Chapter 11

1. Chapter 11 Measurement of Microbial Activities

2. Why measure activity? • Microbial contribution to nutrient cycling • Transfer of energy between trophic levels • Impact of a disturbance to the system

3. Measuring oxygen concentration in aqueous solutions O2 electrode method Colorimetric method

4. Methods used to measure microbial activities Microelectrodes pH H2S O2 O2 + 4e- + 2H2O → 4OH-

5. Ex situ Sealed LaboratoryMicrocosms • Remove gas sample periodically and measure O2 disappearance or CO2 appearance Advantage:Allows one to design complex experiments that can be operated under controlled conditions • standardization of some parameters • soil moisture • temperature • Disadvantage:Destroys soil structure that may be important in controlling microbial activity

6. Dissolved oxygen consumption by microbes degrading organic matter in wastewater Carbon respiration (heterotrophic activity) consumption of oxygen or other terminal electron acceptor Biochemical Oxygen Demand O2 O2 CO2 CO2 CH2O CH2O DO measurement, t = 0 DO measurement, t = day 5 Add water sample, remove sample for DO measurement, seal container, incubate in dark DO0 - DO5 = amount of organic C oxidized to CO2

7. In Situ Field Studies Place a chamber over a plot of surface soil Measure respiration over time advantage minimal physical disturbance to system disadvantage cannot control soil moisture and temperature measurements are more variable Basal respiration Used as an indicator of soil “health” or condition.

8. Sealed (closed) flow-through system Apparatus to determine oxygen demandof sediment in presence of overlying aqueous phase

9. Basal In-situ respiration in an uncontaminated soil Inject air through probe into subsurface. Halt air injection and allow microbes to consume O2. Withdraw gas samples over 2-8h intervals (%O2 used/hr) To measure O2 & CO2

10. Determination of basal rate of oxygen consumption in absence of contaminant Determine basal respiration rate of microbial community by measuring amount of O2 consumed/h at contaminated site over 2-10 h period O2 consumed 1 2 3 4 5 6 7 8 9 10 hours

11. In-situ respiration in a hydrocarbon-contaminated soil Inject air through probe into subsurface. Halt air injection and allow microbes to consume O2. Withdraw gas samples over 2-8h intervals (%O2 used/hr) To measure O2 & CO2 oil

12. Determination of rate of oil biodegradation Determine rate of oil biodegradation by measuring amount of O2 consumed/h at contaminated site over 2-10 h period O2 consumed 1 2 3 4 5 6 7 8 9 10 hours

13. Respiratory gases Radiolabeled carbon sources (14C-acetate) Carbon dioxide measurement Sealed top alkaline solution CO2 trap Saturation kinetics CO2 14C-acetate Water sample bacterium time

14. Liquid Scintillation Counting

15. K + Sn Sn t = = f Vmax v t = incubation time f = fraction of added substrate taken up v = rate of substrate uptake A = amount of substrate added Sn = natural substrate concentration K = substrate concentration at 1/2 Vmax Lineweaver-Burke Plot

16. Incorporation of radiolabeled thymidine into cellular DNA Measure of secondary production in aquatic environments Methods used to measure microbial activities 3H-thymidine Incubate under in-situ conditions Cell replication Scintillation counting 3H 3H

17. Adenylate energy charge (ATP, AMP, ADP) Methods used to measure microbial activities ATP + 1/2 ADP AEC = ATP + ADP + AMP High AEC = >0.8 (active microbial community Low AEC = <0.4 (dead or moribund community

18. Enzyme assays Dehydrogenase assay assesses oxidation-reduction reactions inside cell Methods used to measure microbial activities Active cells Starved cells DAPI-DNA stains all cells Tetrazolium salt- 5-cyano-2,3-ditoly tetrazolium chloride stains only cells that are respiring

19. Enzyme assays Hydrolysis of fluorogenic substrates phosphatase, lipase, esterase enzyme activity Methods used to measure microbial activities

20. Probe for phosphatase enzyme activity • Water soluble • Light blue to no fluorescence

21. Localization of enzyme activity in flocs • Phosphatase activity detected via yellow-green fluorescence of ELF • Activity is: • localized within floc matrix • not associated with protozoans 50 µm

22. Modern Molecular Methods • Stable isotope probing • Which microbial populations are active • Don’t need to cultivate populations • Expression microarrays • Which genes are expressed and at what level? • 2D-polyacrylamide gel electrophoresis • Separates proteins in 2 dimensions • Charge and size • Matrix assisted laser desorption time-of-flight mass spectrometry

23. ACCTG Typical experiment Question: How does a microorganism whose genome has been sequenced respond to a perturbation in its environment? Inoculum of Geobacter sulfurreducens Soil contaminated with cadmium 5-min exposure 10-min exposure Extract mRNA 15-min exposure 25-min exposure Spot on chip containing Reverse-transcription PCR with fluorescently labeled random primers TGGAC* CGGAC* Gene probe

24. Gene chip Each spot contains a probe sequence of a different gene

25. Microarray Investigation Functional Genomics Effect of cadmium on gene expression • How do environmental perturbations influence gene expression in microbes whose genomes have been sequenced? • 674 genes evaluated • Each row represents 1 gene • 4 different time points • Red: up-expression • Green: down-expression • Black: no change

26. Typical experiment Question: How does a microorganism whose genome has been sequenced respond to a perturbation in its environment? Inoculum of Geobacter sulfurreducens Soil contaminated with cadmium 5-min exposure 10-min exposure Extract proteins 15-min exposure 25-min exposure 2-D gel electrophoresis proteomics

27. Proteomics 2-D gel electrophoresis

28. Proteomics Mass spectrometry

30. How do we scale observations and measurements made at the laboratory bench scale to the field scale? Size Sandbox Geoblock Physical model experimental systems scaled to adapt knowledge acquired at the bench for accurate interpretation of observations in the field Control Complexity

31. Computational modeling Small scale, high control minimum complexity Simple algorithms, Simple predictions Experimental system Computer program Results Results Iteration

32. CCl4-saturated grease plug CCl4 Mesoscale Experimental System DNAPL sensors 2m clay layer Evaluate microbial degradation of carbon tetrachloride in unsaturated heterogeneous porous medium bacteria capillary fringe sand 2m 3m macropores saturated zone

33. CCl4-saturated grease plug CCl4 DNAPL sensors 2m clay layer bacteria capillary fringe sand 2m kC ¶ ¶ 2 2 2 3m C U C C C C ¶ ¶ ¶ D D D macropores y x z + = + + - ¶ ¶ ¶ ¶ 2 2 2 ¶ t x y x R R R R z saturated zone d d d d Benefits of mesoscale experimental systems • Large enough size and time scales to allow coupling of hydrological, geophysical, geochemical and biological processes. Accuracy of Biodegradation Rate Constant Influences Prediction Accuracy

34. Computational modeling Medium scale, moderate control increasing complexity Complex algorithms, predictions on a practical scale Experimental system Computer program Results Results Iteration

35. Summary • Many different techniques to measure microbial activities • Different experimental systems can be used to control environmental variables • Scalability of measurements is important • Ultimate goal is to incorporate activity measurements into predictive computational models that are accurate in predicting phenomenon at a relevant scale