i pierwotniaków. Fakt ten sugeruje, że wyniki z wcześniejszych badań nie powinny być automatycznie przenoszone na zespoły mikroorganizmów zasiedlające osady w oczyszczalniach nowego typu.

W niniejszej rozprawie badano, ekologiczną rolę pierwotniaków w osadzie czynnym z podwyższonym usuwaniem biogenów. Sprawdzono również na ile pierwotniaki mogą zostać wykorzystane jako bioindykatory ˗ wskaźniki biologiczne w ocenie efektywności procesu oczyszczania ścieków z zastosowaniem osadu czynnego z podwyższonym usuwaniem biogenów. W tym celu przeprowadzono trzy eksperymenty w skali laboratoryjnej oraz roczny monitoring czterech komunalnych oczyszczalni ścieków.

W badaniach laboratoryjnych przeprowadzonych w bioreaktorach wykazano, że zwiększone zagęszczenie orzęsków pełzających z gatunku Aspidisca cicada nie wpływa na liczebność oraz efektywność działania/pracy bakterii pierwszego etapu nitryfikacji, a co za tym idzie na efektywność redukowania stężenia amoniaku w ściekach. Wpływ pierwotniaków na formowanie osadu czynnego oraz na stopień mineralizacji związków azotu i fosforu w fazie „rozruchu”, nie był jednoznaczny, a wyniki zależały od układu eksperymentalnego i jego skali. W niewielkiej skali i w prostym układzie eksperymentalnym zespół bakterii w obecności pierwotniaków wydajniej mineralizował związki węgla, azotu i fosforu. W bioreaktorach symulujących proces SBR nie wykazano istotnych różnic pomiędzy różnymi zespołami bakterii, pierwotniaków i wrotków w mineralizacji związków węgla, azotu i fosforu.

Zaobserwowano różnice w średniej wielkości kłaczków osadu pomiędzy różnymi zespołami bakterii, pierwotniaków i wrotków. Najmniejsze powierzchniowo kłaczki znajdowały się w zespołach bakterii w obecności zespołu składającego się z wiciowców, ameb nagich, orzęsków i wrotków. Fakt ten można wytłumaczyć zwiększoną presją konsumentów głównie wrotków. W eksperymencie symulującym dwudziestoczterogodzinny deficyt tlenu w bioreaktorach, zaobserwowano niższy stopień redukcji związków azotu i fosforu w bioreaktorach poddanych deficytowi niż w bioreaktorach kontrolnych. Nie odnotowano natomiast wyraźnych zmian w strukturze zbiorowiska bakterii nitryfikacyjnych, akumulujących polifosforany, pierwotniaków i metazoa pomiędzy grupami zabiegowymi. W warunkach laboratoryjnych nie udało się wskazać w zbiorowisku pierwotniaków i metazoa gatunków wskaźnikowych dla deficytu tlenowego. W trakcie badań w bioreaktorach zaobserwowano dużą zmienność w zagęszczeniach i składzie gatunkowym mikroorganizmów wewnątrz grup

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8 eksperymentalnych. Wysoka zmienność wewnątrz grup eksperymentalnych wraz z małą liczbą powtórzeń prawdopodobnie uniemożliwiła wykrycie istotnych różnic pomiędzy zabiegami.

Roczny monitoring zespołu pierwotniaków i metazoa w czterech oczyszczalniach ścieków wykazał, że zespół mikroorganizmów osadu czynnego zależy od indywidualnych cech danego bioreaktora oraz pory roku. Z badanych parametrów procesowych, temperatura najlepiej tłumaczyła zmienność wśród zespołów pierwotniaków i metazoa w osadach czynnych. Na podstawie zebranych danych dotyczących zespołu mikroorganizmów, parametrów procesowych oraz stopnia redukcji zanieczyszczeń nie udało się jednoznacznie wskazać gatunków przydatnych do oceny jakości pracy oczyszczalni z podwyższonym usuwaniem biogenów.

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CHAPTER I Introduction

Brief history and system configurations

The activated sludge (AS) process was first “officially” announced at the beginning of XX^th century to the Society for Chemical Industry at the Grand Hotel in Manchester, England by Ardern and Lockett (1914). Activated sludge nowadays is the most common biological treatment process in the world (Stensel & Mąkinia 2014) and the largest branch of biotechnology (Graham & Smith 2004). AS is used for purifying municipal and industrial wastewater and its applications ranges from small plants for single households to huge facilities serving metropolitan areas. This technology in contrast to traditional biotechnological processes does not operate on stable, strict technological assumptions for pure culture of microorganisms.

The activated sludge process involves culture of different microorganisms in dense flocs forms which are intensely mixed and aerated in biological tanks. The activated sludge system has two main functions:

I) Oxidation of the biodegradable organic matter in the aeration tanks into CO2, H2O, NH4 and simultaneously converted to new cell biomass.

II) Separation of newly formed biomass aggregates from treated effluent, where flocculation and sedimentation processes play main role (Bitton 2011).

Sewage flows into biological reactor and is then mixed with activated sludge biomass. The bacterial flocs very quickly absorb dissolved substances and colloidal particles. Suspended solids also easily adhere to bacterial flocs. Some macro-elements are hydrolyzed by bacterial enzymes secreted outside the cells and monomers could be directly assimilated by bacteria (Figure 1.1).

In secondary clarifier stages bacterial flocs settle and condense. The part of condense sludge called return activated sludge (RAS) is recycled back to the biological reactor and second part of sludge called excessed sludge is removed from the system. The supernatant after sedimentation (effluent) is discharged into water bodies mainly rivers.

The possibility of microbial biomass separation from effluent is very important attribute of activated sludge process. The recycled part of the sludge after sedimentation helps to maintain high concentration of microorganisms in biological reactors. This process allows for a faster acceleration of pollution’s compounds oxidation.

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10 Figure 1.1. The idea of activated sludge process (according to Seviour & Nielsen 2010, changed).

The activated sludge system must cope with domestic and industrial wastes which contain various toxic substances, bioactive pharmaceuticals and heavy metals. In addition, this system needs to work in varied conditions of flow rates and as well as handling large periodic changes in the composition of the waste. The temperature and pH of waste and mixed liquor in biological tanks also fluctuates wide range.

The activated sludge process is a technology that can work well even though it is exposed to above mentioned vast amount of uncontrollable variables.

