Three Biochemical Processes Mediated by Bacteria That are Important in Environmental Engineering

Bacteria demonstrate diverse biochemical metabolic reactions. Describe three biochemical processes mediated by bacteria that are important in Environmental Engineering. How does a fundamental understanding of the microbiology of these processes assist in designing treatment systems for wastes and wastewater?

This essay will look into three processes mediated by bacteria that are important in Environmental Engineering. Also look into how does a fundamental understanding of the microbiology of these processes assist in designing treatment systems for wastes and wastewater.

Clean water supplies can contribute greatly to good ecological health. Water is vital for life, but also an excellent carrier of toxic substances, micro-organism and pathogens which can lead to diseases and bad health. Bacteria have been an important existence to all life forms and are possibly our roots. Bacteria multiply and reproduce at incredible speed, hence more chances of mutations occurring. Bacteria can evolve and adapt to a new environment in little time. Due to this fact, different types of bacteria can be found everywhere, each adapted to its particular environment.
Activated sludge process: Carboniferous oxidation
The Activated Sludge Process utilises a variety of micro-organisms to reduce the organic and toxic matter that is present in our domestic wastewaters and bio-degradable industrial wastewaters into bio-mass. The process is an aerobic and continuous-flow biological. Micro-organisms used are heterotrophic organisms, which obtain the energy necessary for their growth and maintenance functions by breaking down organic food supply.

The Activated Sludge Process (ASP) was put in use in 1914 in England where it was developed. Raw sewage is screened, degritted and primary settled to become primary effluent which is the input of the ASP. The primary effluent is inputted into the aeration tank, this is where most of the biological processes takes place.

Return activated sludge (RAS) is also mixed in with the primary sludge. This mix in the tank contains 1500-2500mg/l of suspensed matter and is known as the mixed liquor suspensed solid (MLSS). The suspended matter consists of both bio-mass and organic suspensed matter which the bio-mass will feed on.

The bio-mass decomposes the organic matter in the primary effluent into simpler products like; carbon dioxide (CO2), water (H20), ammonia(NH3), and new cell biomass.

Air is pumped into the mixed liquor and mixed well by a mechanical mixer, this ensures that the bio-mass are not oxygen staved and promotes aerobic respiration. The mechanic mixer works to distribute the bio-mass in the tank, but often filamentous micro-organisms can cause excess bulking of bio-mass and create a film of sludge floc at the top layer of the mixed liquor. This causes a portion of the bio-mass not getting access to enough oxygen supply. It is important that air is available in the right amount and distributed evenly in the tank. Anaerobic zone inside the floc favors the development of anaerobic bacteria like methanogens or sulphate reducing bacteria, which can lead to odour problems.

The process which bacteria and fungi utilises to decompose suspensed organic matter is as follows:

Soluble organic materials are absorbed through the cell walls of the micro-organisms and into the cells, where they are broken down and converted to energy.
Insoluble (solid) particles are broken down into soluble substances by enzymes secreted by the micro-organisms on the cell walls of the micro-organisms, and then absorbed through the cell wall, where they are also digested and simulated into new cells, CO2, NH3 and H20. (, 2005)

As time passes in the tank, the biological oxygen demand (BOD) will start to decrease with an increase in Bio-mass. As bio-mass continue to increase in the tank until the aeration time has been reach. The content of the tanks is moved to the sedimentation tank, where the sedimentation of the bio-mass forms the microbial flocs (sludge).

To maintain a food-to-micro-organism ratio (F/M), some of this sludge in the sedimentation tank (Clarifier) is recycled back into the aeration tank as RAS. Excess sludge will be extracted and will be treated separately. The remaining effluent in the sedimentation tank will be are transported to facilities for disinfection and final discharge to receiving waters.

There are certain conditions required for the ASP to be efficient. These conditions should be considered in designing and running of an ASP plant, Oxygen concentration, F/ M ratio, pH, temperature and aeration time. Optimal conditions should be used to encourage maximum BOD removal and settlement rate.

