Water or sediment analysis of sewage treatment plants for mobile and sessile ciliates, amoebas, flagellates and filamentous bacteria: qualitative microscopic examination (phase contrast) and after staining; Count per unit volume; Suspended solids; Molecular identification of species (sequencing).

Sludge microorganisms 

Active sludge is not an inert mass, but an active process. In wastewater and industrial waters, air and a biological group consisting of bacteria and protozoa are used to carry out a process of purification of wastewater. They comprise a diverse population of microorganisms that include Eubacteria, filamentous bacteria, rotifers, protozoa of different types and algae. For the sludge activation process to take place properly, it is essential that the resident microflora forms flocs that settle easily, resulting in a clear effluent with a concentration of suspended solids.

The active sludge must be controlled to maintain a relationship between the proportion of wastewater and the feed of the biological group that will process it. To achieve this, it is necessary to control the active sludge in terms of its aeration, the sediments that are generated and the new wastewater load. An alteration of some of these elements or their proportions can lead to an imbalance that is detrimental to the intended purpose.

The active sludge process is an aerobic treatment process that aims to reduce the amount of organic matter in wastewater using microorganisms. Microorganisms that grow in aeration tanks convert dissolved organic matter into their own biomass.

The study of existing microorganisms in a wastewater treatment system may indicate the correct or altered situation that is occurring in the wastewater processing system by the active sludge system.

Next, we describe the main groups of microorganisms involved in this process and the situations in which they can be found in sewage treatment plants by the active sludge system: ciliates, amoebas, flagellate and filamentous bacteria.


The ciliates are a diverse group of heterotrophic protozoa grouped in the phylum Ciliophora, which are present almost anywhere where there is water (lakes, ponds, oceans, rivers and wet soils). They have a size between 10 µm and 4 mm, and their main feature is that their cell is covered with cilia, which allow them to move or feed through ingestion by an oral groove with the participation of them. The cilia have a structure similar to the flagella, but they are shorter, are much more numerous, and are anchored in the basal leather. The movement of the cilia is coordinated by a primitive nerve network that is below the cell surface, so they are considered more organized than flagella. The cilia allow to develop a swimming, fixing, food and sensory function. These protozoa have two types of nuclei, macronucleus and micronucleus. The macronucleus controls non-reproductive functions such as metabolism and homeostasis, while the micronucleus controls the reproductive division. On the surface of these protozoa, the cilia and the oral groove stand out. Inside, in addition to the two nuclei, food vacuoles and contractile vacuoles can be observed.

Not all ciliates are free and mobile, but some are fixed (sessile). Mobile ciliates move thanks to cilia, while sessile ciliates are fixed through a foot or spiral filament (leg) to a substrate. The axial filament, when it exists, is used to extend and contract. The main difference between mobile and sessile ciliates is that mobile organisms can move from one place to another, while sessiles are not able to move from one place to another. Some sessile organisms have immature stages in which they are mobile and viceversa, so this differentiation is only important as a first approximation.

Around 4,500 unique free-living species have been described, and the potential number of existing species is estimated between 27,000 and 40,000. This number includes many ectosymbiotic and endosymbiotic species, as well as some obligate and opportunistic parasites.


The amoebas are protozoa of 100 to 300 µm, without a concrete form, but the form is changing between oval and irregular, with periodic protrusions that extend from the main cellular mass. They move by protusions to foot modes called pseudopods (false feet). The pseudopod expands and then the main cell mass moves into the protusion. Sometimes they can be difficult to detect because they move slowly.

Amoebas can be mobile or cystic trophozoites. The mobile phases are usually frequent in the start-up stages of the installation or in very overloaded treatment plants. Cystic amoebas usually indicate a stable or slightly overloaded treatment system.


Flagellates are small ovoid or pyriform organisms of 5 to 20 µm with one to 4 long flagella for movement, fixed to one of the ends of the cell, which move in the liquid phase. Some flagellates form colonies in which cell bodies are together and their flagella project outward. Some contain chlorophyll and perform photosynthesis, so due to this particular characteristic of resembling plants, they are usually classified as flagellated algae rather than with protozoa. The locomotion of the flagellates is generally fast turning on themselves as the flagella move to propel them. This makes them appear as disorganized active organisms, unlike ciliates that have an organized locomotion mechanism, which can help their identification.