Originally AS system was invented to remove organic substances from domestic waste, this system is called conventional activated sludge system (CAS). Later, in early 90’s when a problem with eutrophication began to be noticeable wastewater treatment plants (WWTPs) were designed and built to remove nitrogen and phosphorous from sewage. The biological nutrient removal systems (BNR) have become more common when multiple governments enforced stricter standards on effluent quality. The BNR systems were designed to create favorable conditions for the growth of bacteria population responsible for nitrogen and phosphorous removal. Compared to CAS, BNR has an anaerobic and anoxic zones in addition to the aeration tank.

There are dozens of different full-scale activated sludge configuration systems. In my thesis I focus on two WWTP configurations (Figures 1.2 and 1.3), which I come upon during my studies, these are commonly found in Poland.

Modified Ludzack-Ettinger Process

The Modified Ludzack-Ettinger (MLE) process has an anoxic zone located in front of aerobic zone. An internal recirculation recycles activated sludge from oxic zone into anoxic zone. RAS is mixed

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11 with the influent and flow into the anoxic zone. The prolonged process of denitrification is connected to the mixed liquor recycle flow rate. Higher recycle rates increase denitrification.

Figure 1.2. Modified Ludzack-Ettinger process configuration.

Three Stage Bardenpho/Phoredox or A2O process (Anaerobic/Anoxic/Oxic)

This system has three zones: anaerobic, anoxic and aerobic respectively. An internal recycle stream returns nitrates from the aerobic zone to the anoxic zone. RAS is recycled to the head of the anaerobic zone along with the secondary influent. With the inclusion of the anoxic zone, the concentration of nitrates in the return sludge is reduced meaning that the anaerobic process is more efficient.

Figure 1.3. Three Stage Bardenpho/Phoredox or A2O activated sludge system configuration.

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Biocenosis – microorganisms in activated sludge

The activated sludge – “semi-natural ecosystem” consist of biotic components such as: archaea, bacteria, viruses and eukaryotes. The microbial community is established in each system during start- up period and subjected to constant fluctuations. Moreover, biological reactors with activated sludge are open systems for the constant colonization of the microorganisms from the surrounding environment (Fiałkowska et al. 2010).

PROCARYOTES ARCHEA

Archaea is the least known and studied group of microorganisms inhabiting the activated sludge.

Archaea are mainly present in anaerobic digestion reactors, which are widely applied to reduce the amount of excess sludge in WWTPs (Ferrera & Sánchez 2016). Archaea seem to be of minor importance for both nitrogen and carbon removal. However, it is still possible that the Archaea have other functions or affect the properties of the activated sludge (Fredriksson et al. 2012). Study conducted by Fredriksson and co-workers (2012) showed that the Archaea community was dominated by Methanosaeta-like species and the Archaea abundance were estimated to make up 1.6% of total cell numbers in the activated sludge and were present both as single cells and colonies.

BACTERIA

Activated sludge bacteria could be divided on the basis of their location into “floc-associated”

bacteria and “free-swimming” bacteria (Bitton 2011). The total number of bacteria present in the activated sludge ranges from 10⁸ cells/ml (Bitton 2011) to 6 × 10⁹ cells/ml (Pike 1975). The number of

“free-swimming” bacteria range between 2−9 × 10⁷ cells/ml (Morgan-Sagastume et al. 2008).

Nielsen and co-workers (2012) presented average abundance of core bacterial community in 25 full- scale Danish enhanced biological phosphorus removal (EBPR) plants based on molecular quantitative fluorescence in situ hybridization (FISH) analysis. Similar study conducted Muszyński (2015) in 6 full- scale Polish EBPR plants. Both studies showed similar results. The most abundant bacterial type was Proteobacteria, the second numerous type was Actinobacteria and the third Chloroflexi. Nitrospira and Firmicutes were the least numerous.

Generally, bacteria could be also grouped based on their role in the purification process as:

 chemo-organo-heterotrophic (heterotrophic) bacteria

 nitrifying bacteria (ammonia oxidizing bacteria and nitrite oxidizing bacteria)

 denitrifying bacteria

 polyphosphate accumulating organisms (PAOs)

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13 The heterotrophic bacteria are the most abundant group of bacteria which are mainly responsible for reduction of carbon substances.

Mainstream nitrogen removal in WWTPs has traditionally based on the processes of nitrification and denitrification. In this strategy, ammonia (NH4+) is oxidized to nitrite (NO2-) and then to nitrate (NO3-) which is then denitrified to nitrogen (N2) gas.

Nitrifiers

In the process of nitrification, ammonia N is oxidized by ammonia oxidizing bacteria (AOB) primarily to nitrite N. Nitrite oxidizing bacteria (NOB) can then convert nitrite N to nitrate N, also under aerobic conditions.

The most abundant AOBs in the Swedish, Danish and Polish EBPR plants represent the genera Nitrosospira and Nitrosomonas (Hallin et al. 2005, Nielsen et al. 2010, Muszyński 2015). In the same EPBR plants nitrate oxidizing bacteria’s (NOBs) was dominated by genus Nitrospira. Other NOB such as Nitrobacter, Nitrococcus, and Nitrospina, were not present in significant numbers in the WWTPs (Nielsen et al. 2010, Muszyński 2015).

Generally nitrifiers are autotrophic organisms, but some could also be potentially mixotrophic and some may grow as heterotrophs under anoxic conditions (Daims et al. 2001, Schmidt 2009). Nitrifiers acquire energy from inorganic chemical sources and utilize CO2 as their primary carbon source. Because of the need to fix CO2, the growth rates and biomass yields of nitrifiers are low compared to heterotrophic organisms (Khunjar et al. 2014).

AOB and NOB density in activated sludge depends on concentrations of oxygen, ammonia and nitrite respectively (Nielsen et al. 2010) and temperature (Downing & Hopwood 1964).

A consequence of the low growth rate and biomass yield is the need to provide sufficient long mean cell residence time (MCRT) for nitrifiers to ensure that biomass does not be washout from the system (Khunjar et al. 2014).

Denitrfers

During denitrification, nitrite N and/or nitrate N are converted to N2 gas in the absence of oxygen. Denitrifers are predominantly heterotrophic bacteria, which use organic carbon compounds for biomass synthesis. The Betaproteobacteria from the genera Thauera, Azoarcus, Zoogloea, Curvibacter and Accumulibacter are the main denitrifers in EBPR plants (Nielsen et al. 2010).