The most important is to maintain the correct F/M ratio. F/M is defined by the organic mass into the activated sludge system and is express in BOD/MLSS per day and indicates the wasting rate of organic matter. For standard ASP it is ideal to have a F/M of 0.2-0.5 (lb BOD5/day/lb MLSS). A low F/M (i.e. High MLSS), promotes decomposition of organic matter and efficient settlement. Sludge settles best when the micro-organisms are short of carbon food and energy source, hence when microbial growth rate is low.
Heterotrophic micro-organisms
The mixed liquor contains a wide variety of micro-organisms, including, bacteria, fungi, prokaryotic and eukaryotic micro-organism.

There are different bacteria breaking down type types of the organic matter. In the activated sludge flocs, the gram-negative bacteria are the major component. These bacteria oxidises organic matter into nutrients and produce polysaccharides and other polymeric materials which promotes flocculation of microbial bio-mass.
Genera most commonly find are Zooglea, Pseudomona, Flavbacterium, Alcaligens, Achromobacter, Corynebacterium, Comomonas, Brevibacterium, Acinetobacter, Bacillus.

There is also fraction of filamentous micro-organism which causes sludge bulking. Some bacteria are responsible for denitrification, by breaking down methane.
Fungi growth in the activated sludge (AS) is not desired but fungi can grow abundantly under conditions of low pH, toxicity, and nitrogen-deficient wastes. Excessive amount of fungi Geotrichum in AS can cause sludge bulking.

Protozoa are prefer aquatics environments and also present in AS as predators of bacteria. Protozoa’s grazing activities can reduce considerable amounts of toxicants like heavy metals in the AS. Protozoa Ciliates is most commonly found in AS. Protozoa flagellates move by using one or more flagella. Stalked ciliate use their stalk to attach themselves to the flocs.

The high levels protozoa flagellates and ciliates indicates high BOD. Whereas high levels of stalked ciliates and rotifers indicate low BOD, hence efficient organic matter removal. Protozoa consumes suspensed organic matter and bacteria and considerably contributes to the reduction of BOD in AS.

Protozoa and rotifers move in the AS by ciliary action. However, rotifers have a stronger ciliary action and this is useful at later stage of ASP, which helps in consuming and removing remaining suspended bacteria. Rotifers remove non-flocculated bacteria and their excretion is surrounded by mucus, which promote settlement of sludge (flocculation).
(Horan, 1996)
(Asis, 2005)
Nutrient removal by ASP: Nitrogen
Aeration tank and settlement tank is capable of removing a decent amount of BOD, suspensed solids and bacteria, but the remaining effluent may still contain other impurities which can cause problems in output sources. The impurities in the effluent can be nutritious to plants and can particularly induce algal growth, which can lead to eutrophication problems. High nitrogen levels in water can also lead to nitrate toxicity problems. Nitrogen and phosphorus are the nutrient in effluent which can cause eutrophication. Human excreta, nucleic acid and proteins are the main organic source of Nitrogen and phosphorus in the effluent. These complex organic matters such as proteins, urea is broken down into inorganic matter, like ammonium or lesser form like ammonia. Bacteria in the aeration tank and settlement tank is capable of removing up to 30% of total nitrogen as biological sludge in mixed liquor due to the presence of the nitrifying bacteria by ammonification. This nitrogen rich effluent is subjected to biological degrading under acidic condition to convert any nitrogen compounds into ammonium NH4.

