G6.

CENGR 422: Environmental Science COMPOSITION AND CLASSIFICATION OF MICROORGANISMS

FUNDAMENTALS OF BIOLOGICAL TREATMENT 3) Cell Components:
a) Cell wall – Provides strength to maintain the cell shape and protects
OVERVIEW OF BIOLOGICAL WASTE WATER TREATMENT the cell membrane.
1) Objectives: b) Cell membrane – controls the passage of dissolved organics and
a) For Domestic Wastewater: nutrients into the cell and the waste materials and metabolic by-
i) Transform dissolved particulate biodegradable constituents products out of the cell.
into acceptable end products. c) Cytoplasm – contains the material within the cell to carry out cell
ii) Capture and incorporate suspended and no settleable functions and includes water, nutrients, enzymes, ribosomes, and
colloidal solids into biological floc or biofilm. small organic molecules.
iii) Transform or remove nutrients, such as nitrogen and d) Cytoplasmic inclusions – contains storage material that can provide
phosphorus. carbon, nutrients, or energy.
iv) Remove specific trace organic constituents and compounds. e) Deoxyribonucleic acid (DNA) - a double-stranded helix-shaped
b) For Industrial Wastewater: molecule that contains genetic information that determine the
i) To remove or reduce the concentration of organic and nature of the cell protein and enzymes that are produced.
inorganic compounds. f) Plasmid DNA – small circular DNA molecules that can also provide
c) For Agricultural Irrigation Return to Wastewater: genetic characteristics for the bacteria.
i) To remove nutrients, specifically nitrogen and phosphorus, g) Ribosomes – particles in the cytoplasm that are composed of
that are capable of stimulating the growth of the aquatic ribonucleic acid (RNA) and protein and are the sites where proteins
plants. are produced.
h) Flagella – protein hairlike structures that extend from the cytoplasm
2) Terms and Definitions: membrane several bacteria lengths out from the cell and provide
a) Metabolic Function: mobility by rotating at high speeds.
i) Aerobic (Oxic) Processes – biological treatment processes that i) Fimbriae and pili – short protein hairlike structures that enable
occur in the absence of oxygen. bacteria to stick to surface.
ii) Anaerobic Processes – biological treatment processes that
occur in the absence of oxygen. 4) Cell Composition:
a) Typical Composition of Bacteria Cells
iii) Anoxic Processes (Denitrification) – the process by which
nitrate nitrogen is converted biologically to nitrogen gas in the Constituent or Element Percent of Dry
absence of oxygen. Weight
iv) Facultative Processes – biological treatment processes in Major Cellular Material
which the organisms can function in the presence or absence  Protein 55.0
of molecular oxygen.  Polysaccharides 5.0
b) Treatment Process  Lipid 9.1
i) Suspended-Growth Processes – biological treatment  DNA 3.1
processes in which the microorganisms responsible for the  RNA 20.5
conversion of the organic matter or other constituents in the  Others (sugars, amino acids) 6.3
wastewater to gases and cell tissue are maintained in  Inorganic Ions 1.0
suspension within the liquid. As Cell Elements
ii) Attached-Growth Processes (Fixed-film Processes) – biological  Carbon 50.0
treatment processes in which the microorganisms responsible
 Oxygen 22.0
for the conversion of the organic matter or other constituents
 Nitrogen 12.0
in the wastewater to gases and cell tissue are attached to
some inert medium, such as rocks, slag, or specially designed  Hydrogen 9.0
ceramic or plastic materials.  Phosphorus 2.0
iii) Combined Processes – term used to describe combined  Sulfur 1.0
processes.  Potassium 1.0
iv) Lagoon Processes – a generic term applied to treatment  Sodium 1.0
processes that take place in ponds or lagoons with various  Calcium 0.5
aspect ratios and depths.  Magnesium 0.5
c) Treatment Function  Chlorine 0.5
