The descriptions in the bullitins below are extracts of process & technology learning material and assets, purposely authored to support WWT process and technology technical personnel attending the process.

Oxidation of reduced carbon as the main nutrient source for WWT bio-culture

Suspended and dissolved in the wastewater influent are bio-degradable nutrient particles and molecules, composed of reduced carbon polymers, oligomers and monomers. The suspended biodegradable particles have to go through additional enzymatic degradation before being available for oxidative processes by the bacteria in the bio-culture. Dissolved biodegradable molecules are immediately available to be used during respiration, and their oxidation release energy to the bacterial cell to power all the metabolic processes.

The key substrate molecules available for immediate digestion and aerobic respiration are molecules like complex sugars and sugars, glucose, fructose and sucrose, fatty acids like acetate, propionate and butyrate and amino acids like, alanine, glycine and lycine.

Digestion of carbohydrate molecules (complex sugars) lead directly to the formation of sugars and fatty acids, whist the digestion of long and medium chain fatty acids form the vital metabolic intermediates of the glycolysis catabolic process. These intermediates are short chain fatty acids like pyruvate and acetate, that immediately enter the citric acid cycle of catabolic breakdown, for energy release.

Digestion of complex sugars (carbohydrate) can be presented through the following reaction:

[CH2O]n + Enzymes => n/6 Glucose-6-Phosphate => 2 Pyruvate => n CO2 +H2O

[C6H10O5]n (Glycogen) + 6n O2 => 6n CO2 + 5n H2O

Digestion of simple sugars can be presented through the following reaction:

Glucose + Enzymes => Glycolysis => Glucose-6-phosphate => 2 Pyruvate => 6 CO2 +H2O

[C6H10O5] (Glucose) + 6 O2 => 6 CO2 + 5 H2O

Digestion of fatty acids can be presented through the following reaction:

CxHyOz + Enzymes => beta oxidation => Acetyl-co-A => CAC => X CO2 +H2O

C15H30O2 (pentadecanoic acid) + 15 O2 => 15 CO2 + 15 H2O

Digestion of amino acids can be presented through the following reaction:

CxHyNzOa + Enzymes => Pyruvate & Acetyl-Co-A => CAC => X CO2 +H2O + NH3

4C6H13NO2(Leucine) + 30 O2 => 24 CO2 + 20H2O + 4NH3

Orthophosphate, are life critical molecules for all organisms, especially the bio-culture communities at work in wastewater treatment systems. Municipal wastewater contains a very high load of orthophosphate due to the presence of digested food in the faecal matter discharged to the treatment works.

New phosphorous permits in phosphate sensitive areas, may well be a lot lower than the minimum of phosphate concentration required to maintain a healthy bio-culture. Several techniques for removal of the phosphate from the discharge water are practiced, with chemical precipitation before and after the secondary bio-processes being most widely used.

Biological Phosphorous Removal utilises bio-culture in the secondary treatment to remove the Phosphorous from the discharge water. The process requires the concentration of Phosphorous Accumulating Organisms (PAO) in the culture, which are capable of polymerising orthophosphate into polyphosphate polymer, as an energy storage mechanism.

Phosphate molecules, as orthophosphate PO43-, play a key role in energy transfer and energy storage during prokaryote metabolism. Phosphate form high-energy anhydride bonds, specifically phospho-anhydride bonds, which are energy-rich P-O-P linkages formed by the dehydration of phosphates. These phospho-anhydride bonds are critical in PAO metabolism, as the two bonds in ATP that release large amounts of free energy (Δ𝐺-30.5 kJ/mol) upon hydrolysis, are equivalent to the P-O-P bonds in polyphosphate.

The accumulated polyphosphate polymer are essentially used by the PAOs as energy supply molecules, that can be used to generate ATP by enzymatic hydrolysis. The enzymes involved are Alkaline Phosphatase (ALP) and Adenylate Kinase (ADK) which will produce ATP for metabolic activity, especially the assembly of carbon polymer, Beta-Hydroxyalkanoates (BHA).

