Wastewater Treatment

CEB 30503
 O2 Fe(III) NO3 SO4
In- Carbon
organic source Electron acceptors

Organic Metabolism

In- Energy
organic source

Radiation
Growth
conditions

T Radiation
p
pH Abiotic
Factors
H2O
Gas
Modern WWTP
What parameters do we need to
consider in order to control the
treatment process?

How does the aerobic and

anaerobic digester work?
The answer to these questions
can be found to some extent in
our next chapter...
 3. Microbial Physiology
Objectives

- In this chapter, a general overview of bioprocesses

will be provided to give you an idea of the width of the
topic.

- Special focus shall be placed on cell physiology and

growth kinetics, since these bioprocesses are
intimately linked to environmental engineering
processes.
 3.1.1 Basic Biological Processes in
Prokaryotes and Eukaryotes
• A biological process is a process of a
living organism. Biological processes are
made up of any number of chemical
reactions or other events that results in a
transformation of matter and energy.
• May include:
– Cell adhesion and aggregation
– Cell communication / signaling
– Morphogenesis (shapes of tissues,
organs and entire organisms)
– Cell physiology
– Reproduction / microbial kinetics
– Interaction between organisms
(population dynamics)
 Cell Physiology
• Physiology (from Greek: physis = “nature, origin”;
and logos = “word, speech“; lit. "to talk about the
nature (of things)") is the study of the mechanical,
physical and biochemical functions of living
organisms.
• Here we shall pay particular focus on biochemistry
and abiotic growth conditions. If we have some
understanding of the chemical basis of life, it will
help in our understanding of the way in which
microorganisms function, how we can make use of
them in treating wastewater, how to avoid upsetting
the biological process etc.
 Carbon & Energy Requirements
• As we have learnt in our previous lecture,
microorganisms can be classified according to
carbon and energy requirements as outlined below.
Table 3.2 Classification of microorganisms according to their energy and carbon requirements

Mode of Energy Source Carbon Source H2S/

Fe2+/
CO2

H2O
Nutrition NH4+

Photoautotroph Light CO2

Chemolithoauto- Inorganic CO2

troph chemicals
SO42-/
Photohetero- Light Organic Fe3+/ H2O
NO3-
troph compounds Biomass

Chemoorgano- Organic Organic

heterotroph compounds compounds
Chemolitho- Inorganic Organic
heterotroph compounds compounds
 Nutrient Requirements
• All microorganisms need O, C, N, H, P and S, which
are the elemental constituents of organic matter.
• They also need minerals such as Na, K, Mg, Ca and
Cl.
• Trace elements that might be required include Mn,
Fe, Co, Cu, Zn, B, Al, V, Mo, I, Si, Sn, Ni, Cr, F and
Se
• Some microorganisms require special growth factors
such as vitamins, amino acids etc.
• Abiotic factors such as pH, presence and
concentrations of anions, T & p can have important
effects on bioavailability and concentration of
nutrients.
 Temperature & pH Range
• Different microorganisms have different temperature
and pH profiles and optimum T and pH for growth.

Figure 3.3 Temperature profiles (left) and optimum pH values for microbial growth (right).
 Oxygen Requirements
• Microorganisms differ in their requirements for
oxygen.
• Some are:
– Obligate aerobes
– Obligate anaerobes
– Facultative aerobes / anaerobes
 Water Requirements
• All microorganism need to be in an environment
where water is present in order to grow. Some fungi
can grow in relatively dry conditions on solid media
but the atmosphere must be damp.
• Solutes (sugar, salts, etc.) dissolved in water have an
affinity for water, and this is therefore unavailable to
micro-organisms. Therefore the more dissolved salts
the less likely it is that microorganisms will survive.
• The availability of water can be expressed as water
activity, which is calculated as the ratio of the vapour
pressure of a solution divided by the vapour pressure
of pure water at the same temperature.
3.1.2 Metabolical Pathways in
Microbial Cells
 Cells and Chemicals

• In the previous section we considered the function of

living organisms at the level of the cell.

• In cell biology we look at the sub-cellular components

and in biochemistry we consider the chemicals that
make up life.

