Since the late 19th century, wastewater treatment heavily relies on microorganisms, primarily bacteria. Microorganisms which can convert the harmful substances to less harmful ones are naturally present in wastewater (Henze et al., 2008). This section lists the most important types and the main reactions that they can catalyse and discusses the most popular method to remove the excess microbial biomass that is inevitably cultivated during treatment.
2.1. Organic matter (COD) removal
Chemo-organo-heterotrophs are organisms that can degrade organic substances (COD) to obtain energy while incorporating the carbon atoms from these substances into cellular compounds. The type that is of most importance for this thesis are aerobic heterotrophic microorganisms. They can degrade organics with oxygen as electron acceptor (a typical combustion reaction), while incorporating some carbon from the organics together with other nutrients into new biomass (Eq. 1.1) (Henze et al., 2008).
COD + O2 + other nutrients → CO2 + H2O + heterotrophic biomass Eq. 1.1 It can be seen that potentially harmful COD is converted into water, which is harmless, carbon dioxide, which is a gas and thus spontaneously leaves the water over time, and some microbial biomass, which still requires removal from the water (see section 2.4). This metabolism is in essence the same as that of humans. We eat organic substances (fats, sugars and proteins) and create energy by burning them with the oxygen we breathe in, while we use a part of the organics to build new cellular material. Some aerobic heterotrophs can first store the organics intracellularly and oxidize them later on (see section 2.3) (Mino et al., 1998).
Some chemo-organo-heterotrophs can also use nitrite or nitrate as electron acceptor for COD degradation and are called denitrifiers (see section 2.2). There are also heterotrophs that can degrade organic compounds under anaerobic conditions, i.e. without oxygen or nitrate/nitrite, by using iron, sulphate or even another organic compound as electron acceptor (Henze et al., 2008). The latter, which are termed fermentative and methanogenic heterotrophs, degrade larger compounds like glucose to smaller ones like acetate and eventually to methane, while simultaneously releasing carbon dioxide. Both methane and carbon dioxide escape as gases, thus cleaner water is also obtained in this case.
2.2. Nitrogen (N) removal
Microorganisms that degrade inorganic substances to obtain energy while incorporating the carbon atoms from inorganic carbon (CO2) into cellular compounds are called chemo-litho-autotrophs. The specific type that can aerobically oxidize ammonium to nitrite via the so-called nitritation reaction are Ammonia Oxidizing Organisms (AOO) (Eq. 1.2), while the ones that oxidize nitrite further to nitrate via the nitratation reaction are called Nitrite Oxidizing Organisms (NOO) (Eq. 1.3) (Corominas et al., 2010, Henze et al., 2008).
NH4+ + O2 + CO2 + other nutrients → NO2- + H2O + AOO biomass Eq. 1.2 NO2- + O2 + CO2 + other nutrients → NO3- + H2O + NOO biomass Eq. 1.3 The complete oxidation of ammonium to nitrate is called nitrification. Recently, so-called comammox organisms have been discovered which can perform both sub-steps (van Kessel
et al., 2015). Overall, nitrification converts ammonium into the ecologically less harmful nitrate, but some much more harmful nitrite can remain (Camargo and Alonso, 2006). Denitrifiers (section 2.1) can remove the residual toxicity by nitrate and nitrite by reducing these compounds with organics as electron donor (Eq. 1.4).
COD + NO3-/NO2- + nutrients → CO2 + H2O + N2 + heterotrophic biomass Eq. 1.4 As such, nitrate and nitrite are converted to harmless nitrogen gas, while organic pollutants are removed.
Even though nitrification and denitrification are the most commonly applied pathways for N removal, there is an alternative. Anaerobic Ammonium-Oxidizing (Anammox) organisms, which are again chemo-litho-autotrophs, can reduce nitrite with ammonium as electron acceptor (Eq. 1.5), with some nitrate as a byproduct (Jetten et al., 1998, Strous et al., 1999).
NH4+ + NO2- + CO2 + other nutrients → N2 + H2O + NO3- Anammox biomass Eq. 1.5 This reaction thus converts two highly toxic N compounds to primarily nitrogen gas directly, which decreases the required oxygen for nitrification (Eq. 1.2 and Eq. 1.3) and does not need organics, opposed to denitrification (Eq. 1.4). Moreover, autotrophs such as Anammox, form less biomass (Eq. 1.5) for the same amount of nitrogen removal, so less waste is produced compared to denitrification (Eq. 1.4).
2.3. Phosphorus (P) removal
P can be removed from wastewater by a specific type of aerobic heterotrophic microorganisms called Polyphosphate Accumulating Organisms (PAO). These have a cyclic metabolism based on the alternating degradation and storage of three different intracellular storage polymers. Under anaerobic conditions (no oxygen, nitrate or nitrite), they are able to store organics from the wastewater intracellularly as polyhydroxyalkanoates (PHA). They obtain the required energy for this storage by degrading other storage polymers, namely polyphosphate (PP) and glycogen. Degradation of PP causes phosphate release into the water (Eq. 1.6).
COD + PP + glycogen → PHA + PO42- Eq. 1.6
Under aerobic or anoxic conditions (in the presence of oxygen or nitrate/nitrite), they are able to oxidize the PHA that was stored earlier. At the same time, they take up phosphate to restore the intracellular PP and synthesize new glycogen (schematically shown in Eq. 1.7).
PHA + O2(NO3-/NO2-) + PO42- + other nutrients →
CO2 + H2O (+ N2) + PAO biomass + PP + glycogen Eq. 1.7 This equation shows that PAO can remove phosphate, organics (under the form of intracellular PHA) and nitrate/nitrite, which makes them highly versatile and useful for wastewater treatment. To obtain a true removal of phosphate, PAO biomass containing the
stored PP should be removed from the wastewater. Otherwise, it can be released again (Mino et al., 1998, Santos et al., 2020). In the next section, it is explained how the P-rich PAO biomass can be removed from wastewater along with the other heterotrophic and autotrophic biomass.
2.4. Biomass separation
Until now, all removal mechanisms that were discussed were based on the capability of different microorganisms to remove different pollutants. However, when these microorganisms are fed, they unavoidably multiply (see Eq. 1.1-Eq. 1.7). This cultivated biomass could still pollute natural waters, e.g. via its COD, N and P content under the form of cellular compounds. Since biomass consists of cells, which are typically heavier than water, they can automatically separate from the water by gravity. This settling is speeded up thanks to the tendency of the cells to aggregate as flocs (Farnsworth and Dick, 1972, Wahlberg et al., 1994). The next section explains how this process is stimulated in wastewater treatment plants.