Wastewater Treatment – The “Biofiltering” action of Activated Sludge floc to remove pollutants, mass transfer and mass conversion, described by Fick’s laws for mass transfer by diffusion.

This content is extracted from learning material for Activated Sludge Process systems, advanced learning a-06, and Mass Transfer, specialised topics s-03 

The pollutant mass transfer by convection is rapid, especially for the particulate fractions, which we can see to right, marked with the subscript sed, for sediment. The fractions in the supernatant, represent the dissolved substances, molecules, colloidal elements and ions. The mass transfer of inert fractions, marked s u p, for supernatant, do not readily transfer by diffusion, as there is usually not a concentration gradient toward the inside of the bioculture. The biodegradable fraction BOsup, diffuse readily toward the inside, as the concentration gradient rapidly regress due to metabolic activity in the bioculture.

Initially the mass transfer of substances in the wastewater takes place by convection, and particles and substances come in contact with the outer layer of the biomass biogel surface. Particulate solids will attach and get trapped in the outer layer, and from there the mass will transfer by physical mechanical means to migrate to the internal spaces of the softer biomass. Very small particles, molecules and dissolved substances will transfer their mass through diffusion. Diffusion mass flux can mathematically be described by Ficks first law, which includes the important measurable parameters of time, area, mass, diffusion coefficient, distance and concentration. The concentration gradient is described by the change in concentration per change in distance in the unit of time. Ficks first law applies more accurately for diffusing substances that are not bio available for metabolic processes of the bioculture.

Dissolved molecules like volatile fatty acids, proteins and ammonia also transfer to the sludge floc boundary layer by convection. In the boundary layer, the development of concentration gradients toward the internal bio-gel spaces, become the net force toward the metabolic centres inside the bio-floc and bacterial cells. Dissolved substances transfer to the inside by diffusion, due to the concentration gradient as described by fick’s second law. Because the biodegradable substances are being converted by the bioculture, their concentration is drastically reduced along the diffusion path x. This means that the change in concentration dE, vary with time by dt, and we have to apply Fick’s second law to find the concentration gradient for the substance E, given by the second derivative for the first law equation.

Slide (1), Wastewater Treatment process & technology – Optimising de-nitrification to minimise nitrous oxide emissions.

This section was extracted from an advanced learning module A-08, anoxic bio-chemistry for activated sludge processes, section 1.

Nitrous Oxide emissions from WWT processes are a concern due to their greenhouse gas potential. Although records of Nitrous Oxide emissions have been shown to minimal, there are conditions in certain WWTw, where these emissions are unacceptably high. In the next section we look at the origin of N2O from the process, and how to re-design the process to minimise them.

The attached diagram shows an Activated Sludge process with an additional tank before the bio-reactor, that can be used to optimised de-nitrification. RAS is recycled into the Anoxic/Dilution tank, and there is an additional FE recirculation system to be used as a further part recirculation loop for nitrate.

To generate favourable conditions in the de-nitrification tank, there should be no dissolved oxygen left in the returned sludge and make-up water. Most of the nitrate that are left in the RAS and the recirculated FE effluent, would serve as the electron acceptor during the de-nitrification process during anoxic conditions.

Bacterial di-nitrification take place via four enzyme catalysed reactions as an electron transport chain, with the different nitrogen-oxygen bonds accepting the electrons sequentially, until the terminal electron acceptor is released as diatomic nitrogen gas N2. In order for a Nitrate molecule to be completely reduced to Nitrogen requires the enzymatic donation of 5 electrons, in total 10 electrons to form 1 diatomic nitrogen.

If we compare the bond energy in nitric oxide, a single triple bond, with the bond energy for the two Nitrous Oxide double bonds, the high activation energy required to overcome the formation of N2O become apparent. Nitrous oxide is also a gas, and the next step in the reduction requires a further 2 electrons from the enzyme catalysing the reduction. The high activation energies dictate a steady supply of electrons from the carbon oxidation reactions in the cell cytoplasm, as well as the presence of adequate alkalinity to maintain the proton balance. If these two conditions are not at an optimum, the reduction to nitrogen is slowed, and nitrous gas may easily escape and emit from the water surface.

