“Degradation”

1. “Degradation” Goals: • To start thinking mechanistically about what controls degradation (and thus preservation) of OM •  to provide a framework for evaluating hypotheses about organic matter cycling and preservation. * To review a number of common indicators of degradation.

2. OM Degradation: Why would we care about this? • Main “control valve” in biogeochemical cycles • Controls: • How much carbon is recycled vs. preserved • Which compounds are preserved • Understanding OM “degradation” tells you about refractory OM composition & how it forms.

3. Degradation: The Junk Yard Analogy What we want to know = how does it all work? What processes control what survives? 2. Which parts to look for that tell you the most?

4. Degradation = “Digenesis”? Not exactly. “Diagenesis“= a broad term for all processes (biological and abiotic) which both break down and alter organic structures. “Degradation”- more or less a subset of these processes- focused on the biological ones. Thus Mostly Degradation = those processes which control progression: large biomolecules => small biomolecules => Monomers CO2(+ New Biomass)

5. Degradation processes = Preservation processes? Not usually. Clearly they are coupled, but.... Thinking about Degradation = thinking about what processes are necessary to get carbon units in macromolcules inside of microbial cells. Thinking about Preservation= thinking about factors, structures, or processes which keep this from happening.

6. Fundamental aspects of OM degradation: 1. Its amazingly efficient: 99.9% of all organic matter produced is degraded and recycled. 2. Its essentially microbial. * Taken as a whole, Macro-organisms are not the major players. Bacteria (everywhere) and Fungi (on land) are.

7. So if bacteria are the keys:What are Key Problems for Microbes? 1.Getting material into their cells: • Size limitations. Most OM is transported actively across membranes. However, Bacterial cell membranes impermeable to compounds > 600-1000 MW.

8. Exact size-limitation differs somewhat, but in general: • an oligopeptide larger than ~6 amino acids in length or a oligosaccharide larger than ~2-3 sugars in length cannot be directly transported into the cell. Implication: most things must be hydrolyzed to smaller components extracellularly! IE: most initial hydrolysis of OM must occur outside the cell.  The first “bottleneck” of degradation is breaking polymeric material down to monomers

9. Which leads to key problem II: How do you hydrolyze material outside your cell? Either: • Send enzymes out to get it. (“exoenzymes”) or • Wait for it to come to you, “trap it” – membrane-bound enzymes.

10. Considerations if you send enzymes out: a) Will the hydrolyzed material make its way back to you? (by diffusion- ie random walk processes?) B) Will material out there be “available” for enzymatic attack? Recall: enzymes are highly stereospecific- ie, need to be able to randomly collide with substrate in correct orientation.

11. Some Considerations if you “wait”: • Is there enough soluble material diffusing to you? • If not, can you colonize a surface or particle, and hydrolyze surface- attached material?

12. An Broad Ecological Issue Controlling Degradation: “will it be worth it”? ..on balance, for microbe to produce and “throw away” all the the energy, nitrogen, and carbon to send these enzymes out there?  A consequence: thought that exoenzyme degradation is more common in sediments (limited space, high OM) than in dissolved environments (although it happens in dissolved environments too!).

13. Many Proposed mechanisms of OM preservation are predicated on interfering with enzymatic processes-

14. Consequence: What is “refractory” OM? Often thought/ talked about as “non-degradable” OM. BUT- from above discussion- to be “refractory” it does not have to be impossible to degrade .. Just not worth it..in a given environment

15. Consequence: If you want to study effects of different structures or conditions on degradation- Its really a study of “rates” Ie, not “can x be degraded”, but how does a change affect the rate of degradation of x.

