1. NIMBioS Investigative Workshop Individual-Based Ecology of Microbes: Observations and Modeling June 8-10, 2011 Knoxville, Tennessee Q3: Interactions with the environment Elucidating the bacteria responsible for utilization of dissolved organic matter in streams Philips Akinwole Philips Akinwole

3. Personal Introduction MSc in Environmental Science and Technology (2003-2005) International Institute for Infrastructural, Hydraulic, and Environmental Engineering, (UNESCO-IHE), Delft, the Netherlands Project: Litter decomposition across multiple spatial scales in German stream networks so…I’m broadly trained in stream and microbial ecology using stable isotope probing and phospholipid techniques (a multidisciplinary approach) to link microbial community structures to functions … presently PhD Candidate in Microbial Ecology/ Department of Biological Sciences University of Alabama, Tuscaloosa, AL (2007…) … interested in linking microbial community structures to functions Presenting Projects: Stable Isotope Probing of Dissolved Organic Matter Processing in Streams Spatial Patterns of Microbial Signature Biomarkers in Stream Networks Measuring dissolved organic matter spiraling lengths: whole stream releases

4. Elucidating the bacteria responsible for utilization of dissolved organic matter in streams  Streams and rivers can be found everywhere.  Bodies of flowing water moving in one direction from headwaters (springs, snowmelt or even lakes) and then travel all the way to their mouths, usually another water channel or the ocean.  The characteristics of a river or stream change during the journey from the source to the mouth.  Full of life; from microbes to plants (algae and macrophytes) and animals (fish and macroinvertebrates). … some concepts Dissolved organic matter (DOM), the largest pool of carbon in aquatic ecosystems, is heterogeneous in nature and supplies the needed energy and carbon to microbes; in streams it may meet the majority of heterotroph carbon demand (Kaplan et al., 2008). …bacterial are the main heterotrophic microorganisms in stream responsible for the breakdown of organic biomass and the recycling of various key elements (N,P,S,C) Neversink River, NY

5. Elucidating the bacteria responsible for utilization of dissolved organic matter in streams One of the methods (biochemical and molecular) of studying microbial diversity/community structure is the Phospholipid Fatty Acid (PLFA) Analysis  PLFAs are a major component of the cell membrane of all microbes (bacteria and eukaryotes) and they only remain intact in viable cells.  are relatively easily extracted from natural samples.  PLFA analysis as biomarkers provides direct information on the whole microbial community including quantitative information on viable (living) biomass, community structure (Population “Fingerprint”) and microbial activity.  Fatty acids are identified and quantified by gas chromatograph (GC).

6. Elucidating the bacteria responsible for utilization of dissolved organic matter in streams Stable Isotope Analysis Isotopes are forms of an element that differ in the number of neutron (H, C, N, O & S) - functions as natural dyes/colors, generally tracking the circulation of elements (discrimination between heavy and lighter isotopes). -- trace ecological connections at many levels, from individual microbes to whole landscapes Direct links between microbial identity and biogeochemical processes are now possible with stable isotope analysis of biomarkers (lipids, amino acids & nucleic acids) Usually, stable isotope ratios are given in the δ -notation, which for carbon is defined as: … measured with the gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS).

7. In stream ecosystems, monomeric constituents of DOM are most labile while polymeric /humic substances of terrestrial origin are the major constituents of DOM pool (40-70% of DOM in rivers) and have been considered biochemically refractory (Benner 2003) … and poorly used by microbiota due to the apparent chemical recalcitrance of humic substances. Elucidating the bacteria responsible for utilization of dissolved organic matter in streams Recent studies (Frazier et al.2005, Kaplan et al., 2008 etc) have suggested the susceptibility of humic substances to microbiological and photolytic oxidations and could provide a supplement to heterotrophic metabolism.

8. This study was framed within the general question “Does streamwater DOM contribute significantly to stream ecosystem metabolism?” and particularly addressed the question “Which microorganisms within the stream community utilize humic-DOM?” Elucidating the bacteria responsible for utilization of dissolved organic matter in streams -generate a tracer model that is more reflective of natural stream DOM, contrasting the use of bicarbonate or acetate as tracers (e.g. Pace et al., 2004; Johnson & Tank 2009), - we synthesized a 13 C-DOM tracer from tulip poplar tree-tissues with size and lability fractions approximating streamwater DOM (Wiegner et al., 2005). …to do this