The design of the process as shown on the diagram, allow for a stringent anoxic environment, with high quantities of nitrate as electron acceptors present, as well as the highest possible concentration of SVFAs for fast fermentation and quick supply of electrons to drive the reduction. Alkalinity dosing can easily be affected by making sure the pH of the reaction water remain basic or at the optimum pH of 7.5.

Slide (2), Wastewater Treatment process & technology – Optimising de-nitrification to minimise nitrous oxide emissions.

This section was extracted from an advanced learning module A-08, anoxic bio-chemistry for activated sludge processes.

The conditions that will ensure rapid de-nitrification without stressing the enzymes, as mentioned in section 1, are

1 the presence of the nitrate as electron acceptors

2 a steady supply of short chain fatty acids to provide electrons

3 and sufficient bicarbonate alkalinity to maintain the proton gradient across the membrane

On the attached diagram the sequential reduction steps are shown, but this academic method of writing introduces confusion, because the redox reactions take place randomly, at the same time and without prejudice on a first come first served basis. Although the Nitrate electron transport chain have 4 unique enzymes that can only bind its specific reactant, the overall functioning of the energy management and transfer, share the enzyme activity with other enzymes and enzyme complexes form the aerobic electron transport process. 

The nitrate molecules are reduced to nitrite with the enzyme Nitrate Reductase, with the active site of the enzyme facing toward the cytoplasm of the cell. This orientation is crucial for the overall energy yield of the complete reduction to nitrogen, on the opposite side of the membrane anchoring most of the enzymes. Nitrate, with its three nitrate-bound oxygen bonds, has roughly the same redox potential as molecular oxygen, the main electron acceptor for the aerobic electron transport chain. In practise though, the energy from nitrate reduction is substantially less than for normal aerobic electron transport.

As a consequence of the orientation, when the enzymes that are catalysing the formation of water, for both electron acceptors, nitrate and oxygen, release water, the reaction products are released on the basic side of the membrane, having used (removed) 2 X hydrogens (acidity) from the cytoplasm. Nitrite is also released in the cytoplasm, and have to migrate through a special channel to the acidic side for the rest of the reduction reactions to occur.

Excess energy transferred to the trans-membrane enzymes during the redox reactions, is used to induce (pump) hydrogen ions across the membrane, from the basic side, to the acidic side. The hydrogen ions on the outside of the cell in the periplasma, cause the more reductive environment needed for the three other reactions with higher activation energy to proceed. The hydrogen ion concentration and charge gradient, is an electrochemical gradient (Proton motor force) that is used by the enzyme ATP synthase to generate ATP in the cytoplasm, by allowing the flow of protons back through the membrane.

Slide (3), Wastewater Treatment process & technology –Optimising de-nitrification to minimise nitrous oxide emissions.

This section was extracted from an advanced learning module A-08, anoxic bio-chemistry for activated sludge processes.

Nitrate, with its three nitrate-bound oxygen bonds, has roughly the same redox potential as molecular oxygen, the main electron acceptor for the aerobic electron transport chain. In practise though, the energy from nitrate reduction is substantially less than for normal aerobic electron transport.

The nitrate molecules are reduced to nitrite with the enzyme Nitrate Reductase, on the cytoplasm side of the membrane. Nitrite is released into the cytoplasm, and have to migrate through a special channel to the acidic side for the rest of the reduction reactions to occur. In the periplasm, with its higher concentration of hydrogen ions, the enzymes catalyse the N-O bond cleavage and the addition of two hydrogens, in one step, holding the oxygen in its most favourable position until the reaction is complete.

All three the enzymes remove two hydrogens for each catalysed reaction of a N-O bond cleavage, the net result is the removal of 6 hydrogens from the periplasmic matrix. This affects the energy yield from Nitrite reduction by reducing the hydrogen ion concentration and therefore the electrochemical gradient to the cytoplasm side of the membrane.

