Biological treatment

Bacterial power! Know about their demands and how to influence them

With biology it is about biodegradability of organic matter. Mostly bacteria are used to purify wastewater. Bacteria are maintained in (bio)reactors.

All such bacteria and microbiology in general + other ‘solids’ together is named sludge.

With eating and growing biomass wastewater will be purified. On the other hand, the amount of sludge will increase over time. For biological treatment it’s important to have a specific amount and quality of sludge in the bioreactor:

  • For the amount of sludge we basically talk about Mixed Liquid Suspended Solids (MLSS). In several g/l for example.
  • For quality it’s about the type or species of bacteria selected in the sludge, and how active they are. Furthermore, not all MLSS is organic. To deviate such the abreviation MLVSS is used as well. It’s the volatile part of MLSS = the organic part. Often about 60-80% of the sludge is organic.

After biological treatment the sludge will be separated from the water, resulting in:

  1. purified water = (final) effluent. To be discharged (to the environment) or further purified (and reused).
  2. sludge. To be returned to the bioreactor (= return activated sludge, RAS) + to prevent an increasing amount of sludge in the bioreactor (from growth), part of it must be removed as well (= waste activated sludge, WAS).
Basic setup of a WWTP. More ‘sophisticated’ concepts are described below; see (an)aerobic treatment.

Over the years many types of sludge and species of bacteria are addressed. From that, many types of treatment concepts are developed as well. Below we have simplified it to two main types:


With oxygen.

For the best purified water. Low in carbon, nitrogen, phosphorus, and more. Compliant with discharge limits.

More sludge produced.

Larger footprint.


Without oxygen.

For the best ‘energy balance’. Much lower energy consumption then aerobic. Production of biogas!

Less sludge produced.

Smaller footprint.

Bacteria to grow in a different ecology

suspended growth

Bacteria grow in flocs.

All flocs/sludge is mixed in the water (mixed liquid suspended solids, MLSS).

Such sludge (from outlet) can be separated by settling, flotation or filtration.

  • with a treated water = effluent.
  • with the sludge to be recirculated back to the bioreactor (RAS) and partly removed (WAS).

attached or biofilm growth

Bacteria grow in biofilms.

The biofilm/sludge is fixed on a medium. Medium is fixed or trapped in the bioreactor. And so sludge will stay in bioreactor as well.

Excess sludge is ‘srubbed’ from the medium, to washout with the water. Such sludge (from outlet) can be separated by settling, flotation or filtration too.

  • with a treated water = effluent.
  • with the separated sludge to be removed (WAS). Basically no return sludge applicable.

granular growth

Bacteria grow in granules.

All granules/sludge is mixed in the water (MLSS). With special designed reactors such sludge will stay in reactor too.

  • with a treated water = effluent.
  • with a sludge (from bioreactor) to be partly removed (WAS).

These concepts are further described below, with aerobic and anaerobic technologies.

Bacteria to grow under different redox conditions

aerobic or oxic

With oxygen.

Ideal for removal of carbon (COD/BOD), and for nitrification1.

1 conversion of nitrogen (from ammonium NH4 to nitrite NO2 and nitrate NO3).


Without oxygen. With nitrate.

Ideal for nitrogen removal by denitrification2, and indirectly removal of carbon (COD/BOD).

2 conversion of nitrogen (from nitrate NO3 and nitrite NO2 with consumption of carbon, to nitrogen gas N2).


Without oxygen. Without nitrate.

Ideal for removal of carbon (COD/BOD) by biogas production, phosphorus by biological phosphorus removal (bio-P)3, and nitrogen by deammonification4.

3 with specifically adapting redox conditions over time, the final waste activated sludge (WAS) will contain much more phosphorus, the so called ‘luxury uptake’.

4 by Anammox. Conversion of nitrogen (from ammonium NH4 with nitrite NO2 to nitrogen gas N2).

These concepts are further described below, with aerobic (including each redox condition) and anaerobic technologies.

As well as more information is given with carbon, nitrogen or phosphorus removal.

Bacteria to remove 3 main components


formation of new cell material, removed with the sludge.

conversion to carbon dioxide CO2, emitted to the sky.

conversion to bio methane CH4, utilised as biogas.

other, like formation and extraction of extracellular polymeric substances (EPS). As ‘new’ concept in Aerobic granular sludge (AGS) plants.


formation of new cell material, removed with the sludge.

conversion to nitrogen gas N2, emitted to the sky.


formation of new cell material, removed with the sludge.

biologically ‘luxury update’ for extra phosphorus ‘captured’ in and removed with the sludge.

non-biologically, but chemicals to precipitate phosphorus. Also ‘captured’ in and removed with the sludge.