Flagellate feeds on soluble organic matter and scattered bacteria. They are more frequent in very overloaded wastewater treatment plants or during the start-up stages of the facilities. They predominate when there is a high density of isolated bacteria, and they are related to turbid effluents produced at times of greatest toxicity. They also increase when there is inadequate aeration that causes anaerobic conditions in the treatment tanks. Its presence in the wastewater treatment system indicates high levels of soluble biochemical demand, low concentrations of dissolved oxygen and a high organic load. The first life forms are considered after a chemical alteration occurs in the wastewater system. In this way, they are useful to indicate when the system is improving or if it continues with the alteration.

In wastewater treatment plants they are surrounded by bacteria. These unicellular protozoa are one of the first indicator microorganisms seen in wastewater treatment plants. In the treatment system they consume bacteria and absorb soluble organic compounds for their nutritional requirements.

In general, flagellates are observed in the initial phases of wastewater treatment or when there is a stage of logarithmic growth of bacteria.

In activated sludge, the sudden appearance of flagellates may indicate an increase in organic load or a toxic event that kills a significant portion of bacteria, so it must be studied what is happening in order to increase its population.

Filamentous bacteria 

Filamentous bacteria are an important component of the treatment process by the active sludge system. Low concentrations of filamentous bacteria are required to sediment the solids in healthy active sludge. As the population of filamentous bacteria in the system increases, they cause the formation of brown adherent viscous foam.

The active sludge process depends on an ecosystem balance between the bacteria communities present in the system, Aeromonas, Achromobacter, Alcaligenes, Bacillus, Flavobacterium, Micrococcus, Pseudomonas, Thiobacillus, Acinetobacter, nitrifying organisms, Nitrosomonas and Nitrobacter.

The main problems related to the active sludge system are the increase in volume, deflocculation, foaming and the increase in solids (denitrification). Of the many filamentous microorganisms indicated in the literature, only some such as Microthrix parvicella, Nostocoida limicola, Nocardia spp., Gordonia amarae and Eikelboom species types 021N and 0041, have been considered repeatedly related to increased volume and foams.

The thick foam layer is formed on the surface of the mud sedimented by mycolic acid producing bacteria. The composition of wastewater is one of the significant factors that affect foaming since it has been found that slow-degrading organic material and lipids favor the growth of both Microthrix parvicella and Gordonia amarae. This occurs when the age of sludge is greater than 10 days, and it is suggested that it occurs in wastewater treatment plants that operate at low temperatures.

The filamentous bacteria form the macrostructure of the flocs, resulting in a clear effluent with a low concentration of suspended solids. To do this, the macro structure of the floc facilitates the adhesion of the floc. The majority of activated sludge plants around the world suffer from the problem of bulging and/or foaming, caused by the proliferation of some filamentous bacteria. Foaming is a problem that was initially thought to be caused by Nocardia (Gordonia) amarae. Other more recent studies have identified a large number of filamentous bacteria in foam samples. These include other species of Nocardia, Microthrix parvicella and various morphological types of Eikelboom. The five dominant types are usually types 0092, 0675, 0041 and 0914, and Microthrix parvicella.

Tests performed in IVAMI: 

  • Microscopic examination in contrast of phases and after staining (Gram and Neisser) for mobile or sessile ciliates, free or cystic amoebas, flagellates isolated or in clusters, filamentous bacteria and their characteristics.
  • Count per unit volume of the microorganisms mentioned.
  • Suspended solids.
  • Molecular identification of species (sequencing).

Sample recommended:

  • Wastewater from treatment plants (1,000 mL in sterile polypropylene container), foams (250 mL in sterile polypropylene container).

Preservation and shipment of the sample:

  • Shipment within 24 hours to the laboratory under refrigeration conditions (white cork container -expanded polystyrene- with frozen packs).

Delivery of results:

  • Microscopic examination in contrast of phases and after staining (Gram and Neisser) for mobile or sessile ciliates, free or cystic amoebas, flagellates isolated or in clusters, filamentous bacteria and their characteristics: 48 to 72 hours.
  • Count per unit volume of the microorganisms mentioned: 48 to 72 hours.
  • Suspended solids: 48 to 72 hours.
  • Molecular identification of species (sequencing): 4 to 5 working days.

Cost of the test:

  • Microscopic examination in contrast of phases and after staining (of Gram and Neisser) for mobile or sessile ciliates, free or cystic amoebas, flagellates isolated or in clusters, filamentous bacteria and their characteristics (each group): Consult to ivami@ivami.com.
  • Count per unit volume of the microorganisms cited (each): Consult to ivami@ivami.com.
  • Molecular identification of species (sequencing) (each): Consult to ivami@ivami.com.