There are many processes present to reduce complex nitrogen compounds in effluent, but biological treatment in wastewater treatment plants is undoubtedly the most cost efficient.
(Orhon, Artan, 1994)
Biological nitrification: Autotrophic

After the effluent has been treated by the aeration tank and settlement tank, nitrogen is present mainly in the form of ammonium NH4. The ammonium must go through a two step process to complete the nitrogen removal process (nitrification followed by denitrification). Nitrification is the biological process under aerobic conditions using autotrophic bacteria. Autotrophic bacteria are able to provide their own energy by oxidising inorganic ions. There are two types of bacteria required to complete the process of nitrification.
Nitrosomonas oxidise ammonia to nitrite ions, Nitrobacters oxidises nitrite ions to form nitrate ions with the oxygen source is from water H2O. Nitrifiers synthesise their energy from this process in forms of energy, carbonate or bicarbonate.

Nitrification can be expressed by the following formula.
NH4+ +1.83O2 + 1.98HCO3- --> 0.021C5H7NO2 + 1.041H2O + 0.98NO3- + 1.88H2CO3
As indicated in the formula, nitrification process is:
A process that has an incredibly large oxygen demand, 1.83 moles is required per 1 mole ammonium removed.
C5H7NO2 is the product bio-mass of nitrifying bacteria; cell growth yield is low compare to cell growth of heterotrophic micro-organism. There are only 0.021 moles of new cells per 1 mole ammonium removed. Nitrification removal of ammonium (alkaline) and bicarbonate (HCO3) will lower the pH in the mixed liquor.

Nitrification can be implemented in “single-stage” or “two-stage” ASP.
(Orhon, Artan, 1994)
Single stage nitrification in activated sludge:
To maintain good nitrification, the growth of heterotrophic organism must not exceed that of nitrifying bacteria. In ASP, nitrification often occurs towards the end of the aeration time, because then, the BOD and F/M is low (<0.3), so the growth of the heterotrophic organism is limited. To ensure decent growth of nitrifiers, other conditions to consider are the temperature, nitrifiers’ concentration, pH and the oxygen supply. Usually, the sludge age or the retention time of the mixed liquor can be used to determine whether the conditions are suitable for decent nitrification, for a given temperature.

Temperature have an immense effect on the sludge age required, at warmer temperature the sludge age required is a lot lower than a cooler temperature. Hence climate changes can effect the quality of nitrification treatment. Winter nitrification treatment is expected to be less efficient compare to summer. Typical effluent ammonia concentrations are 2mg/L in summer and 5mg/L in winter.

The rate of removal of nitrogen compounds is not depending on the concentration of nitrogen compounds in the effluent. But concentrations of nitrogen compounds vary in effluent, a safety factor can be applied to ensure the system will cope even at abnormal concentration levels of nitrogen compounds. The safely factor is expressed by
Safety factor = Peak concentration of nitrogen concentration
Mean concentration of nitrogen concentration

In practise, it is best not to use a safety factor larger than 2.5. This is because a large safely factor will require a larger sludge age, causing the mixed liquor volume to build up over time, hence larger aeration tanks are required. Safety factor larger than 2.5 is problematic and not cost efficient. If effluent source has unstable nitrogen concentrations, it will be more effective to use flow-balancing tanks to stabilise concentrations.
(Barnes, Bliss,1981)
Two-stage nitrification in activated sludge:
(see fig 3)
This process is the same biological procedure as single-stage nitrification, but uses an aeration for nitrification and one for denitrification. This enables the heterotrophic and autotrophic bacteria to growth in separate tanks and in their own desired environment. This two-stage process favors the growth of the nitrifiers, as they can growth without the competition of heterotrophic bacteria and in its anoxic conditions. The two-stage process is more versatile compare to single-stage.
There are minor setbacks with using a two-stage system mainly in the nitrification aeration tank. Nitrification has a considerably low bio-mass yield which can lead to poor sludge settlement and foaming. Due to a more efficient nitrification, pH of the mixed liquor is likely to lower than the single-stage system. Often there is a need to correct the low pH of over acidic mixed liquor in the two-stage system.
(Barnes, Bliss,1981)
Denitrification – heterotrophic
Denitrification is an anaerobic process and is used to remove the nitrogen compounds from the effluent of the nitrification process (Nitrate (NO3-)). Denitrification utilises a variety of heterotrophic bacteria (Alcaligenes, Achromobacter, Micrococcus and Pseudomonas) in an anaerobic process called Nitrate respiration to reduction the nitrogen in nitrogen compounds. The process uses a carbon source (methanol source from industrial and agriculture wastes) as electron donor in reduction process. Nitrate has a redox state of +5, for every reduction process taken place, one electron is passed on the nitrogen compound. By repeating the process, the nitrogen in compound will have enough electrons and will be able to let go of the oxygen. Then finally be reduced to gaseous nitrogen, which will leave the effluent and into the atmosphere.