Below in short some information about microbiology. We are interested in understanding the metabolism of especially bacteria. Metabolism can be defined as “the chemical processes that occur within a living organism in order to maintain life” or more shortly “sum of all reactions in a cell”.

More generally, we are interested in how to ‘manipulate’ the conditions for bacteria in such way that single or ‘cooperating’ species of bacteria will convert ‘food’ into targeted ‘waste products’.

As simplified mass balance of the metabolism of a bacteria, we could define:

Carbon source + Energy source + Oxygen source = ‘waste products’ + new cell material

Autotrophic vs Heterotrophic:


Using inorganic carbon source (usually CO2).

Slow growing organisms.


Using organic carbon source (organic matter).

Fast growing organisms.

Matrix of processes:

Carbon removal

Most wastewaters contain all kind of organic matter. Organic matter is mostly carbon.

Carbon in wastewater is basically expressed as COD (chemical oxygen demand), BOD (biological oxygen demand) or TOC (total organic carbon).

In many countries discharge limits of 125 mg/l COD and 25 mg/l BOD5 are applicable.

Removal of COD/BOD/TOC can be done highly efficient with bacteria. As explained before we focus on 3 main concepts:

  1. Aerobic, with oxygen, to produce CO2 + (more) sludge
  2. Anaerobic, without oxygen, to produce biogas CH4 + (less) sludge
  3. biosorption related technologies (in pre-treatment), to embed more ‘energy’ in the sludge. For example for disposal or later digestion (to biogas CH4).

With COD it’s a test where chemicals are used to oxidise the organic material. Basically it’s an 2-hour quick test.

Where BOD is the oxygen demand related to what is biologically removed in a specific time frame. Often a 5-day test is used (at 20°C): BOD5. Other tests relay on 7 or 20 days for example. Such longer duration will give higher BOD values, while microorganisms had more time to consume ‘all’ organic material.

COD > BOD20 > BOD7 > BOD5.

Deviated from BOD and COD a third method is available: TOC (total organic carbon). It’s a very fast (a few minutes only) and reliable test as well. The production of CO2 is measured upon complete oxidation of organic material through combustion at high temperature. The CO2 mass is indicative of the mass of organic carbon that was initially present in the sample. The ratio TOC/COD is not a fixed value, unfortunately. Although it’s sophisticated equipment it has the benefit of the high speed, with ability to use it as an online and realtime measurement.

Nitrogen removal

In wastewater treatment nitrogen became a very import parameter to be removed. Like with phosphorus it’s about protection of the environment. Protection from unwanted eutrophication of surface waters.

In many countries discharge limits of 30 mg/l total-N are applicable. Limits still going down; sometimes already as low as 10 mg/l total-N only.

Below we described some aspects:

Nitrogen balance and cycle

Nitrogen is an important compound in biology. Bacteria need it for growth. Bacteria can be very helpful for us to convert it from one to another appearance. Often started from an organic nitrogen (org-N), and step wise converted to nitrogen gas (N2) as aimed result, to emit that to the sky. In parallel, with bacterial growth organic, some nitrogen will be assimilated as well, embedded in and disposed with the (waste) sludge. And so, nitrogen is removed from the water.

With each nitrogen form expressed as N you can calculate it.
Total N = Organic N + Inorganic N
Kjeldahl TKN = Organic N + Ammonia N
NOx-N = NO3-N + NO2-N
Source: Ecological Energetic Perspectives on
Responses of Nitrogen-Transforming
Chemolithoautotrophic Microbiota to
Changes in the Marine Environment (2017)

Different species of bacteria

In text below several abbreviations will be used. Some explained here. Classified in 2 groups:


slow growing. sensitive. prefer higher temperature, in the range of 15-38°C and basically higher is better.

  • AOB = Ammonia Oxidising Bacteria. Nitrosomonas sp.
  • CMX or Comammox = COMplete AMMonia OXidising bacteria. Research ongoing; recently ‘discovered’. Nitrospira sp.
  • NOB = Nitrite Oxidising Bacteria. Nitrobacter sp.
  • AMX or Anammox = ANaerobic AMMonium OXidising bacteria. Anammox sp.


heterotrophic. fast growing. easy well behaviour.