The hydrogen ion concentration and charge gradient, is an electrochemical gradient (Proton motor force) that is used by the enzyme ATP synthase to generate ATP in the cytoplasm, by allowing the flow of protons back through the membrane.

Interruptions in the supply of electrons from the metabolic centres in the cytoplasm, may be the cause of most Nitrous Oxide release from the WWTp during denitrification. When the process is operated with a sufficient pre-acidification (anaerobic zone for fermentation), before the anoxic zone, the steady supply of SVFAs are possible, without the need for additional carbon source.

Low bicarbonate alkalinity impacts the electrochemical gradient if the cytoplasm, where substantial quantities of hydrogen ions are continuously generated from the SVFAs fermentation is allowed to become acidic. Bacteria have mechanisms to actively transport bicarbonate ions into the cytoplasm, and these larger ions can’t enter the periplasm by diffusion. Bicarbonate alkalinity is at its highest throughout the water and cell matrix at a pH of 7.5

Slide (4), Wastewater Treatment process & technology –Optimising de-nitrification to minimise nitrous oxide emissions.

This section was extracted from an advanced learning module A-08, anoxic bio-chemistry for activated sludge processes, section 4.

Although records of Nitrous Oxide emissions have been shown to be minimal, there are conditions in certain WWTw, where these emissions are unacceptably high. Let us look a bit closer at some of these conditions.

The attached graphic shows a activated sludge process designed with the approach of optimising de-nitrification in a dedicated anoxic zone, and minimising spontaneous anoxic-denitrification in the other anoxic & anaerobic zones that may exist in the bio-reactor or final settlement tanks.

The design shows the process with an additional tank before the bio-reactor, that can be used to optimise the de-nitrification. RAS is recycled into the Anoxic/Dilution tank, and there is an additional FE recirculation system to be used as a further part recirculation loop for nitrate. The anoxic tank is positioned to receive influent which has been subject to pre-acidification, to allow for the necessary levels of fermentation products to be available as a SVFA carbon source.

The anoxic tank can handle solids trough the bottom to allow for the RAS to be efficiently returned to the main reactor after de-nitrification has been completed. The anoxic tank can be operated with a variable volume by emptying the flow through the bottom hopper with the solids. This aspect allows for flexibility for adjusting the HRT of the anoxic conditions to requirements, and to adjust the concentration of incoming ammonia before entering the main reactor, by part recirculating the FE. (Improved de-nitrification with high concentration)

Nitrous oxide emissions are known to become excessive when the enzymatic reduction processes of de-nitrification are incomplete, stressed or interrupted. This may happen when de-nitrification happen in the reactor and FST, when the dissolved oxygen is low (high carbon load, high concentration NH4, inadequate DO & aeration capacity), when there are not enough SVFAs available (late in the lane and in the FST), and when the alkalinity of the system is to low.

Biological Phosphorous Removal (BPR) is a challenging process with detailed knowledge of biochemistry processes and micro-biology required by attending technical support personnel.

BPR rely on the principle that Poly-phosphate Accumulating Organisms (POAs) will remove ortho-phosphate from the water, and accumulate Poly-phosphate (PP) inside their cell bodies during aerobic/starvation conditions.

When the bacteria are then cycled to anaerobic conditions, with available VFAs & acetate, the bacteria use energy released from PP to polymerise acetate and form polyhydroxyalkanoates (PHA). This can then be used as a reduced carbon food source during future starvation periods in the next aerobic cycle, where PHA is metabolised with the use of available dissolved oxygen.

The process has four distinct sequential phases, all designed to favour the metabolic cycles of POAs, thereby increasing their competitive advantage from alternative metabolic pathways.

Raw influent enters into the anaerobic zone, flows over into the anoxic zone and further into the aerobic zone, as indicated by the coloured arrows on the process schematic. After the aerobic zone, water is separated from the bio-culture in the FST as standard for an ASP system.

