1. CEE 210 ENVIRONMENTAL BIOLOGY FOR ENGINEERS Lecture 6: Quantifying Microorganisms Instructor: L.R. Chevalier Department of Civil and Environmental Engineering Southern Illinois University Carbondale

2. Environmental Biology for Engineers 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

3. Environmental Biology for Engineers Composition 70-90% Water Dry weight inorganic and organic On average, carbon is 50% of this dry weight

4. Environmental Biology for Engineers Chemical Formula of Microorganisms Most commonly used ◦ C 5 H 7 O 2 N ◦ Useful simplification, but not a true chemical formula nor an exact stoichiometric expression ◦ Compare these values to the elemental composition of E. coli ElementAtomic Weight % Cell Dry Weight Element Ratio Formula C12504.25 H1887 O16201.32 N14 11 Other elements include, but are not limited to, phosphorus, sulfur, potassium, calcium and magnesium

5. Environmental Biology for Engineers Chemical Formula of Microorganisms Element% Dry Weight General function 3Constituent of nucleic acid, phospholipids, coenzymes 1Constituent of proteins and coenzymes 1Major cation in cell processes 1 0.5Major cation in cell processes and enzyme cofactor 0.5Major cation in cell processes, cofactor in ATP reactions 0.5Major anion in cell processes 0.2Constituent of cytochromes and other proteins, enzyme  trace elements 0.3Inorganic constituents of special enzymes

6. Environmental Biology for Engineers Chemical Formula of Microorganisms C 5 H 7 O 2 N We can use this formula ◦ Estimate nutrient requirements ◦ Convert gravimetric cell mass measurements into THOD of cell tissue Consider the following example that determines the THOD of microbial cells

7. Environmental Biology for Engineers 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).

8. Environmental Biology for Engineers Bacterial Growth Binary fission ◦ 2 0, 2 1,2 2,2 3 …..2 n 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 Organism°CDoubling time, h Vibrio natrigens37 Escherichia coli40 Vibrio marinus15 Nitrobacter agilis27 Source: Stanier et al., 1986

9. Environmental Biology for Engineers Exponential Growth N = number of cells per volume of medium t=time k=specific growth rate N o = number of cells per volume when t=0 t d = doubling time

10. Environmental Biology for Engineers 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 ◦ Lag phase ◦ Exponential growth phase ◦ Stationary phase ◦ Death phase

11. Environmental Biology for Engineers 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=N o e -bt

12. Environmental Biology for Engineers Bacterial Growth Curve Cell numbers (log) Time

13. Environmental 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 10 6 /L after 24 hours. Assuming a negligible lag phase, estimate: a)The specific growth rate b)The initial number of cells

14. Environmental Biology for Engineers Some Methods used to measure bacterial growth MethodApplicationComments Direct microscopic count Enumeration of bacteria in milk or cellular vaccines Cannot distinguish living from nonliving cells Viable cell count (colony counts) Enumeration of bacteria in milk, foods, soil, water, laboratory cultures, etc. Very sensitive if plating conditions are optimal Turbidity measurement Estimations of large numbers of bacteria in clear liquid media and broths Fast and nondestructive, but cannot detect cell densities less than 10 7 cells per ml Measurement of total N or protein Measurement of total cell yield from very dense cultures Only practical application is in the research laboratory Measurement of Biochemical activity e.g. O 2 uptake CO 2 production, ATP production, etc. Microbiological assays Requires a fixed standard to relate chemical activity to cell mass and/or cell numbers Measurement of dry weight or wet weight of cells or volume of cells after centrifugation Measurement of total cell yield in cultures Probably more sensitive than total N or total protein measurements able 1. Some Methods used to measure bacterial growth

15. Environmental Biology for Engineers 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

16. Environmental Biology for Engineers References 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) http://www.textbookofbacteriology.net/index.html Stanier, R.Y. et al., 1986, The Microbial World, Prentice- Hall.

17. Environmental Biology for Engineers Sources of photographs and images in sidebar Human brain ◦ http://www.healthnak.com/mind/ http://www.healthnak.com/mind/ X-rays images ◦ http://martingallerycharleston.com/index.html http://martingallerycharleston.com/index.html Cold Virus (altered in Photoshop) ◦ http://medphoto.wellcome.ac.uk/ http://medphoto.wellcome.ac.uk/ About the Instructor 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