Small volatile fatty acids (VFAs) and amino acids seem to be substances which determine the presence of the various denitrifiers (Nielsen et al. 2010). Some species are more versatile than others. Some have a large polyhydroxyalkanoate (PHA) − accumulating capacity, which may be competitive in some system layouts (Nielsen et al. 2010). None of the important denitrifiers can consume glucose, galactose or mannose (Nielsen et al. 2010). The structure of the denitrifying bacteria population in some WWTP

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14 highly depends on addition of external carbon substrate (e.g. methanol) for maintaining sufficient denitrification capacity in full-scale plants (Hagman et al. 2008).

Polyphosphate accumulating organisms (PAOs)

Polyphosphate accumulating organisms (PAOs) are the important group of bacteria responsible for phosphate removal in activated sludge process. PAOs constitute only a minor part of an EBPR bacterial community in full-scale plants, typically 15–25% of all bacteria (Nielsen et al. 2010).

PAO achieve dominance under anaerobic/aerobic conditions having a selective advantage over the other bacterial populations present in their abilities to synthesize intracellular storage compounds under the

‘feast-famine’ conditions which characterize EBPR systems. Majority of phosphate removal from the EBPR process is often achieved through anaerobic–aerobic recycling conditions (Seviour et al. 2003).

Under anaerobic conditions PAOs can take up carbon sources as organic substrates like acetate or/and as short chain volatile fatty acids (VFAs) and store them intracellularly as carbon polymers called poly- b-hydroxyalkanoates (PHAs). During this anaerobic stage, PHA levels in cells biomass increase in parallel with the assimilation of carbon source. Intracellular polyphosphate content decrease and at the same time increase in phosphate levels in the bulk liquid. In aerobic phase in the absence of any organic compounds PAOs use their stored PHA as the energy source for biomass growth, simultaneously they uptake phosphate from liquid to synthesize and storage polyphosphate intracellularly. Final P removal from the wastewater is achieved through the removal of waste sludge containing a high polyphosphate content in PAOs biomass (Mino et al. 1998, Seviour et al. 2003, Oehmen et al. 2007).

The genus Accumulibacter is abundant PAOs in most EBPR plants. The Actinobacteria genus, Tetrasphaera probably contains species belonging to PAOs (Kong et al. 2005). Tetrasphaera are generally more abundant in EBPR plants than Accumulibacter (Nielsen et al. 2010). This is confirmed by the Muszyński (2015) study, in all six investigated WWTPs, where genus Tetrasphaera was dominating group of PAOs.

The presence of different organic substrates in influent is probably the key parameter that determine the occurrence of the various PAOs in activated sludge. Most Tetrasphaera can feed on starch, glucose and grow under anaerobic conditions. By contrast, the primary substrates for Accumulibacter are acetate, propionate and some amino acids. However, Tetrasphaera do not store PHA, so their physiology is still a mystery and various Tetrasphaera ecotypes may play different roles in full scale EBPR plants (Nielsen et al. 2010).

Glycogen accumulating organisms (GAOs)

According to recent reports (Filipe et al. 2001, Saunders et al. 2003, Oehmen et al. 2005), GAOs are able to take carbon under anaerobic conditions and compete for substrate with PAOs. Apparently, GAOs exhibit a similar metabolism as PAOs, but they do not store poly-P. As consequence, GAOs metabolism does not include anaerobic P-release and subsequent aerobic (and/or anoxic) P-uptake

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15 likewise PAO. Consequently, GAOs do not contribute to the EBPR performance. On the contrary, proliferation of GAOs at laboratory and full-scale wastewater treatment plants has been identified as one of the main reasons for EBPR deterioration (Saunders et al. 2003, Thomas et al. 2003) and failure (Satoh et al. 1994, Filipe et al. 2001).

The competition with GAOs seems to depend on the temperature and the relative composition of acetate/propionate (Oehmen et al. 2006, Lopez-Vazquez et al. 2008). GAOs are not abundant and reached less than 1% of bacteria in Danish plants, perhaps due to the generally low temperatures (Nielsen et al. 2010) and also are not abundant in Polish plants where their abundance were less 2.5% of bacteria (Muszyński 2015).

VIRUSES

Viruses are considered to be important part of natural aquatic microbial ecosystems. In water environments viruses are abundant and can affect bacterial communities through the lysis of bacterial cells or through horizontal gene transfer (Fuhrman 1999, Wommack & Colwell 2000). Suttle (1994) estimated that 10–20% of the bacteria are lysed daily by viruses. In turn, Fuhrman and Noble (1995) calculated that viruses contribute similarly like protists to bacterial mortality in coastal seawater. Viral genes and viral activity generate genetic variability of prokaryotes and are a driving force for ecological functioning and evolutionary change in aquatic ecosystems (Weinbauer & Rassoulzadegan 2004).

The concentrations of viruses in the activated sludge, quantified by epifluorescence microscopy, ranged from 4.2 × 10⁷ to 3.0 × 10⁹ individuals/ml and this concentration is as high as, or even higher, than those in other natural environments (Otawa et al. 2007). Study performed by Khan and co-workers (2002) and Lee and co-workers (2004a, 2006) showed that 30–60% of the bacterial isolates from activated sludge had related bacteriophages. These findings suggest, that viruses are part of the microbial systems in activated sludge, although their roles have not yet been clarified (Otawa et al. 2007). Our knowledge about the dynamics and abundance of the viruses associated with microorganisms in activated sludge is still poor and more investigations are required (Otawa et al. 2007, Seviour & Nielsen 2010).

EUKARYOTES

The Eukaryotes which inhabited activated sludge are usually divided according to taxonomy and historical reasons into three groups: fungi, protozoa and metazoa.

FUNGI

Generally the popular view is that fungi are not important members of activated sludge microbial community (Seviour & Nielsen 2010). Some authors claimed that fungi could be potentially important contributors to various functions in activated sludge like: organic compound biodegradation, construction of sludge flocs and detoxification (Niu et al. 2017). On the other hand, Jenkins et al. (2003)

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16 gave examples that in WWTPs where pH droped below 4.5 fungi out-competed bacteria and caused bulking problems.

The application of molecular methods gave a markedly different view of fungal community composition compared to that based on previously used culture-dependent methods. Evans & Seviour (2012) research showed, that many of the fungi detected using molecular methods in five WWTPs located in Canada and Australia were novel uncultured representatives of the major fungal subdivisions, particularly the newly recognized Cryptomycota.

Niu et al. (2017) used pyrosequencing and qPCR methods and showed that the members of the municipal WWTP fungal communities in China were assigned to 7 phyla and 195 genera. The most abundant phyla were Ascomycota and Basidiomycota. Additionally, the 23 core genera were shared by all studied WWTPs and account for 50% of the total abundance. Multivariate analysis showed that DO and the C/N ratio were the two most dominant contributors to the variation in fungal community structure (Niu et al. 2017). High difference between results obtained from culture-dependent technique and molecular methods indicates that these second group of tools should be used in future to explore fungal community and their potential role and function in activated sludge.