Returning the activated sludge is also sequentially to the anoxic contact zone, then further back to the anaerobic contact zone, and thirdly back to the aerobic reactor. The anaerobic and anoxic tanks are designed like settlers to optimise the solids handling capabilities, crucial for cycling the bio-culture back and forth.

The anoxic zone is of significant importance and arranged in this sequence to allow for rapid de-nitrification by the bulk bacteria, using the acetate and available nitrate to support their metabolism. (See Nitrate electron transport chain elsewhere on these pages)

To optimize the BPR process, strict adherence to provide the PAOs with a competitive advantage is crucial, and the process may perform poorly or fail completely in the event of an uninformed change to an asset, sequence or biochemical reactant.

The fifth element in the process sequence on this schematic, is a additional chemical phosphate precipitation system.

Wastewater Treatment process, anaerobic, anoxic and oxic zones, the bio-chemistry and important redox reactions in activated sludge process systems. This demonstration presentation contains content from advanced learning modules for the bio-process chemistry of wastewater treatment process and technology.

The reaction conditions that prevail in different areas of activated sludge systems, may be described as anaerobic, anoxic and oxic, or aerated. Before we look at the reactions that take place in these different zones, lets quickly define how the conditions are deliniated. In the anaerobic zones, there are no dissolved oxygen, or oxygen bound species like nitrate or sulphate present. In the anoxic zones, there are no dissolved oxygen present but substantial amounts of oxygen bound species, most notable nitrogen bound oxygen, like nitrate. The oxic zone is where most of the carbon and nitrogen oxidation take place and in the aeration reactor the main purpose of the technology, is to make dissolved oxygen available as terminal electron acceptors in the bio-culture.

During reduced nitrogen oxidation reactions the metabolite nitrate is formed, one nitrate molecule for each molecule of ammonium oxidised. Nitrate contains three oxygen atoms attached to the oxidised nitrogen. These three oxygen atoms destabilise the Nitrogen-Oxygen bonds in three consecutive enzyme catalysed reactions as the electrons are transferred down the nitrate electron transport chain. If there are no molecular oxygen available bacteria with nitrate reducing enzymes, can utilise the nitrate-oxygen bond to accept electrons and ultimately produce di-nitrogen and water as reaction products. These conditions are the anoxic conditions as defined, and deliberately prepared with the anoxic tanks, channels and zones in wastewater treatment processes. The nitrogen reduction reactions take place with the use of several catalytic enzymes and most of the bulk populations of bacteria in activated sludge systems are capable of respiring using these metabolic pathways. 

This section was extracted from WWTp, Advanced module (A-07), for Wastewater Treatment Process and Technology, Biological Phosphorous Removal, BPR systems, process biochemistry knowledge for process design, commissioning and optimisation.

Biological Phosphorous Removal (BPR) is a challenging process with detailed knowledge of biochemistry processes and micro-biology required by attending technical support personnel.

The BPR process relies on the principle that Poly-phosphate Accumulating Organisms (POAs) will remove ortho-phosphate from the water, and accumulate Poly-phosphate (PP) inside the cell bodies during aerobic/starvation conditions. When the bacteria are then cycled to anaerobic conditions with available VFAs & Acetate, the bacteria use the PP to release energy and store a carbon polymer, poly-hydroxyalkanoate (PHA), for future starvation periods.

During anaerobic conditions, Poly-phosphate Accumulating Organisms (POAs) can utilise the stored Poly-phosphate (PP) with its high energy phosphate-anhydride bonds, to generate ATP. In the absence of Oxygen, the bacteria can’t metabolise the available fatty acids aerobically to generate large amounts of ATP, so that the PP energy store is vital for the PAOs, to accumulate acetate in carbon polymers of PHA. During the anaerobic phase, the PAOs release ortho-phosphate in the bulk water.

In downstream processes where the acetate and fatty acid concentration is low, PAOs can use the stored PHA, as a carbon source, metabolising it aerobically with the use of oxygen as the electron acceptor. During the aerobic phase, the PAOs use PHA as energy source, to assemble poly-phosphate polymer (PP), and for cell growth.