• Hierarchies based on size and complexity of the

molecules are shown in the next slide
 Metabolism
• Chemical Reactions
– Within a living cell, there is a constant turn-over of
biochemicals (carbohydrates, lipids, proteins, nucleic acids
and combinations thereof) in many hundreds, possibly
thousands, of enzyme-catalysed chemical reactions. This is
metabolism, i.e. all the chemical reactions that occur within
a cell or organism.
– Metabolism has many different sets of chemical reactions
known as metabolic pathways. In order to simplify this
complex situation, two major types of pathways can be
considered:
• Catabolism is the breakdown of large molecules into small ones to
release energy.
• Anabolism is the building-up of small molecules to produce large
molecules, which requires energy.
 Metabolism

Fig. 3.4 Catabolism and anabolism

• Catabolism and anabolism are related as shown in Figure 3.4.

• During anabolism, adenosine triphosphate (ATP) is broken down to ADP
and inorganic phosphate, liberating energy that can then be used in the
synthesis of biochemicals.
 Aerobic respiration by chemotrophs
• Organisms that obtain energy from the oxidation of inorganic compounds
are called chemotrophs.
• Chemothrophs have many sources of inorganic electron donors (N2,
NH4+, NO2, S0, S2-, S2O3, Fe0, Fe2+, H2, CH4).
• Volcanic activity, agricultural and mining operations, burning of fossil fuel
and biological activity are the main sources of these inorganic e donors.

Table 3.3 Inorganic electron donors used in aerobic respiration by chemolithtrophs

Electron donor Electron acceptor Example
Fe2+ → Fe3+ + e- O2 → H2O Acidithiobacillus ferrooxidans,
Leptospirillum ferrooxidans
NH4+ → NO3+ e- O2 → NO3 Nitrosomonas
NO2- → NO3+ e- O2 → NO3- Nitrobacter
H2S → SO42- + 2H++ e- O2 → SO42- Thiobacillus spp.
HS- → S0+ e- O2 → SO42- Thiobacillus spp., Beggiatoa
S0 → SO42-+ e- O2 → SO42- Thiobacillus spp.
S2O3 → 2 SO42-+ e- O2 → SO42- Thiobacillus spp.
H2 → H2O+ e- O2 → H2O Ralstonia eutropha
 Anoxic respiration by chemolithotrophs

• In the absence of oxygen, inorganic electron donors can still

be oxidised via a process known as anoxic respiration.
• A list of chemotrophs known to oxidise inorganic compounds
is shown in table 3.4.

Table 3.4 Inorganic electron donors used in anoxic respiration by chemotrophs

Electron donor Electron acceptor Example

Fe2+ → Fe3+ NO3- → N2 Thiobacillus denitrificans
NH4+ → N2 NO2- → N2 Brocadia anammoxidans
S0 → SO42- NO3- → N2 Thiobacillus denitrificans
S2O3 → 2 SO42- NO3- → N2 Thiobacillus denitrificans
H2 → H2O NO3- → N2 Paracoccus denitrificans
 Anaerobic Respiration
• If electron acceptors other than O2 are
used during oxidation of organic
matter, then the process is called
anaerobic respiration.
• Other electron acceptors that are near
the O2 / H2O pair are Fe3+ / Fe2+ and
NO3- / NO2-.
• More electronegative acceptors are
SO42-, S0 and CO2.
• A summary of the most common types
of anaerobic respiration is given in
Figure 3.7.

Figure 3.7
3.2 Microbial Growth Kinetics
 Introduction
• Effective control of any system using biological processes is based on
an understanding of the basic principles governing the growth of micro-
organisms.

• We have already looked at the environmental conditions, which have an

important effect on the survival of micro-organisms, like pH,
temperature, electron donors and acceptors, water, nutrient and energy
requirements.

• Now we shall begin to look at the growth of microorganisms and in

particular the kinetics of bacterial growth which is a key parameter in
the design of biological treatment processes.
 Growth in batch culture
• Bacteria reproduce by binary fission, that
is one cell simply divides into two.
• The growth of a bacterial culture can be
represented by a curve that consists of
four stages or phases:
– Lag phase - growth and reproduction are
just beginning
– Log phase - reproduction is occurring at an
exponential rate
– Stationary phase - environmental
surroundings and food supply cannot
support any more exponential growth
– Death phase - when all of the nutrients
have been exhausted, the population dies
off

Fig. 3.9 Phases of growth

(top) in batch culture (right).
 Specific growth rate
• In a batch reactor where there is no nutrient limitation or depletion and no
loss of cells due to endogenous metabolism or death, growth of biomass
will be exponential during the unlimited growth phase.