One of the most interesting fungi group which inhabits activated sludge and can directly influence another biological component are predatory fungi.

Zoophagus and Lecophagus are two genera of predacious fungi that feed on metazoa. The fungi from genus Zoophagus feed mainly on loricated rotifers (Monogononta), whereas species from genus Lecophagus trap mainly bdelloid rotifers and tardigrades (Pajdak-Stós et al. 2016).

Only one species Lecophagus vermicola feed exclusively on nematodes (Zhang & Hyde 2014).

Predatory fungi Zoophagus insidians capable of captured and killing rotifers in activated sludge in a full-scale WWTP have been mentioned by Sladká and Ottová (1973). Cooke and Pipes (1970) claim that predatory fungi are very rare in activated sludge because they did not found any predatory fungi in samples took from 19 different wastewater treatment plants. Sladká and Ottová (1973) suggested that this was due to the MCRT because short MCRT are not optimal for rotifers and nematodes, which are the main food source of predatory fungi. The newest observation made by Fiałkowska and co-authors (2016b) suggested that activated sludge of WWTPs in Poland are surprisingly often inhabited by predatory fungi. The role of predatory fungi in activated sludge is not well recognized due to the lack of research. But last observation made by Pajdak-Stós et al. (2016) showed that fungi Zoophagus sp. can strongly affect the rotifer community in activated sludge. This topic is also open to new investigation.

PROTOZOA

The last years of investigations and discoveries in the field of the taxonomy of the eukaryotes completely rebuild old classification (Adl et al. 2012). Modern-day protozoa is a historical term but still common used by scientists working on unicellular organisms inhabited activated sludge. This term helps

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17 to classify distant phylogenetically related microorganisms. In scientific terminology of activated sludge microbiology and in my thesis protozoa are: flagellates, amoebas and ciliates.

Protozoa are commonly found in the mixed liquor and very often reach concentration from 10⁵−10⁶ cells/ml to 3−20 × 10⁶ cells/ml according to Curds and co-authors (1968) and Madoni (2011) respectively. A first complete list of 228 species of ciliated protozoa found in activated sludge published Curds and Cockburn (1970a). Revisions of the list of species have been made during last 40 years by many authors (Madoni & Ghetti 1981, Augustin & Foissner 1992, Salvadó 1994, Foissner & Berger 1996, Martin-Cereceda et al. 1996, Amann et al. 1998, Ettl 2001, Chen et al. 2004, Hu et al. 2013b, Zornoza 2017).

Flagellates

Flagellates in activated sludge ecosystem are usually divided according to size into two groups:

small flagellates (<20 µm), from which the most abundant representative is Bodo saltans, and large flagellates (>20 µm), notably such genera as Peranema, Entosiphon, Euglena, and Notosolenus (Parada- Albarracín et al. 2017). Both groups of flagellates are heterotrophs that feed primarily on free bacteria and are capable of using soluble organic compounds (Seviour & Nielsen 2010).

Poole (1984) observed flagellates in different types of conventional activated sludge systems (CAS), with or without nitrification, with different values of MCRT and with different values of sludge loading.

In turn Madoni (1994), links high concentrations of small flagellates in CAS with instability, poor aeration, organic overload, low nitrification capacity, low performance in the treatment process and start-up phase of WWTP. Several researchers have reported that small flagellates could indicate shock loadings of organics and nitrogen compounds (Drzewicki & Kulikowska 2011) or the high concentration of biochemical oxygen demand (BOD5) in effluent (dos Santos et al. 2014). Bodonids were related with effective nitrogen removal (Perez-Uz et al. 2010). Large flagellates are frequently found in activated sludge (Madoni 2011), although they are not usually correlated with the effectiveness of the process.

Perez-Uz et al. (2010) observed a negative relation between large flagellates such as Peranema and the nitrogen removal, caused by the possible grazing on aggregates of nitrifying bacteria. However, Hu et al. (2013a) detected the opposite relation with nitrification. These same researchers correlated the higher density of large flagellates with poor results in the organic matter removal.

Among the bacterivorous protists, the nanoflagellates are known as a major consumers influencing both the bacterial community structure and the bacterial density (Posch et al. 1999, Šimek et al. 1999, Hahn

& Höfle 2001). Study conducted by Šimek and Chrzanowski (1992) showed that nanoflagellates were size selective. Hahn and co-authors (1999, 2000) demonstrated that bacterial filament and microcolony formation in some species is a defense mechanism against flagellate grazing (Hahn & Höfle 2000).

Pernthaler et al. (1997) observed in a two-stage chemostat that flagellate grazing is the driving force forming filamentous bacteria and a community dominated by Betaproteobacteria. Flagellates selectively fed on Alphaproteobacteria which had high growth rates and were always present as a minor

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18 fraction of total bacteria. These studies however were conducted in laboratory chemostats or in fresh water lakes and the results should not be transferred directly into the active sludge ecosystems.

Amoebas

Amoebas observed in activated sludge have been divided into two groups on the basis of their appearance: naked amoebas (without a shell) also called free-living amoebas and testate amoebas (with a shell). In the scientific literature there is a lack of works which together investigated the community of naked and testate amoebas.

Naked amoebas

Naked amoebas are almost always found in activated sludge. Sydenham (1971) observed that the genus Flabellula was a characteristic and numerically dominant organism in the activated sludge in two WWTP in London area. In a recent investigation Ramirez and co-workers (2014) studied naked amoebas inhabiting WWTP for the textile industry. They isolated representatives of fourteen naked amoeba genera: Acanthamoeba, Echinamoeba, Korotnevella, Mayorella, Naegleria, Platyamoeba, Saccamoeba, Stachyamoeba, Thecamoeba, Vahlkampfia, Vannella, Vermamoeba, Vexillifera and Willaertia. The highest numbers of amoebas were noted in samples from the aeration tank and seasonal distribution during one year was not observed. The most frequently present amoebas were representatives of Acanthamoeba and Vermamoeba which were found in all treatment system stages (influent, aeration tanks, secondary clarifier, effluent). The factors that probably determined the presence and distribution of naked amoebae in the activated sludge system were their capacity to form cysts, which allowed them to survive in unfavorable conditions such like lack of food, extreme temperatures and low dissolved oxygen levels (Ramirez et al. 2014). Naked amoebas are bacterivorous or predators of other protists and even small metazoans (Berger 2009).