  • OHO = Ordinairy Heterotrophic denitrifiers. Many species.

Ammonification first

Conversion of organic nitrogen (org-N) to ammonium (NH4).

Ammonification is the biochemical degradation of organic nitrogen into ammonium. It’s basically done by bacteria consuming organic carbon. These are called heterotrophic bacteria. These bacteria can transform the nitrogen either in the presence of oxygen (aerobic conditions) or without oxygen (anaerobic conditions).

In a wastewater system ammonification of organic nitrogen is already (partly) done in the sewer and septic, buffer, equalisation tanks. Further on more will be converted in the bioreactors as well. Some of the organic nitrogen, however, will not be degraded and remain as low residual value in the (final) effluent.

Traditional nitrification-denitrification

Conversion of ammonium (NH4) to nitrogen gas (N2).

  • Nitrogen to be converted from ammonium (NH4) via nitrite (NO2) to nitrate (NO3), named nitrification. That process requires aerobic conditions (with oxygen). AOB and NOB organisms doing the job.
  • To also remove those nitrogen compounds (NOx) from the water, denitrification is needed. That process will execute under the absence of oxygen, with the availability of carbon (COD, BOD, TOC). Denitrification is to convert nitrate (NO3) via nitrite (NO2) to nitrogen gas (N2), which will be emitted to the sky.

Depending on the chosen process steps, a compliant final effluent is then possible, having all forms of nitrogen at low levels: low organic-N + low NH4 + low NOx = low total nitrogen (TN).

In parallel from such removal from the water to the air, some nitrogen will also be removed with the grown biomass and sludge.

Over the years several alternatives are developed. Specially to deal with scenarios of limited availability of carbon. The so named C/N ratio must be high enough to remove enough nitrogen from the water via nitrification/denitrification processes.

See some alternatives below:

External addition of carbon source

It has the idea to overcome the carbon deficit for the denitrification process.

Nitrite shunt

More efficient conversion of ammonium (NH4) to nitrogen gas (N2).

In some wastewaters the conditions are not in favor for a full nitrification like above.

  • The idea with this ‘shunt’ is elimination of both the second nitrification step and the first denitrification step.
  • The same organisms doing the job, except the NOB to be eliminated, by disfavoring their conditions.
  • It can save 25% on oxygen demand + 40% on carbon consumption!

Anammox (ANaerobic AMMonium OXidation)

Most efficient conversion of ammonium (NH4) to nitrogen gas (N2).

In some high loaded nitrogen rich wastewaters, or sidestreams from sludge digesters, the amount of nitrogen is very high (in relation to available carbon or COD/BOD).

  • Here the idea is to eliminate the second nitrification step as well. Furthermore to minimize first nitrification step + to eliminate even the full denitrification.
  • AMX is introduced in doing the job. Furthermore, process conditions are favored for AOB as well.
  • Conditions for NOB, CMX and OHO to be disfavored.
  • The benefit of these Anammox bacteria is it’s ability to anaerobically (without oxygen) convert ammonium (NH4) to nitrogen gas (N2) directly, by consumption of nitrite (NO2). That nitrite originates as result of the first step of nitrification by AOB.
  • It can save 62% on oxygen demand + 100% on carbon consumption!

Automation or Advanced Control

Optimising solutions (like above), in favor of nitrogen removal. Instead of carbon removal.

Phosphorus removal

In wastewater treatment phosphorus became a very import parameter to be removed. Like with nitrogen it’s about protection of the environment. Protection from unwanted eutrophication of surface waters.

In many countries discharge limits of 2 mg/l total-P are applicable. Limits still going down; sometimes already as low as 0.15 mg/l total-P only.

Differently from carbon and nitrogen removal, phosphor doesn’t have compounds that can be emitted to the sky. Therefor all is related to cell growth, to be removed with the excess sludge. Unfortunately, many wastewaters contain more phosphate then needed by the ‘standard’ bacteria.

Therefor some alternatives are developed over the years:

Bio-P or Enhanced Biological Phosphorus Removal (EBPR)

  • Luxury uptake of phosphorus by bacteria. More specifically by the poly-phosphate accumulating organisms (PAO). Those types of bacteria have the ability to take more phosphorous with them then they strictly need. It’s a process depending on having the different environmental conditions of anaerobic (without O2 and NOx) and aerobic (with O2).
  • It can remove up to about 5x more phosphorus then by ‘standard’ sludge growth only. The captured phosphate will be removed with the excess sludge.