The removal of ortho-phosphate from the bulk water during the aerobic phase, and the accumulation poly-phosphate inside the bacterial cells, make the phosphorous removal from the system possible though the customary sludge wasting cycle.

When designing a BPR process, it is important to generate strict conditions for the PAOs, to be able to maximise their different metabolic cycles, and compete for the available resources in the wastewater influent.

Gram negative bacteria, form the bulk of bacterial species in the bio-culture of wastewater systems, estimated to be more than 90 percent in total. With a strong negative charge on the outer surface of the membrane, caused by negatively charged phosphates, carboxyl groups and lipo-polysaccharides that are integral to the membrane structure. Calcium with its double positive charge, is vital to attract the negative charge of bi-carbonate, anchoring it to the negative charge of the cell membrane, to be ready for transport into the interior of the bacterial cell, via the trans-membrane transporter.

Bicarbonate is a highly reactive inorganic carbon molecule, and a important metabolic intermediate that can be incorporated into the organic molecules, as an addition of an extra organic carbon during protein, carbohydrate or structural molecule assembly. In addition to this, bicarbonate alkalinity is vital for the maintenance of the proton gradient across cell membranes, the mechanism through which the organism generates energy from the electron transport chain during respiration.

To guarantee bicarbonate supply and the pH control, the transport of bicarbonate to the cytoplasm of the cell, specialised mechanisms of control and transport evolved in procaryotes. Bicarbonate can be formed from carbon-dioxide by the carbonic anhydrase enzyme, delivering the bicarbonate to the bicarbonate transporter complex binding site on the extracellular side of the membrane. Bicarbonate is delivered to the cytoplasmic side of the membrane where the reaction can be reversed by the C A enzymes to deliver the bicarbonate in the cytoplasm. 

In the cell cytoplasm, a large quantity of carbon dioxide is formed as a result of the metabolic breakdown of reduced carbon molecules, with the citric acid cycle. The protons from the carbon dioxide acidity, are continuously induced across the inner membrane to the periplasmic space, by electron transport chain complexes I and III. The mechanism of the proton pump generate ATP with the membrane bound enzyme ATP Synthase, which deliver the protons back in the cytoplasm. The bicarbonate cannot enter the periplasmic space due to the transporter enzyme mechanism, and through the control of the C A enzymes, the proton gradient across the inner membrane is maintained. If the pH in the cytoplasm is allowed to drop, the proton gradient is reduced and the ATP synthase, producing ATP, slows down.

This graphic is an extract from the “Diffusion Mass Transfer” learning module S-10, describing the central concept in Activated Sludge systems determining the efficiency with which the bio-process affect the removal of pollutants from wastewater.

Initially the mass transfer of substances in the wastewater takes place by convection, and particles and substances come in contact with the outer layer of the biomass bio-gel surface. Particulate solids will attach and get trapped in the outer layer, and from there the mass will transfer by physical mechanical means to migrate to the internal spaces of the softer bio-floc. Very small particles, molecules and dissolved substances will transfer their mass through diffusion. Diffusion mass flux can mathematically be described by Ficks first law, which includes the important measurable parameters of time, area, mass, diffusion coefficient, distance and concentration. The concentration gradient is described by the change in concentration per change in distance in the unit of time. Ficks first law applies more accurately for diffusing substances that are not bio available for metabolic processes of the bio-culture.

Dissolved molecules like volatile fatty acids, proteins and ammonia also transfer to the sludge floc boundary layer by convection. In the boundary layer, the development of concentration gradients toward the internal bio-gel spaces, become the net force toward the metabolic centres inside the bio-floc and bacterial cells. Dissolved substances transfer to the inside by diffusion, due to the concentration gradient as described by Fick’s second law. Because the biodegradable substances are being converted by the bio-culture, their concentration is drastically reduced along the diffusion path x. This means that the change in concentration dE, vary with time by dt, and we have to apply Fick’s second law to find the concentration gradient for the substance E, given by the second derivative for the first law equation.