Testate amoebas

The most common genera of testate amoeba found in biological wastewater treatment are:

Arcella, Euglypha, Centropyxis, Trinema, Bullinularia, Difflugia, Cochlipodium (Ortiz et al. 2009, Sydenham 1971). Arcella spp., Centropyxis sp. and Euglypha spp. were found both in aerobic and anaerobic zones in biological systems (Ortiz et al. 2009). Testate amoebas were commonly found in wastewater treatment systems with nitrification and low organic load, long MCRT, and high dissolved oxygen level (Poole 1984, Madoni et al. 1993, Madoni 1994). Testate amoebae are more abundant in plants with a high sludge age because of low growth rate. In summer period they are more common in activated sludge when temperature is higher and in consequence the growth rates of testate amoebas increase. Sasahara and Ogawa (1983) found that Euglypha and Difflugia were always abundant in WWTP from brewery industry with low sludge load and good quality of the effluent.

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19 The roles of amoebas in activated sludge systems are poorly understood. Most studies on protozoa in activate sludge have focused on ciliates (Salvadó 1994, Martin-Cereceda et al. 1996, Ettl 2001, Dubber & Gray 2011a, b). Ciliates and flagellates are very effective bacterial feeder in activated sludge and very often dominate in protozoa community (Seviour & Nielsen 2010). For that reason, amoebas probably play a marginal role in these specific wastewater treatment systems. Amoebas are non-swimmers nor filter feeder and the surfaces and the volumes accessible to them are reduced in comparison with those available for free-swimmers and filtering protozoa (Rodriguez-Zaragoza 1994).

Different opinion had Sydenham (1971) which claim that the amoebas in activated sludge probably play equal ecological important role as the ciliates. Fiałkowska and Pajdak-Stós (2008) observed that testate amoebas can ingest filamentous bacteria.

On the other hand, testate amoebas show rapid response to environmental changes, which makes them useful as possible bioindicators of purification process and effluent quality (Rodriguez-Zaragoza 1994).

Amoebas and flagellates in biological nutrient removal systems probably play a different role than in conventional systems. These groups are clearly associated with nitrogen elimination (Perez-Uz et al.

2010).

Ciliates

The ciliates usually dominate the protozoan community in biomass and very often in density.

They can reach up 10% of the total prokaryotic and eukaryotic cells dry mass (Pauli et al. 2001). Ciliates have been divided into “ecological” groups on the basis of their feeding strategy and mode of locomotion (Curds 1965, Madoni 1994, Ettl 2001):

1) bacterivorous ciliates*

 free-swimming ciliates

 attached (sessile) ciliates

 crawling ciliates

2) carnivorous (predatory) ciliates 3) omnivorous ciliates

*normally only bacterivorous ciliates are categorized in relation to mode of locomotion

In activated sludge tanks, ciliates community is dominated by attached and crawling species (Pauli et al. 2001). Conducted studies suggests that ciliates community inhabiting activated sludge are similar worldwide (Curds & Cockburn 1970a, Sudo & Aiba 1984, Salvadó 1994, Amann et al. 1998, Ettl 2001, Perez-Uz et al. 2010, Drzewicki & Kulikowska 2011, Hu et al. 2013b, Zornoza 2017).

According to Curds (1982), all authors of publications concerning the role of protozoa in the activated sludge process agreed, that clear effluent appeared when high density of ciliates were present in biological reactors. The one of the well-known experiment which directly demonstrates effect of the ciliates in activated sludge process were performed by Curds and co-workers (1968). Under ciliates free

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20 conditions, all laboratory scale activated sludge units produced very turbid effluent with high density of free-swimming bacteria and high concentration of biochemical oxygen demand (BOD5), chemical oxygen demand (COD), organic nitrogen and suspended solids (SS). Cultures of ciliates protozoa were added to part of the experimental plants and after stabilization of ciliates population, the quality of effluent parameters from these plants was significantly improved.

The main roles of ciliates in activated sludge system are:

 reduction and elimination of suspended particles and bacteria cells

In activated sludge tanks and secondary clarifiers present ciliates community has sufficient time to filter effluent and effectively remove big part of bacteria and abiotic suspended solids with similar size to bacteria (Pauli et al. 2001). Research conducted by Eberl and co-workers (1998) clearly showed that predation by protozoa was the main mechanism for bacterial cells removal from AS systems.

 reduction of the total biomass

Between 25 to 48% of the bacteria can be consumed by ciliates and this correspond to 10–19% reduction of the accumulated sludge (Ratsak et al. 1994).

 flocculation

Luxmy et al. (2000) observed that higher density of attached and free-swimming ciliates significantly reduced number of small flocs (<10 µm) and dispersed bacteria (<1 µm) in membrane-separation activated sludge process. Experiments performed by Arregui and co-workers (2007, 2008) demonstrated that crawling and attached ciliates can contribute to aggregation/flocculation by the active secretion of polymeric substances. Aggregates produced in the presence of ciliates were more compacted than those produced by bacteria alone. For the other hand the role of protozoa in floc formation in activated sludge could be slender because wastewater itself contains a high concentration of different particles and bacteria which can produce extracellular polymeric substances and collide and glue-together (Pauli et al. 2001).

METAZOA

Still little is known about role of metazoans in activated sludge. Generally, metazoans like nematodes, rotifers, annelids, gastrotrichs and tardigrades are probably important predators of bacterial cells (Seviour & Nielsen 2010). The abundance of metazoa in activated sludge is usually not very high.

Rotifers and nematodes are the most common while annelids and tardigrades are rather rarely found in activated sludge samples.

Greater abundances of metazoa can reduce sludge production (Luxmy et al. 2000, Lapinski &

Tunnacliffe 2003, Ratsak 2001, Song & Chen 2009). On the other hand some authors claim that grazing pressure from metazoan can negatively affect nitrification (Lee & Welander 1994, Puigagut et al. 2007).

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21 Rotifers

Rotifers consist of classes Bdelloidea and Monogononta, and in activated sludge representatives of both classes occurred. Bdelloidea rotifers are usually filter-feeders, while Monogonota are filter- feeder and grazers. Rotifers in activated sludge were studied in detail by Klimowicz (1970, 1972, 1973), Doohan (1975) and Sudzuki (1981). Rotifers occurred in WWTP with a long MCRT because their reproduction rate is relatively slow compared to protozoans and they are washed out with the excessive sludge from plants with a short MCRT (Sládeček 1983). Nowadays in BNR plants, MCRT is adjusted to favor growth of nitrifying bacteria population which also has low growth rate what is suitable also for rotifer growth and presence in activated sludge.