16. Some mechanisms limiting degradation: Sediments: Surfaces, or micro-pore”protection”- * Keeps enzymes away, or lessens steric availability “Encapsulaton” (or: the “french onion soup hypothesis”) * Hydrolyzable material is “sequestered” inside aliphatic material

17. More examples DOM: Concentration effect in DOM: no surface to colonize, and not enough material out there to make it “worth-while” to send out exoenzymes. Everywhere: Structural modification: minor or major structural changes, foils enzyme mechanism

18. A second main degradation: Energy yield • Once polymers are broken down and taken up, the “real” degradation ( i.e. redox reactions inside cell) gets going. * essentially an electron transfer reaction from reduced carbon (CHx) to an electron acceptor (O2, SO4, etc)

19. Which introduces another variant of “is it worth it”: The O2 factor: Which Electron Acceptor is available and does this matter? Or as most question is most commonly put: Is Aerobic degradation any faster / slower than anaerobic degradation?

20. Aerobic vs Anaerobic which is faster? Answer?:remains debated. Biological view: has been shown that anaerobic degradation can be just as fast as aerobic degradation. Geochemical view: continues to be that oxygen is very important rate-limiting factor, especially in very long term. But are contrasting such views comparing apples and oranges.. ? Probably.. In part, due to a huge time-scale problem.

21. Aerobic vs. Anaerobic which is faster? But are contrasting such views comparing apples and oranges.. ? Probably.. In part, due to a huge time-scale problem. • OVER Short time scales, biology wins. • OVER very LONG time scales, thermodynamics wins (02 is critical..)

22. Interesting subtleties around O2: Capabilitiesfor degradation may differ in oxic vs anoxic regimes. • Different species, but also anaerobic degradation tends to proceed via consortia of bugs- which apparently can degrade things that single bugs cannot. • If this is true for some degradation pathways, implies that some molecule types might mainly be degraded deep in sediments, or else in anaerobic microzones.

23. Part II: Measuring & testing degradation How fast is it going? Which parts tell you the most?

24. Measurement I: How fast is it going? * Key question = Rates 1. Why? 1) = Balancing Carbon cycle processes. IE: is OM being preserved in a given environment. 2. Putting structures in a context- ie: assigning “liability” to certain structural classes, or looking to structures to explain fast or slow bulk degradation.

25. Major issue: The “Time-Scale Problem” 1.Problem: how do you do an experiment to model or test degradation processes which occur in deep ocean sediments over, say , 1000 years (ie typical organic geochemical questions)? eg: measuring enzymatic activity is of little use when trying to understand interactions occurring on thousand-year time scales

26. Major issue: The “Time-Scale Problem” 2. Conversely: measuring total organic carbon contents may be of little value when estimating short-term degradation  In general: For any given technique, the reaction rates that can be investigated probably should not be extrapolated much beyond a factor of 10 of the time scale of the experiment..

27. That said, approaches taken can be grouped by time scale:Short term assays: a) usually the province of microbiologists, or photochemists. Eg: Labeling or tagging assays. Use an internally- 1) tagged (e.g. radioactive) or 2) externally tagged (eg special functional group) Classic internal Example: addition of radiolabeled glucose to samples in order to measure respiration rates (14CO2).

28. External tag tests: 1.Goal:, want to create either a precursor or product molecule tied directly to process of interest that is very easy to measure Eg: MCA-peptides: have a special “peptide” bond at the end that is hydrolyzed similarly to other protein bonds, but when the MCA is released it becomes fluorescent, making the buildup of fluorescence an indicator of protease activity. Disadvantages: Labels can have an effect on the process being studied.

29. External tag tests: 1.Goal:, want to create either a precursor or product molecule tied directly to process of interest that is very easy to measure Eg: MCA-peptides: * have a special “peptide” bond at the end • when hydrolyzed: MCA is released and becomes fluorescent • Measure fluorescence buildup = protease activity. Disadvantages: Labels can have an effect on the process being studied.

30. Approach to study Structural modification: C. Arnosti- many studies of tagged carbohydrates in the environment- have shown that Small changes in molecular structure can tremendously affect (speed up, slow or even stop) the ability of microorganisms to degrade a substance.

31. Hydrolysis rates of different carbohydrates in sediment vs. seawater • Major rate differences are seen in sediment vs. Seawater • But also: different related structures are NOT hydrolyzed at same rates! •  biological communities are very specific (on short time scales.)