i) Biological Nutrient Removal – the term applied to the removal
 Iron 0.2
of nitrogen and phosphorus in biological treatment processes.
 Other trace elements 0.3
ii) Biological Phosphorus Removal – the term applied to the
biological removal of phosphorus by accumulation in biomass
5) Environmental Factors
and subsequent solids separation.
Environmental conditions of temperature and pH have an important
iii) Carbonaceous BOD Removal – biological conversion of the
effect on the selection, survival and growth of microorganisms. In general,
carbonaceous organic matter in wastewater to cell tissue and
optimal growth for a particular microorganism occurs within a fairly narrow
various gaseous end products. range of temperature and pH.
iv) Nitrification – the two-step biological process by which
ammonia is converted first to nitrate and then then to nitrate. Type Temperature Optimum
v) Denitrification – biological process by which nitrate is reduced Range (◦C) Range (◦C)
to nitrogen and other gaseous end products. Psychrophilic 10 – 30 12 – 18
vi) Stabilization – biological process by which the organic matter Mesophilic 20 – 50 25 – 40
in the sludges produced from the primary settling and
Thermophilic 35 – 75 55 – 65
biological treatment of wastewater is stabilized, usually by
conversion to gases and cell tissue.
6) Microorganism Identification and Classification:
vii) Substrate – term used to denote the organic matter or
a) Taxonomic Classification - Conventional taxonomic methods used to
nutrients that are converted during biological treatment or
identify a bacterium rely on physical properties of the bacteria and
that maybe limiting in biological treatment.
metabolic characteristics.
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b) Phylogenetic Classification - The characterization of microorganism i) govern the oxidation of substrate and the production of
based on genetic information and evolutionary location in time is biomass, which contributes to the total suspended solids
termed phylogeny, which is more current method of identification and concentration in a biological reactor.
classification.
c) Use of Molecular Tools - Besides the ability to identify and classify 14) Rate of Utilization of Soluble Substrates
specific bacteria, molecular tools can be used to provide information a) Removal of Substrate
previously unavailable about active microbial communities, and to i) One of the principal concerns in wastewater treatment
study water or wastewater treatment plant effluents for specific b) Substrate Utilization Rate
pathogenic organisms. i) When the substrate is being used at its maximum rate, the
bacteria are also growing at their maximum rate.
INTRODUCTION TO MICROBIAL ORGANISMS ii) The maximum specific growth rate of the bacteria is related to
7) Carbon and Energy Sources for Microbial Growth - To continue to reproduce the maximum specific substrate utilization rate.
and function properly, an organism must have sources of energy, carbon
for the synthesis of new cellular material, and inorganic elements 15) Other Rate Expressions for the Utilization of Soluble Substrate
(nutrients) such as nitrogen, phosphorus, sulfur, potassium, calcium, and a) Particular Rate Expression to Use
magnesium. i) Depends mainly on the experimental data available to fit the
kinetic equations and the application of the kinetic model.
8) Carbon Sources - Microorganism obtain their carbon for cell growth from
wither organic matter or carbon dioxide. Organism that use organic 16) Rate of Soluble Substrate Production from Biodegradable Particulate
carbon for the formation of the new biomass are called heterotrophs Organic Matter
while organism that derive cell carbon from carbon dioxide are called a) Rate Expressions for Substrate Utilization
autotrophs. i) Based on the utilization of soluble substrates