Laboratory experiments conducted by Lapinski and Tunnacliffe (2003) shown, that rotifers have two distinct effects on suspended particles. They can consume biomass and improve settling, probably due to enhanced aggregation. Ding et al. (2017) has confirmed hypothesis of Lapiński and Tunnacliffe, showing that bdelloid rotifer commonly observed in activated sludge − Philodina erythrophthalma, improves the flocculation performance of two bio-flocculating bacteria and promotes the formation of sludge flocs. Rotifer secretion promotes the bacterial growth and extracellular polymeric substances (EPS) production. The rotifer secretion functions as a chemical signal rather than as the bioflocculant or nutrient for bacterial growth.

Monogonota rotifers from genus Lecane can control activated sludge bulking by consuming different type of filamentous bacteria from WWTP (Fiałkowska & Pajdak-Stós 2008, Kocerba-Soroka et al.

2013a, Drzewicki et al. 2015, Fiałkowska et al. 2016a, Pajdak-Stós et al. 2017). According to Puigagut et al. (2007) rotifers from Lecanidae family reduce mean floc surface area due to its grazing activity and may had in consequence some negative effect on overall nitrogen removal process. On the other hand Kocerba-Soroka et al. (2013b) experiment showed that high density of representatives of Lecanidae family – species Lecane inermis does not affect nitrification process.

Nematodes

The role of nematodes in wastewater treatment processes has been barely studied. It has been suggested that nematodes are important bacterial feeders (Salvadó et al. 2004). Nematodes density in activated-sludge systems generally is less than 1% of the total microfauna. Regarding nematodes slow growth rate, their presence and higher density are related with the high values of MCRT (Salvadó 1994).

Annelida (Worms)

Annelida are the largest organisms observed during the microscopic investigation of activated sludge (Eikelboom 2000). The most common worms appearing in conventional activated sludge (CAS) processes are Aeolosomatidae and Naididae (Liang et al. 2006). Inamori et al. (1983) after Ratsak and Verkuijlen (2006) reported the presence of large quantities of Aeolosoma sp., Pristina sp., Nais sp., Dero sp., and Chaetogaster sp. in WWTPs. Elissen et al. (2008) monitored four WWTPs in Netherlands

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22 during 2.5 years and they found species belonging to the Aeolosomatidae (Aeolosoma hemprichi, A.

variegatumand A. tenebrarum) or Naidinae (Tubificidae) such as: Nais spp., Pristina aequiseta, Chaetogaster diastrophus. Many researchers investigate possibility of application Annelida to reduce the sludge production (Wei et al. 2003, Elissen et al. 2006, Song & Chen 2009, Tamis et al. 2011).

Elissen et al. (2008) tried to relate worm composition and densities with process parameters and they suggest similar to Ratsak and Verkuijlen (2006) that the population peaks of Annelida are phenomena that are hard to explain and control. Additionally, they suggest that potential effects of worms on WWTP process performance, such as waste sludge reduction, should be interpreted with much care.

Tardigrades

Out of all metazoans tardigrades have been recorded least frequently. For example, Chen et al.

(2004) found tardigrades only in 4 out of 200 samples (2%) taken from five WWTPs in Beijing during 1 year, whereas rotifers, nematodes, gastrotrichs and annelids occurred, respectively, in 83%, 21%, 20%

and 9% of collected samples. According to Utsugi (2001), the tardigrade Isohypsibius myrops observed in Japanese treatment plants, seemed to live and reproduce in aeration tanks as a scavenger. Mainly species Thulinius ruffoi was observed during investigation of activated sludge samples taken from Polish WWTPs (Sobczyk et al. 2015). According to Jenkins et al. (2003) tardigrades are indicators of a low food-to-microorganism ratio (F/M). Eikelboom (2000) claimed that tardigrades were occasionally observed at low sludge loading levels (below 0.1 kg BOD/kg MLSS/day). The role of tardigrades in the activated sludge process is also unclear because of the poor knowledge of their biology and very low occurrence rates in aeration tanks.

Gastrotrichs

The gastrotrichs is the least known group of metazoa that inhabits activated sludge. In research conducted by Poole (1984) on 13 WWTPs, gastrotrich occurred rarely, only in 6% of collected samples and only in nitrifying plants. So far, all gastrotrichs found in the activated sludge belonged to the genus Chaetonotus (Poole 1984, Leal et al. 2013). Because of lack the information about their ecology, the role of gastrotrichs in the activated sludge process similarly to tardigrades is still unclear.

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23

Ecology (Ecological aspects in activated sludge technology)

From the ecological point of view in wastewater treatment plants, the only difference from natural processes is that part of metabolism transformation take place in technically controlled environment. Summarizing we can treat the activated sludge process and biocenosis as a semi-natural aquatic ecosystem. According to Pauli an co-workers (2001) The biocoenosis in wastewater treatment plants should not be regarded as a community with a rigid composition and constant characteristics but rather as an artificial but biological segment of natural self-purification processes, the composition of which is influenced by ecological conditions and physical-chemical factors, thus differing from plant to plant and even within a plant over time.

The similarity of activated sludge flocs to the marine snow

Activated sludge flocs that are formed in biological tanks in WWTP do not differ distinctly from bacterial aggregates occurring in natural water bodies. Alldredge and Silver (1988) described aggregates in marine systems as “marine snow” which is composed of organic detritus, living photosynthetic and heterotrophic microorganisms, and inorganic particles. Comparable aggregates could also occur in the pelagic zone of lakes as “lake snow” (Paerl 1973, Grossart & Simon 1993) and in lotic systems as “river snow” (Böckelmann et al. 2000). Study conducted by Grossart & Simon (1998) suggests that lake snow aggregates and activated sludge flocs have similar functions in their environment. Lake snow aggregates act as hotspots of enhanced microbial particulate organic matter (POM) decomposition and their microbial community was dominated by Betaproteobacteria, especially during aggregate aging when filamentous and thus grazing resistant bacteria dominated. The main difference between flocs from activated sludge and flocs from natural water environments is that activated sludge flocs are devoid of photosynthetic microorganisms.

Both natural and engineered flocculated aggregates are usually colonized by numerous heterotrophic flagellates, amoebas, ciliates, and small metazoans. In spite of the obvious similarity, the two systems have been studied by different research teams and treated as separate research fields. In particular, active sludge appears to be a research field often overlooked by ecologists.