0 1 2 3 4 5 6 Time [hr]

• The specific growth rate is defined as the quantity of new biomass formed
per unit of original biomass per unit of time. Following equation can be
used:
X 
ln  t  Xt – Population at time t, e.g. number of cells per mL
  X0 
X0 – Population at time zero
t1  t0
t – time [hr]
 t
X t  X 0e
 – specific growth rate [hr-1]
 Doubling time

0 1 2 3 4 5 6 Time [hr]

• The doubling time, td, is analogous to the half-life for first order decay. It is
defined as the time taken for the population to double in number (Xt = 2X0)
and is related to the specific growth rate, .

X   2X0 
ln  t  ln  
td   0    0  
X X ln 2
  
Calculate the specific growth rate and doubling time for a
culture of bacteria from the following data.

T [hr] Bacterial conc.

[No. / mL]
1 4.0 x 104
2 1.6 x 105
3 6.3 x 105
4 2.5 x 106
5 1.0 x 107
6 4.0 x 107
 Nutrient limitation
• Nutrient limitation occurs when the
growth of the organism is restricted by a
single nutrient, for example, C, N, or P.
• The way in which the specific growth
rate falls from its maximum value during
the deceleration phase as the substrate
concentrate becomes limiting, was first
reported by Jacques Monod (1910-1976)
and is known as the Monod equation:
Fig. 3.10 Nutrient concentration, s vs.
growth rate, .

s – Nutrient concentration [mg L-1]

s
  m m – Maximum specific growth rate [hr-1]
Ks  s Ks – Saturation coefficient for the growth limiting nutrient, a
measure of the ability of the organism to uptake the nutrient
Substrate Utilisation
Substrate Utilisation
Substrate Utilisation
 Production of Biomass in Continuous
Cultures

Fig. 3.11 Continuous culture system.

• Our analysis of bacterial growth in fixed volume batches is only of limited

value in many engineering application, where continuous flow processes
are used (e.g. wastewater treatment)
• In a cont. process biomass is retained inside a reactor and the stream to
be treated (e.g. wastewater, industrial effluent, contaminated groundwater)
is fed continuously to the reactor, and treated product is withdrawn at the
same rate.
Need to Balance Organic Load (lbs BOD)
With Number of Active Organisms in
Treatment System

Food to Microorganism Ratio

F
F:M or
M
 Mass Balance on Biomass

• Although fresh nutrients are added continually, all other factors will remain
constant. We can define the mean residence time (or hydraulic retention
time, HRT) as:
V V – reactor volume [m3]
HRT 
Q Q – Feed flow rate [m3 h-1]

• Sometimes it is convenient to refer HRT to dilution rate, D, which is the

reciprocal of the HRT with units [h-1].
 Mass Balance on Biomass

• If D ≤ m , biomass will be retained in reactor

• If D > m, biomass wash-out will occur

• The mass of cells, m, produced in the reactor per unit time is given by:


m  YQ si  s  Y – Yield coefficient, kg biomass per kg nutrient
Q – Feed flow rate [m3 h-1]
si – Concentration of limiting nutrient in influent [kg m-3]
s – Concentration of limiting nutrient in reactor [kg m-3]
 Oxygen limitation
• In order to achieve a high productivity from an aerobic continuous culture,
like the activated sludge process, it is necessary to run at a high dilution
rate, D (or low HRT), and high biomass production, m. In order to increase
m, a high concentration of the limiting nutrient, si, is required. However, as
m is increased there will be a maximum value at which oxygen supply will
become limiting!
• For aerobic organisms, a specific oxygen uptake rate (q0) can be
determined with the units of kg O2 per kg cell and hr. The rate at which
oxygen is transferred into the water must exceed this minimum.
• The equation for oxygen transfer into solution is given by:

n = kLa(cDO*-cDO) n – Rate of O2 mass transfer [kg h-1]

kLa = absorption coefficient [m3 h-1]
cDO – Dissolved oxygen concentration in influent [mg L-1]
cDO* - Equilibrium DO concentration [mg L-1]