ii) 20 to 50 percent of the organic material enters as soluble
9) Energy Sources - The energy needed for cell synthesis may be supplies by compounds.
light or by a chemical oxidation reaction. Bacteria can oxidize organic or b) Particulate Substrate Conversion Rate
inorganic compounds to gain energy. i) Rate-limiting process that is dependent on the particulate
a) Phototrophs – organisms that are able to use light as an energy substrate and biomass concentrations.
source.
i) Heterotrophs – sulfur-reducing bacteria 17) Rate of Biomass Growth with Soluble Substrates
ii) Autotrophs – algae and photosynthetic bacteria a) Biomass Growth Rate
b) Chemotrophs – organisms that derive their energy from chemical i) Proportional to the substrate utilization rate
reactions. b) Biomass Decay
c) Chemoautotrophs – obtain energy from the oxidation of reduced i) Proportional to the biomass present
inorganic compounds.
d) Chemoheterotrophs – derive their energy from the oxidation of 18) Total Volatile Suspended Solids and Active Biomass
organic compounds. a) Total Volatile Suspended Solids
i) Determined using a volatile suspended solids (VSS) test to
10) Nutrient and Growth Factor Development - Nutrients, rather than carbon determine the concentration of a VSS in an aeration
or energy sources, may at times be the limiting material for microbial cell stabilization basin system.
synthesis and growth. The major growth factor falls into three classes: b) Active Biomass
a) Amino Acids i) Part of a living system that is actively growing or reproducing.
b) Nitrogen Bases
c) Vitamins 19) Net Biomass Yield and Observed Yield
a) Net Biomass Yield
BACTERIAL GROWTH AND ENERGETICS i) The ratio of the net biomass growth rate to the substrate
11) Bacterial Reproduction - Bacteria can reproduce by binary fission, by utilization rate.
asexual mode or by budding. Generally, they reproduce by binary fission, b) Observed Yield
in which the original cell becomes two new organisms. The time required i) Accounts for the actual solids production that would be
for each division, which is termed the generation time, can vary from days measured for the system.
to less than 20 min.
MODELING SUSPENDED GROWTH TREATMENT PROCESS
12) Bacterial Growth Patterns in a batch reactor - Bacterial Growth Patterns in 20) Description of Suspended Growth Treatment Process
a batch reactor is characterized by identifiable phases. a) Complete-Mix Reactor with Recycle
a) The lag phase - Represents the time required for the organisms to i) Can be considered as a model for suspended growth
acclimate to their new environment before significant cell division processes
and biomass production occur. ii) A similar complete-mix reactor may be used in laboratory
b) The exponential-growth phase - During this phase, bacterial cells are studies to assess wastewater treatability.
multiplying at their maximum rate, as there is no limitation due to
substrate or nutrients. 21) Biomass Mass Balance
c) The stationary phase - During this phase, the biomass concentration a) Simplified Word Statement:
remains relatively constant with time. In this phase, bacterial growth i) Accumulation = inflow – outflow + net growth
is no longer exponential and the amount of growth is offset by the
22) Substrate Mass Balance
death of cells.
a) Simplified Word Statement:
d) The death phase - In this phase, the substrate has been depleted so i) Accumulation = inflow – outflow + generation
that no growth is occurring, and the change in biomass
concentration is due to cell death. 23) Mixed Liquor Solids Concentration and Solids Production
a) Mixed Liquor Solids Concentration
MICROBIAL GROWTH KINETICS i) It is the concentration od suspended solids in an aeration tank
13) Microbial Growth Kinetics Terminology during the activated sludge process, which occurs during the
a) The Microbial Growth Kinetics treatment of waste water.