32. Intermediate-term Indicators (days to a week Experimental Incubations: Variations on similar theme: A sample is collected and then incubated, either in situ or in a laboratory, under “natural” conditions, and the fate of organic matter is followed over time. Classic Example: a sample is collected, placed in a sealed container, and the buildup of CO2 (or the draw down of OC) is monitored over time. (BOD and COD assays).

33. Intermediate-term Indicators (days to a week Example 2: Studies of OM degradation in seawater usually entail isolation of microorganisms (and OM in question) from sources via filtration and then incubation under natural conditions. The macromolecule is tracked over time and degradation rates are assumed (hoped?) to be equivalent to that of the natural system

34. Example: combination of internal tag + bottle experiment 3H Nagata et al, 2003, L&O 1 Question: Does Peptidoglycan really degrade any slower than protein in dissolved phase?. Experiment: Grow bacteria using 14C- labeled components (eg amino sugars, or peptides, which you can buy). Then isolate the Peptidoglycan, and do a bottle feeding experiment. Grow up bugs, isolate Peptidoglylcan Feed to cultures in seawater HPLC & Size separations: where did radioactivity go?

35. Nagata Experiment Found: • Protein – labeled material degrades totally, and is respired. (all in H20) • Peptide-labeled material does degrade, but LMW fragments hang around. • Sugar-labeled material also degrades, BUT a much larger percentage than others hangs around!

36. Nagata Experiment Overall • Peptidoglycan does degrade slower than protein. • BUT also, use of creative labels tells you that apparently it’s mostly the sugar-portion of the peptidoglycan structure that is most “refractory” and builds up. .

37. Nagata Experiment Further: • Implies that an important “bottleneck” in degradation is not the hydrolysis into fragments, but instead the “uptake” and utilization of the fragments. • Overall: pretty darn detailed view of relative fates of a complex macro-molecule!

38. Intermediate-term Indicators (days to a week e- acceptor Studies Instead of tracking OM, degradation is tracked by following the depletion of an electron acceptor such as O2 or NO3-. In field: can in principle track the entire process of degradation- eg: Landers or probes measuring total depletion (or buildup) of given species from sediment column.

39. Some Landers ready to be deployed

40. They go from very simple to very very complex

41. “Long-term” Indicators (Months to millennia) Experiments ranging from • longest possible controlled lab simulations, or • via measures of various proxies in sediment cores. •  Can try to draw conclusions about what has occurred over very long time scales.

42. Eg 1: Artificial Ecosystems Every decade or so, a research institution will create a huge set of incubators in which many aspects of carbon cycling can be monitored simultaneously of several years. • Eg: “MERL” tanks at URI. • Advantages and disadvantages: entire system closed and controlled, but is it realistic?

43. Eg 2: Sediment profiles Monitoring and Modeling Changes in Organic Carbon Content (e.g. Multi-G Models). Models of down-core diagenesis usually assume first-order decay of organic matter and relate decay rates to molecular-level compositions of materials Rate-constant modeling approach to sediment TOC

44. Eg 2: Sediment profiles • Advantages and disadvantages: allows an estimate of reactivity over time, • but typically are constrained by poor fits to real data and • a lack of understanding of what organic matter fits into what rate- compartment and why. Rate-constant modeling approach to sediment TOC

45. Finally: “Indicators” of Degradation and Diagenesis(sometimes also called “proxies”)

46. Categories of Common Indicators: Microbial by-products of degradation: Eg: Some non-protein amino acids and fermentative by-products accumulate in samples during degradation.

47. Some other categories of Common Indicators: Bulk indicators of degradation: changes in, or unusual distributions of, • bulk OM composition (e.g. Van Krevelen plots and C:N ratios). Chirality: biologically-mediated production of compounds always favors one enantiomer over another, thus an abiotic products will show no or few signs of preferential isomerization.

48. END