The activated sludge flocs are complex heterogeneous structure (Liss et al. 1996) consist of bacteria and protozoa and sometimes fungi, organic and inorganic particles bounded extracellular polymeric substances (EPS) (Li & Ganczarczyk 1990). The flocs have wide size range and can reach more than 1000 µm (Jenkins et al. 2003). The EPS chemical composition is variable and the major components are proteins and polysaccharides but lipids and humic, fulvic and nucleic acids are also found (Frølund et al. 1996). The origin of these substances is probably microbial but some fraction of proteins and DNA may originate from waste food incoming with sewage. The EPS matrix could constitute more than 50% of the organic matter in floc structure where bacterial cells constitute only 5−20% (Nielsen et al. 2010). The EPS determine floc properties and play main role in floc formation

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24 (Frølund et al. 1996), but also the abilities of microorganisms to stick to each other and to nonbiological particles is very important in floc formation process (Jenkins et al. 2003). Additionally, divalent cations Ca²⁺, Mg²⁺ in model of flocculation proposed by Higgins & Novak (1997) bind lectins (proteins) within the floc matrix, lectins with their multiple binding sites bind polysaccharides within the biopolymer network. The polysaccharides can then bridge between neighboring lectins, and these bridges stabilize the entire biopolymer network.

The filamentous bacteria maintain the internal structure of the flocs, forming a “backbones” for the remaining elements of flocs (Jenkins et al. 2003). Flocs stability depends also on both filamentous bacteria abundance and on the balance between filamentous and floc-forming bacteria (Burger et al.

2017). Flocs play also an important role in removal of bacterial cells and other particulate matter from medium. Free-swimming bacteria and other particles entering the reactors adsorb very quickly to existing flocs (Olofsson et al. 1998). Zita & Hermansson (1997) showed that hydrophobic bacteria attach in higher rate than hydrophilic ones.

Not only biotic factors affect the activated sludge flocs structure but also the abiotic factors, mainly operational parameters like: hydraulic retention time (HRT) and mean cell residence time (MCRT).

Shorter HRT favors bacterial community which more efficiently and faster aggregate and settle. This direction of selection with short HRT is used in granular sludge technology (Morgenroth et al. 1997).

Microbial loop

Energy transfer and materials cycling through different trophic levels are fundamental to ecosystem function (Odum 1968). The fact that bacteria link flow of energy and matter in so called

“microbial loop” is well known fact in ecosystem theory (Pomeroy 1974, Azam et al. 1983).

The microbial loop is a diverse community of bacteria, archea, fungi, heterotrophic nanoflagellates viruses and protozoa which in natural ecosystems work alongside, or is embedded within the classical food chain composed of organisms, such as algae, zooplankton, fish and other large consumers. This microbial loop has great significance not only in seas, oceans and lakes but also in streams and rivers.

In wastewater treatment plants based on activated sludge technology, only the microbial loop is present and is responsible for all of the essential waste treatment processes (Graham & Smith 2004).

Top-down or Bottom-up control

Another important ecological aspect which is directly connected with food web present in treatment plants with activated sludge are variants of control method of this ecosystem called: top˗down and bottom-up control. The question how food webs are resource- (bottom-up) or predation- (top-down) controlled is one of the most fundamental and still actual research questions in ecology (Lynam et al.

2017). When looking for analogies to natural freshwater bodies, we can assume that activated sludge tanks resemble highly eutrophic lakes deprived of classical food chain elements such as algae, zooplankton (mainly cladocerans and copepods) and fish. If we treat activated sludge as semi-natural

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25 aquatic ecosystem we could transfer mentioned below results from natural aquatic ecosystems to activated sludge bioreactors, but with some cautions.

Experimental studies in lakes support the importance of resource regulation and reveal little top˗down control from protozoans (Pace & Cole 1994). Gasol & Vaque (1993), Tzaras and Pick (1994), Wieltschnig et al. (2001) and Özen (2012) results support the idea, that under eutrophic conditions compared to oligotrophic systems, between heterotrophic nanoflagellates and bacteria weak or no coupling is visible.

From the other hand laboratory microcosm experiments demonstrated that protozoans selection grazing pressure can affect the bacterial community, where grazing – resistant cells would have a strong selection advantage. This fact could explain to some extent domination of filamentous and aggregates growth bacteria forms in activated sludge (Güde 1979, 1982). Not only grazing by protozoa but also filtration mainly done by ciliates eliminates free-swimming bacteria (Pauli et al. 2001) and as consequence favors filamentous and aggregates form of bacteria. As mentioned by Hahn and Höfle (2001) in pelagic ecosystems predation by bacterivorous protists can influence the morphological structure, taxonomic composition and physiological status of bacterial communities.

The enormous predator and selection pressure exerted on the bacteria in activated sludge by filtrating ciliates showed Pauli and co-authors (2001). Their calculation showed that average ciliates densities of 10 000 ind./ml in activated sludge with average filtration rate of 100 nl/h each, can filtrate whole biological reactor in less than one hour.

As in case of other aquatic ecosystems, biological treatment processes are best viewed as exhibiting simultaneous bottom˗up (resource supply-driven) and top˗down (food web structure-driven) control of ecosystem structure and function. The incoming sewage stream supplies resources for the microorganisms. These resources stimulated the growth and reproduction of a diverse microbial community, which are then grazed by protozoans and metazoans and also killed by viral infections. The size and species composition of the microbial community in activated sludge are regulated simultaneously by the amount and composition of the sewage, by predation and by complex interactions between the present organisms (Graham & Smith 2004).

Additionally, operational parameters: HRT and MCRT which are present only in engineered ecosystems affect the microbial community in biological reactors.

These parameters are included into concept of permanence capacity/“ability to stay” originally in Spanish: El concepto de capacidad de permanencia demonstrated by Salvadó and Canals (2014). Their concept is attempt to explanation how combination of the most important abiotic and biotic factors occurring in treatment plants may influence on protozoan and metazoan community. The main assumptions of this idea are presented in details below.

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26 The concept of permanence capacity/“ability to stay” (Salvadó & Canals 2014)

The specific characteristics of wastewater treatment process entail a series of limitations in defining the ecological niches for microorganisms appearing in activated sludge system.

Microorganisms, in order to colonize and establish in activated sludge environment, must cope with (along with chemical conditions of water, toxicity and food availability) two major limiting parameters of the physical environment: the hydraulic retention time (HRT) and the mean cell residence time (MCRT). Both parameters, according to the presence of a substrate to adhere to, directly limit microorganisms with different colonization strategies and determine which strategies may be useful for occupation new niches and which may not. The concept of permanence capacity of species can be understood as the set of mechanisms that organisms present to overcome these limitations. For typical microfauna populations in activated sludge treatment systems it consists basically of two factors: the affinity to the substrate/floc surface (e.g. crawling type of movement or structures like stalks which allows adhering to flocs) and the reproduction rate.