b) Solids Production

i) Sludge Production – refers to the residual, semi-solid material
left from industrial wastewater, or sewage treatment
processes.
 Primary Sludge – produced by settleable solids
removed from raw wastewater in primary settling
 Secondary Sludge – produced by biological
processes
 Chemical Sludge – produced by precipitation of
specific substances
24) The Oxygen Requirements
a) For biodegradation of carbonaceous material:
i) Determined from a mass balance using the bCOD
concentration of the wastewater treated and the amount of
biomass wasted from the system per day.
25) Design and Operating Parameters
a) Food to Microorganism (F/M) Ratio
i) It is a process control number that helps you to determine the
proper number of microorganisms for your system.
b) Specific Substrate Utilization Rate
c) Organic Volume Loading Rate
i) Defined as the application of soluble and particulate organic
matter.
26) Process Performance and Stability
a) Domestic wastewater may have significant variability in its composition
and may not always be treated as a single waste type.
27) Modeling Plug-Flow Reactors
a) Plug-Flow System with Cellular Recycle
i) Can be used to model certain forms of the activated-sludge
process.
ii) In a true plug-flow model, all the particles entering the reactor
stay in the reactor in an equal amount of time.
iii) Some particles may take more passes through the reactor
because of recycle, but while they are in the tank, all pass
through in the same amount of time.
SUBSTRATE REMOVAL IN ATTACHED GROWTH TREATMENT PROCESSES
28) Introduction
a) In an attached growth treatment process, a biofilm consisting of
microorganisms, particular material, and extracellular polymers is
attached and covers the support packing material, which may be plastic,
rock, or other material.
29) Substrate Flux in Biofilms
a) Substrate Flux Across the Stagnant Layer to the Biofilm
i) The thickness of the stagnant layer will vary with the fluid
properties and fluid velocity.
ii) Higher velocities result in thinner films with greater substrate
flux rates.
30) Substrate Mass Balance for Biofilm
a) Simplified Word Statement:
i) Accumulation = inflow – outflow + generation
31) Substrate Flux Limitations
a) The Substrate & Surface Flux Limitation
i) The substrate limitation may be due to the reaction rates
within the biofilm or to bulk liquid concentrations and
diffusion rates across the stagnant layer.
AEROBIC BIOLOGICAL OXIDATION
32) Process Description
a) Initial Biological Uptake
i) During the initial biological uptake of the organic material,
more than half of it is oxidized and the remainder is
assimilated as new biomass, which may be further oxidized by
endogenous respiration.
ii) For both suspended and attached growth processes, the
excess biomass produced each day is removed and processed
to maintain proper operation and performance.
G6.3
33) Microbiology
a) Aerobic Heterotrophic Bacteria
i) They are able to produce extracellular biopolymers that result
in the formation of biological flocs (or biofilms for attached
growth processes) that can be separated from the treated
liquid by gravity settling with relatively low concentrations of
free bacteria and suspended solids
b) Protozoa
i) By consuming free bacteria and colloidal particulates,
protozoa aid effluent clarification.
ii) Their presence is a good indicator of a trouble-free stable
process operation.
34) Growth Kinetics
a) Design Parameters
i) With the exception of some difficult-to-degrade constituents
in industrial wastewaters, the kinetics for aerobic oxidation of
organic substrates seldom control the SRT design value for the
activated-sludge process.
ii) For good floc formation, sufficient time is needed for the
biomass in the activated-sludge aeration tank to develop
extracellular polymers and a floc structure.
35) Environmental Factors
a) For Carbonaceous Removal:
i) pH in the range 6.0 to 9.0 is tolerable, while optimal
performance occurs near a neutral pH
b) For Industrial Wastewaters:
i) Care must be taken to assure that sufficient nutrients (N and
P) are available for the amount of bsCOD to be treated.
BIOLOGICAL NITRIFICATION
36) Nitrification
a) Biological nitrification is the biochemical oxidation of ammonia
nitrogen by means of autotrophic aerobic bacteria. This process
consists of two stages that involves two distinct groups of bacteria,
each responsible for one of the stages.
b) NITROSOMONAS bacteria, which oxidize ammonia nitrogen (N-NH4+)
to nitrite (N-NO2-):
2NH4+ + 3O2 ==> 2NO2- + 4H+ + 2H2O
NITROBACTERS bacteria, which oxidize nitrite (N-NO2-) to
nitrate (N-NO3-):
2NO2- + O2 ==> 2NO3-
Therefore, the total oxidation reaction is NH4+ + 2O2 ==>
NO3- + 2H+ + H2O
37) Process Description
a) Suspended Growth – a more common approach is to achieve
nitrification along with BOD removal in the single-sludge process,
consisting of an aeration tank, clarifier, and sludge recycle system.
Attached Growth System – used for nitrification, most of the
BOD must be removed before nitrifying organisms can be
established. Nitrification is accomplished in an attached
growth reactor after BOD removal or in a separate attached
growth system designed specifically for nitrification.
38) Microbiology
a) Starting with classical experiments on nitrification by Winogradsky
(1891), the bacteria genera commonly noted for nitrification in
wastewater treatment are the autotrophic bacteria Nitrosomonas and
Nitrobacter, which oxidize ammonia to nitrite and then to nitrate,
respectively.
BIOLOGICAL DENITRIFICATION
39) Denitrification
a) Is the biological process by which nitrate is converted to nitrogen and
other gaseous end products
b) The requirements for the denitrification process are:
i) nitrogen present in the form of nitrates;
ii) an organic carbon source, and
 G6.4