Simultaneously precipitation

  • In other words, addition of chemicals like FeCl3.
  • It will react with ortho-phosphate to form flocs. Those flocs will be captured in the sludge, to be disposed with the excess sludge as well.

Aerobic treatment

Different from the anaerobic solutions (without input of oxygen) these aerobic solutions require oxygen to remove the carbon (and nitrogen).

With aerobic solutions we define the bioreactors where we need oxygen for the removal of pollutants. More precise it’s about the systems where we aim to oxidise carbon and nitrogen to CO2 and N2 respectively on one hand. On the other hand, in such systems we will produce a sludge also containing carbon and nitrogen compounds. Furthermore, phosphorous and many other compounds will be in the sludge too.

Resulting in emitting to the sky + removal with the sludge = clean effluent to be discharged or reused.

Over the years different types of reactors and processes are developed, like:

suspended growth

Suspended growth; bacteria grow in flocs.

Continuous (CAS) or batch (SBR) reactors.

Sludge separation by settling or flotation.

Alternative: Membrane bioreactor (MBR) to make such systems more compact + for superb effluent quality

attached growth

Attached growth; bacteria grow in biofilms.

Moving bed (MBBR) or Fixed bed (FBBR) Bioreactors.

Sludge separation by settling, flotation or filtration.

Benefits: more compact + less sensitive for calamities.

granular growth

Granular growth; bacteria grow in granules.

Anammox (deammonification) or Aerobic granular sludge (AGS)

Sludge separation by density or size difference.

Benefits: more compact + superb for biological nutrient removal (BNR)

Schematised such systems can be simplified till some main hardware concepts. Sketches below are generalised as well.

From these many more concepts can be configured. Some examples:

  • Where the bioreactor is drawn as 1 reactor here, it could be several in reality. For example several reactors for different purpose and different redox condition (an)oxic vs (an)aerobic.
  • Systems can be designed as batch reactor as well. In batch systems often all steps, redox phases, and even sludge/water separation, are time-shifted in 1 reactor only.

Anaerobic treatment

Different from the aerobic solutions (with input of oxygen) these anaerobic solutions are fully operated without addition of oxygen.

It makes these systems highly energy efficient. Significant less (electrical) energy is needed + Biogas is produced = Nett energy produced solutions!

Where these solutions are extremely energy efficient in removing COD, other compounds like nitrogen and phosphorus will basically NOT be removed. It will even be released from an organic to reactive form.

  • Organic-N → NH4-N
  • Organic-P → PO4-P

Such compounds must be removed in a following (aerobic) treatment step. Also the effluent BOD or COD values often are not yet low enough to comply with discharge limits. Therefor additional aerobic treatment is needed as well.

Since decades it’s known that conversion of organic matter to biogas (by anaerobic processes) has significant benefits above oxidation till carbon dioxide (by aerobic processes). Over the years different concepts are developed.

mixed sludge

Suspended growth; bacteria grow in flocs.

Anaerobic Contact Reactor (ACR) or Anaerobic Digester (AD).

Medium loaded system.

Due long HRT (often 1-3 weeks) basically no sludge recirculation needed.

Basically used in the sludge line.

Alternative: Membrane bioreactor (anMBR) or other sludge retaining solutions, are used to make such systems more compact.

granular sludge bed

Granular growth; bacteria grow in granules.

Upflow Anaerobic Sludge Blanket (UASB) reactors.

High loaded system, typically 10-15 kg COD per m3/d. About 1 m/h upflow.

HRT of hours only.

Sludge separation by settling (3-phase separator).

Used in the water line.

Benefits: much more compact.

expanded granular sludge bed

Granular growth; bacteria grow in granules.

Expanded Granular Sludge Bed (EGSB) or Internal Circulation (IC) reactors.

Highest loaded system, typically 15-30 kg COD per m3/d. Over 6 m/h upflow.

HRT of hours only.

Sludge separation by settling (3-phase separator).

Used in the water line.

Benefits: most compact + improved water/sludge contact + enhanced segregation of small inactive SS from sludge bed + low strength influent possible.

Schematised overview of the above reactor concepts is nicely illustrated in figure below.

Source: “Biological Wastewater Treatment: Principles, Modelling and Design (2008)”