For a given species to maintain in the system, the nutritional status of the wastewater, the tolerance of that species to the physicochemical conditions of the medium (T, pH, O2, NH4+, heavy metals concentration) and trophic interactions (e.g. predation, competition) must be considered. The HRT and the MCRT act as physical constraints, determining if species remain or not in the system. Summarizing combination of certain values of HRT, MCRT, reproduction rate and the affinity to the substrate/floc surface of the species will eventually determine the ability of the species to be present in the reactor.

Another important factor affecting protozoan, metazoan and bacterial community is sludge load called also food to microorganism ratio (F/M). Study performed by Curds and Cockburn (1970b) showed that the sludge load was an important factor which partially determines the protozoan population in activated sludge tanks. Sludge load affects the number of ciliate species. The largest numbers of species were found in sludge loaded between 0.2−0.3 gBOD5/g MLSS/day (Curds & Cockburn 1970b). Additionally, ciliates species number decreased with increasing loading. In sludge maintained at low loadings (0.09−0.3 g BOD/g MLSS/day) were found wider range of number of ciliates species. Salvadó and Gracia (1993) in their research found strong negative relationship between ciliates diversity index and organic loading rate. They clearly showed that higher ciliates diversity index were related with lower values of organic loading rate.

Summarizing, the activated sludge flocs composition and structure of microbial community is a result of biotic (predator pressure, competition and other interaction between microorganisms in food web) and abiotic factors, which could be grouped to environmental: temperature, pH, dissolved oxygen concentration, influent composition and operational: HRT, MCRT and F/M.

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27 Protozoa as bio-indicators of wastewater treatment plant performance

Curds and Cockburn (1970b) were probably the first researchers who used protozoa community as bio-indicators of effluent quality of activated sludge system plants.

Later there have been many attempts to relate the physical-chemical parameters of effluent or the activated sludge with the present species of ciliates and other protozoa (Morishita 1976, Madoni &

Getthi 1981, Al-Shahwani & Horan 1991, Esteban et al. 1991, Salvadó et al. 1995, Perez-Uz et al. 2010, Hu et al. 2013a). The general conclusion from these researches is that each plant may be expected to develop its own distinctive protozoan community which depends on the specific nature of the plant itself (Seviour & Nielsen 2010) and no general and clear pattern exists. Attempt to explain this phenomenon was described by Salvadó and co-authors (1995). They observed that relation between various physical- chemical parameters and a particular species does not follow a linear model. Whereas, statistical analyses used to relate physical-chemical parameters and protozoa are generally based on linear models.

Another problem was the range of investigated parameters. If values of these parameters was above or below the values of the optimal range for microorganisms it was easy to find good correlation coefficients with positive or negative values for the same species (Salvadó et al. 1995).

Additionally, in systems with biological nutrient removal compared to conventional systems the total protozoan abundances were lower but simultaneously more diverse (Liu et al. 2008, Dubber & Gray 2011a). Research conducted by Perez-Us et al. (2010) and Dubber and Gray (2011a) showed that results from previous studies from 80’s and 90’s on conventional activated sludge plants cannot be directly extrapolated to new treatment plants configurations and further research in this field is needed.

Still, one of the best known and popular “old” methods to estimate wastewater treatment plant performance based on observation of protozoa community is Sludge Biotic Index (SBI).

Sludge Biotic Index (SBI)

SBI method was invented and presented by Paolo Madoni in 1994. SBI method was inspired by the Extended Biotic Index (Woodiwiss 1980) and according to Madoni it is applicable to all types of activated sludge plants. SBI had set up on the basis of the results obtained by the numerous research conducted on the activated-sludge microfauna during more than 20 years’ period (Madoni 1994). This method based on two principles: i) the dominance of the protozoa key group and ii) the number of morphological species, because both factors are related to the environmental and operational condition of the treatment plant (Madoni 1994). The main advantage of this method is that SBI values are numbers from 0 to 10, grouped into four quality classes and these values can be easily used in reports or record sheets and operators can easily follow changes of the sludge conditions.

In recent years group of researchers tested the applicability of SBI method in treatment plants with different sewage e.g. Leal et al. (2013) from petrochemical industry, Pedrazzani et al. (2016) from piggery slaughterhouse. Drzewicki and Kulikowska (2011) presented some SBI limitation in WWTP

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28 working with shock organic and ammonium loadings caused by periodic sewage delivered from septic tanks.

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29 THE GOAL OF THIS STUDY

The purpose of this research was to determine the role of protozoa in ecological context in activated sludge systems with biological nutrient removal. In this thesis activated sludge was treated as a specific, semi-natural aquatic ecosystem, and the concentration of organic compounds, nitrogen and phosphorus in treated wastewater was a measure of the ecosystem's functioning. Generally, I would like to present activated sludge process from ecologist’s point of view.

This PhD thesis focused on microorganisms interaction in activated sludge food web and possibility to use protozoa community as bioindicators of the condition and the efficiency of process performance in wastewater treatment plant with biological nutrient removal.

For this purpose, three experiments in laboratory scale bioreactors were designed and conducted and four full scale municipal wastewater treatment plants were monitored during one year.

The research was based on the following hypotheses:

 Protozoa are the main consumers of bacteria in activated sludge ecosystem and for that reason they have a strong influence on bacterial community composition.

 Protozoa affect groups of bacteria responsible for biological nutrient removal process – ammonia oxidizing bacteria, nitrite oxidizing bacteria and polyphosphate accumulating organisms.

 The activated sludge ecosystems with different protozoa communities function in different way.

From these hypotheses, the specific goals of the work were formulated:

 Check how increased crawling ciliates density affects the populations and functioning of ammonia oxidizing bacteria – the bacteria group responsible for one of the most important process in nitrogen cycle.

 Check how protozoa community interacts with bacteria community during the formation of activated sludge in start-up period.

 Check how protozoa and bacterial community react to “unforeseen” events that occur during operation of wastewater treatment plants.

 To investigate if changes in protozoa community composition can be related to changes in operational or environmental parameters.

More specific research hypotheses are included in chapters in the description of each experiment.

Analysis of the possibility of using protozoa inhabiting activated sludge to evaluate the efficiency of wastewater treatment

Wydział Biologii Instytut Nauk o Środowisku