### CEE 210 Environmental Biology for Engineers

```Lecture 6: Quantifying Microorganisms
CEE 210 ENVIRONMENTAL
BIOLOGY FOR
ENGINEERS
Instructor: L.R. Chevalier
Department of Civil and Environmental Engineering
Southern Illinois University Carbondale
Objectives
Review the composition of microorganisms
 Calculated the THOD of bacterial cells
 Understand the bacterial growth curve
 Calculate the specific growth rate of bacteria
 Review methods for measuring bacteria

Environmental
Biology for
Engineers
Composition
Ash, 15%
Volatile, 85%
70-90% Water
Dry weight inorganic and organic
On average, carbon is 50% of this dry weight
Environmental
Biology for
Engineers
Chemical Formula of Microorganisms

Most commonly used
◦ C5H7O2N
◦ Useful simplification, but not a true chemical formula nor an exact
stoichiometric expression
◦ Compare these values to the elemental composition of E. coli
Element
Atomic
Weight
% Cell Dry
Weight
Element
Ratio
Formula
C
12
50
4.2
5
H
1
8
8
7
O
16
20
1.3
2
N
14
14
1
1
Other elements include, but are not limited to, phosphorus, sulfur, potassium, calcium and magnesium
Environmental
Biology for
Engineers
Chemical Formula of Microorganisms
Element
Nitrogen,
14%
Carbon, 50%
S trace
elements
Environmental
Biology for
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% Dry
Weight
General function
3
Constituent of nucleic acid, phospholipids,
coenzymes
1
Constituent of proteins and coenzymes
1
Major cation in cell processes
1
Major cation in cell processes
0.5
Major cation in cell processes and enzyme cofactor
0.5
Major cation in cell processes, cofactor in ATP
reactions
0.5
Major anion in cell processes
0.2
Constituent of cytochromes and other proteins,
enzyme
0.3
Inorganic constituents of special enzymes
Chemical Formula of Microorganisms
C 5 H7 O 2 N
 We can use this formula

◦ Estimate nutrient requirements
◦ Convert gravimetric cell mass measurements into THOD of cell
tissue

Environmental
Biology for
Engineers
Consider the following example that determines the
THOD of microbial cells
Example: THOD of Bacterial Cells
Determine the theoretical oxygen demand of 1 g of
microbial cells using the empirical formula for
microbes. Assume that the organic nitrogen in the
cells is not oxidized (remains in the -3 oxidation
state).
Environmental
Biology for
Engineers
Bacterial Growth

Binary fission
◦ 20, 21,22,23…..2n where n is the number of generations
◦ Generation time a.k.a. Doubling time
 Time it takes for two cells to form from the parent cell
 It is also the time it takes to double the cell numbers
◦ This varies by species and growth conditions
Environmental
Biology for
Engineers
Organism
°C
Vibrio natrigens
37
Escherichia coli
40
Vibrio marinus
15
Nitrobacter agilis
27
Source: Stanier et al., 1986
Doubling time, h
Exponential Growth
dN
 kN
dt
N
t
dN
N N  0 kdt
o
 N 
  kt
ln
 No 
N  N o e kt
N  2No
Environmental
Biology for
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ln 2
k
td
N = number of cells per volume of medium
t=time
k=specific growth rate
No = number of cells per volume when t=0
td = doubling time
Bacterial Growth Curve

Exponential growth can only be carried out up to a
certain point
◦ Limited by environmental conditions, e.g. nutrients depleted

Closed batch systems consistently show a bacterial
growth with 4 distinct phases
◦
◦
◦
◦
Environmental
Biology for
Engineers
Lag phase
Exponential growth phase
Stationary phase
Death phase
Bacterial Growth Curve

lag phase
◦ Microorganisms initially adjust to the new environment
◦ Indicative of microbe’s ability to degrade waste

exponential phase
◦ Microorganisms start dividing regularly by the process of binary fission

stationary phase
◦
◦
◦
◦
◦

Exhaustion of available nutrients
Limited oxygen
pH changes due to build up of CO2
Accumulation of end products;
Limited space
death phase
◦ Number of viable cells decreases geometrically (exponentially), essentially
the reverse of growth during the exponential phase
◦ N=Noe-bt
Environmental
Biology for
Engineers
Cell numbers (log)
Bacterial Growth Curve
Time
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Biology for
Engineers
Example of Exponential Growth
Given the bacterial cell numbers in a batch reactor
measure 34,000/L in 4 hours after incubation, and 5.2 x
106/L after 24 hours. Assuming a negligible lag phase,
estimate:
a) The specific growth rate
b) The initial number of cells
Environmental
Biology for
Engineers
Some Methods used to measure bacterial growth
Method
Direct microscopic count
Viable cell count (colony
counts)
Turbidity measurement
Measurement of total N or
protein
Environmental
Biology for
Engineers
Application
Enumeration of bacteria in
milk or cellular vaccines
Enumeration of bacteria in
milk, foods, soil, water,
laboratory cultures, etc.
Estimations of large
numbers of bacteria in clear
liquid media and broths
Measurement of total cell
yield from very dense
cultures
Measurement of
Biochemical activity e.g. O2
Microbiological assays
uptake CO2 production, ATP
production, etc.
Measurement of dry weight
or wet weight of cells or
Measurement of total cell
volume of cells after
yield in cultures
centrifugation
Cannot distinguish living
from nonliving cells
Very sensitive if plating
conditions are optimal
Fast and nondestructive, but
cannot detect cell densities
less than 107 cells per ml
Only practical application is
in the research laboratory
Requires a fixed standard to
relate chemical activity to
cell mass and/or cell
numbers
Probably more sensitive
than total N or total protein
measurements
Objectives
Review the composition of microorganisms
 Calculated the THOD of bacterial cells
 Understand the bacterial growth curve
 Calculate the specific growth rate of bacteria
 Review methods for measuring bacteria

Environmental
Biology for
Engineers
References




Environmental
Biology for
Engineers
Chapter 11: Quantifying microorganisms and their
activity
Bioremediation Principles, 1998, Ewies, J.B., Ergas, S.J.,
Chang, D.P.Y., Schroeder, E.D., WCB McGraw Hill.
Todar’s Online Textbook of Bacteriology, K. Todar,
http://www.textbookofbacteriology.net/index.html
(accessed March 2010)
Stanier, R.Y. et al., 1986, The Microbial World, PrenticeHall.
Sources of photographs and images in sidebar

Human brain
◦

X-rays images
◦

http://www.healthnak.com/mind/
http://martingallerycharleston.com/index.html
Cold Virus (altered in Photoshop)
◦
http://medphoto.wellcome.ac.uk/
Environmental
Biology for
Engineers

Professor, Civil and Environmental Engineering

Fellow, American Society of Civil Engineers (ASCE)

Diplomat, Water Resources Engineering, American Academy of Water Resources Engineering (AAWRE)

Board Certified Environmental Engineer, American Academy of Environmental Engineers (AAEE)

Licensed Professional Engineer, State of Illinois
```