There is the order of reduction of nitrate:

NO3- -> NO2- -> NO -> N20 –> N2
Nitrate -> Nitrite -> Nitric oxide -> Nitrous oxide -> Nitrogen

Of which nitric oxide, nitrous oxide and nitrogen are gaseous denoting complete remove from aqueous mixed liquor, which completes the process of Nitrogen Removal in Activated Sludge.

Unlike nitrification, denitrification has no oxygen demand and also has a much higher bio-mass yield. Denitrification bacteria use up protons in the reduction process, and cause the pH to rise, which the opposite of nitrification. The rate of denitrification is dependent on the nitrate concentration, the electron donors’ concentration and temperature.
(Barnes, Bliss,1981)

Phosphorus removal
Phosphorus is the limiting factor of eutrophication, by limiting phosphorus in wastewater to under 10μg/L, will cease the process of eutrophication even in high levels of nitrogen compounds. Previous biological phosphorus removal processes were inefficient in removing sufficient proportions of phosphorus, and chemical process were often preferred over biological ones. With research and new development in phosphorus removal processes, there are now several new processes that are available and moderately efficient in use.
(Horan, 1996)

Phosphorus can be removed in a Biological Phosphorus Removal Process (BPR) in which phosphorus is taken up in new cell growth, but this has simply transferred phosphorus from the mixed liquor to the sludge, phosphorus have not been removed.
Bacteria Acinetobacter spp is a Phosphorus Accumulating Organism (PAO) and is able to use phosphorus to synthesise ATP. Under anoxic conditions, Acinetobacter spp uses up poly-phosphorus (poly-P) and BOD present to synthesise phosphorus and a polymer (PHP – Carbon storage) which it stores away. Then in aerobic conditions, now where BOD concentrations are low and phosphorus concentrations are high. Acinetobacter spp then degrade its PHP store as a carbon source and release ATP also reducing phosphorus concentrations at the same time. Hence for POA to efficiently remove Phosphorus concentrations , anaerobic conditions should be maintained.
(Derin, Nazik, 1994)

(Grady, Daigger, Lim, 1999)

Biological excess phosphorus removal (BEFR) is another biological process able to remove phosphorus from wastewaters. This process requires dissolved oxygen concentrations of 2mg/l or above in the late stages of aeration tank and also to maintain aerobic conditions in the clarifier, to achieve effective phosphorus removal. In this process a part of the underflow from the clarifier from the activated sludge reactor is fed to a Gravity Sludge Thickener. Under aerobic conditions in the thickener, phosphorus is released. The phosphorus-rich supernatant from the thickener is treated by phosphate precipitation using lime. The effluent from the thickener is returned to the aerations tanks of the activated sludge reactor.
(Grady, Daigger, Lim, 1999)

Designing Treatment processes
There are other treatments that can made ASP effluent purer and safe by removing sulphur and pathogens, ASP can be tailored to suit any type of effluent concentration and environment. ASP is widely used in domestic, and industrial water treatment systems. Only by fully understanding the biological process involved in treatment, can one design a treatment plant that will be efficient in waste removal and cost efficient. One must evaluate the content of impurities and their concentrations in the effluent in order to determine what process, conditions and reactor types to treat the effluent in. The variable components in ASP are the aeration tanks/reactor, aeration systems, settlement tank (clarifier) and amount of return activated sludge.