iii) an anaerobic environment. The processes currently used for
biological denitrification are presented as follows.
40) Process Description
a) Assimilating Nitrate Reduction – involves the reduction of nitrate to
ammonia for use in cell synthesis. Assimilation occurs when NH4-N is
not available and is independent of DO concentration.
b) Dissimilating Nitrate Reduction or Biological Denitrification – is
coupled to the respiratory electron transport chain and nitrate or
nitrite is used as an electron acceptor for the oxidation of variety of
organic or inorganic electron donors.
b) Aceticlastic methanogens – split acetate into methane and carbon
dioxide.
c) Hydrogen-utilizing methanogens – use hydrogen as the electron donor
and carbon dioxide as the electron acceptor to produce methane.
BIOLOGICAL REMOVAL OF HEAVY METALS
49) Metal removal
a) In biological treatment processes is mainly by adsorption and
complexation of the metals with the microorganisms.
b) Heavy metals like Zn, Cu, Ni, and Ar are known to have toxic effects at
very low concentrations as well as very high concentration.

41) Microbiology 50) Methods for Heavy Metal Removal

a) Bacteria capable of denitrification are both heterotrophic and a) Biosorption - Sorption process is transfer of ions from solution phase
autotrophic. to the solid phase, actually describes a group of processes, which
includes adsorption and precipitation reactions.
BIOLOGICAL PHOSPHORUS REMOVAL b) By Bacteria and Microorganism - Removal of chromium, lead and
42) Phosphorus Removal copper ions by microorganisms from industrial wastewater. They
a) Is generally done to control eutrophication because phosphorus is a investigated the effect of pH, temperature, initial concentration on the
limiting nutrient in most freshwater system. metal removal.
b) Requirements c) Activated Sludge Process - Is the most common option uses
i) Readily biodegradable BOD in the form of volatile fatty acids microorganisms in the treatment process to break down organic
ii) Phosphorus material with aeration and agitation, and then allows solids to settle
iii) Cycling between anaerobic and aerobic environments out.
c) Advantages d) Trickling Filters - Which consist beds of coarse media (often stones or
i) Low operational costs plastic) 3-10 ft. deep help to grow microorganisms. Wastewater is
ii) Improved treatment performance sprayed into the air (aeration), then allowed to trickle through the
d) Disadvantages media and microorganisms break down organic materials in the
i) Capital cost wastewater. Trickling filters drain at the bottom and the wastewater is
ii) More complicated treatment collected and then undergoes sedimentation.
e) Phosphorus accumulating organisms (PAOs) are encouraged to grow and e) Stabilization ponds or lagoons - Are slow, cheap, and relatively
consume phosphorus removal is comprised of an anaerobic tank having inefficient, biological method that can be used for various types of
a ꚍ value of 0.50 to1.0 h that is placed ahead of activated sludge aeration wastewater. They rely on the interaction of sunlight, algae,
tank. microorganisms, and oxygen.

43) Microbiology
a) Phosphorus accumulating organisms (PAOs)
i) Store excess phosphorus inside cells
ii) Release phosphorus for energy in anaerobic environment
iii) Take in phosphorus in aerobic environment

ANAEROBIC FERMENTATION AND OXIDATION

44) Anaerobic fermentation and oxidation
a) Processes are used primarily for the treatment of waste sludge and
high-strength organic wastes.
b) Anaerobic fermentation processes are advantageous because of the
lower biomass yields and because energy, in the form of methane, can
be recovered from the biological conversion of organic substrates.

45) Process Description

a) Three basic steps are involved in the overall anaerobic oxidation of a
waste:
i) Hydrolysis
ii) Fermentation (also known as acidogenesis)
iii) Methanogenesis

46) Hydrolysis
a) The first step for most fermentation processes, I which particulate
materials is converted to soluble compounds that can then be
hydrolyzed further to simple monomers that are used by bacteria that
perform fermentation, is termed hydrolysis.

47) Fermentation
a) The second step is fermentation (also referred to as acidogenesis).
b) In the fermentation process, amino acid, sugars and some fatty acids
are degraded further.
c) The principal products of fermentation are acetate, hydrogen, carbon
dioxide and propionate and butyrate.

48) Methanogenesis
a) Is carried out by a group of organisms known collectively as
methanogens.