9. Elucidating the bacteria responsible for utilization of dissolved organic matter in streams … research activities Collection of streambed sediment from WCC Sediments were incubated in mesocosm chambers with 175 µg C L -1 13C – labeled DOM Microbial biomass and community structure were analyzed by PLFA The fatty acid methyl esters (FAMEs) obtained from PLFA analyses were identified and quantified by GC, and analyzed for their δ 13 C values using the GC-IRMS. For each experiment, treatments were identified as: T0 – sediments collected from WCC at the beginning of the experiments; T13C – sediments incubated for 48hrs in streamwater amended with 13 C-labeled leachate and, T50 – sediments incubated for 48hrs in streamwater. Four experiments were conducted in the light (unfiltered sunlight) and dark (chambers were covered with styrofoam and black plastic)

10. Results Elucidating the bacteria responsible for utilization of dissolved organic matter in streams br17:1a, a17, 18:1w5, i17, cy17:0, br18:0a, 10me16, i16, cy19, a15 22:6w3, 16:1w13t, 20:5w3, 16:3, 20:4w6, br18:0c, 14:0 16:1w7c br17:0, 16:4w1 17:0, 22:0, br19:1a, 20:0, 18:0, 18:1w9 PCA to evaluate changes in total microbial community structure during mesocosm incubation. Red arrows emphasize a comparison between T0 and T48/50 data (mesocosm controls) while blue arrows emphasize a comparison between T0 and T13C data ( 13 C-DOM treatment). [ ● (Exp1), ♦ (Exp 2), ▲ (Exp3), ■ (Exp4)]

11. Results Elucidating the bacteria responsible for utilization of dissolved organic matter in streams Stable carbon isotopic information for PLFAs from Experiment 1 (Dark) sediment incubated with and without 13C-labelled DOM T0 = natural stream sediments T48 = natural steam sediments incubated with recirculating streamwater for 48 hrs T13C = natural steam sediments incubated with recirculating streamwater amended with 13 C-DOM for 48hrs

12. FAME Bio marker EXP 1 (Dark) EXP 2 (Light) EXP 3 (Light) EXP 4 (Light) i15:0Prokaryote√√√√ a15:0Prokaryote√X√√ 16:0Non specific√√√√ 16:1w7c, 16:1w5cProkaryote√Depletion√√ 16:1w9Non specific√√√√ 10me16:0ProkaryoteXXXX cy17:0Prokaryote√XXX 18:2w6EukaryoteXX?X 18:3w3Eukaryote√X?X 18:1w9c, 18:1w7cNon specific√√√√ cy19:0Prokaryote√XXX 20:4w6Eukaryote√X?? 20:5w3Eukaryote√√√ X 22:6w3Eukaryote√XXX √= Enrichment X = No enrichment ? = Potential enrichment Summary of incorporation of 13 C-labeled DOM into microbial lipid by experiment Results Incorporation of label into PLFA was studied in light and dark incubations. Dark incorporation was fully sensitive to 13C-labelled DOM utilization

13. Conclusions  The PCA analysis of microbial biomass and community structure validate the robustness of mesocosm-based experimental design for determining the role of sediment microbes in processing streamwater DOM via incorporation of 13 C-DOM into microbial fatty acids.  PLFA analysis indicated that both phototrophic and heterotrophic microeukaryotes were present in the sediment community. For the dark experiment, we assume that the 13 C- labeled DOM uptake by prokaryotes is direct and not facilitated by photolysis.  The consistently labeled fatty acids: i15:0. 16:0, 16:1w9, 18:1w9c and 18:1w7c in our study are found in bacteria often associated with aerobic or facultative anaerobic metabolism (Findlay 2004).  Fatty acids 10me16:0 (showed no labeling) and cy17:0 (only showed moderately labeling when sediments were incorporated in the dark) are consistently found in bacteria associated with anaerobic metabolism, such as sulfate reducing bacteria.  Eukaryotic marker fatty acids (20:4w6, 20:5w3) were most constantly labeled in the dark experiment suggesting that microeukaryotic predators are consuming bacteria utilizing 13 C-labeled DOM.

14. … motivation for and challenges in individual base approach Individual based approach use information at the level of individual organisms and derive characteristics of the population as an integral of all individuals represented in the population. - Successfully applied in conservation biology …however, they include many parameters which are difficult to estimate for natural populations and which rely on very detailed knowledge on the species and the spatial structure of its populations. - I’m interested in how to apply/use individual base model in my work, - advances made in microbial ecology, - applications and uses in other areas

15. Thank you