Size and dimensions of reactors will depend on the flow rate / aeration time, sludge age required, the volume of the return sludge. By increasing these factors, larger reactors will be required, both the manufacture and running of larger reactors costs more.
Single sludge systems
Single sludge systems’ aeration tank systems have BOD removal process, nitrification and denitrification occurring simultaneous in the same reactor. Single sludge systems use alternating aerobic and anoxic tanks to provide alternate conditions for the oxygen demanding and non-oxygen demanding organism (nitrifiers). It is possible to create anoxic and aerobic zones in one tank and have both processes occur simultaneously by disabling some portion so aerators in the tank.

Single sludge systems begin with an anoxic condition tank to provide a kick-start source of nitrate to prepare for denitrification in the second tank (aerobic tank), and then third tank being anoxic and so fore. Mixed liquor recirculation (MLR) is to returned the nitrate generated in the aerobic tank to the first anoxic tank for nitrification. After is series of aeration tanks, the effluent will finish in a single clarifier where the sludge settles, effluent is removed and portion can be returned to first tank. The waste activated sludge (WAS) can be removed to maintain good substrate concentrations. Overall single tank systems are more cost efficient and less maintenance required.
(Barnes, Bliss,1981)

(Grady, Daigger, Lim, 1999)
Multi-sludge systems
Multi-sludge systems, uses three separate tanks each with it’s own clarifier, to carry our BOD removal process followed by nitrification then denitrification. Return activated sludge is returned within every tank.
Nitrification and denitrification by hand in hand in the nitrogen removal process. Nitrification process has a lower yield than denitrification; hence the removal of nitrates exceeds that of nitrate input, so large portions of sludge are returned to the aeration tank to restore nitrate levels.
But it is more efficient to use two aeration tanks to carry out denitrification after nitrification and achieved higher removal rates. Higher removal can speed up the flow rate through the systems and so smaller reactors can be used, although two tanks system requires two settlement tanks. Nitrification occurs at a low BOD, extra electron donors (carbon source) will be required to give decent denitrification yield. In the denitrification tank, there is likely to be alkaline condition problem, hence pH control will be required.

Variable conditions in aerations that need to be considered are; the F/M, aeration time, sludge age, amount of aeration, safely factor of nitrification or use of flow-balancing tanks. By increasing these factors/parameters listed above will increase the cost of the activated sludge process. Detail knowledge of the biological processes requirements is required to set these parameters.

(Barnes, Bliss,1981)
(Horan, 1996)

(Horan, 1996)
ASP is an extremely wide and unique process, where the responsibility
Fundamental knowledge of basic biology of micro-organism will be absolute vital in understanding the outline stages of the treatment. Knowledge and understanding of the life cycle and requirements of micro-organism is essential to basic design ideas of the ASP reactor. Extensive knowledge and understanding of combinations of the biological processes is required to set parameters for the ASP reactor. An under designed ASP plant can result in severe problem in receiving waters. Also poor designs of the ASP plant can lead to uneconomical solutions and in industrial scale of ASP plant, it can be very expensive.


Derin Orhon; Nazik Artan
Modeling activated sludge systems
Technomic publishing company

Barnes, Bliss; Gould & Vallentine
Water and wastewater engineering systems
Pitman international

C. P. Leslie Grady, Jr; Glen T. Daigger; Henry C. Lim
Biological Wastewater Treatment, 2nd edition
Marcel Dekker

Horan, N. J.
Biological Wastewater Treatment Systems, theory and operation,
John Wiley and Sons
Electronic source:

What is Environmental Engineering?

Bergen county utilities
Virtual Tour of Wastewater Treatment Process Activated Sludge Process
2005 WPC_VT_WasteWaterActivatedSludgeProcess.htm

Activated Sludge, Process Control, Activated Sludge, Microscopy, Filamentous Organisms,bulking, scum