Bacterial diversity along an oxygen gradient in Hood Canal, Washington February 22, 2005

Transcription

1 1 Bacterial diversity along an oxygen gradient in Hood Canal, Washington February 22, 2005 Tiffany R. A. Straza University of Washington, Seattle School of Oceanography (206)

2 2 Project Summary Bacteria are now recognized to be an integral part of the marine ecosystem, processing much of the primary production and accounting for much of the respiration within the water column. Previous studies of bacterial diversity have described depthspecific bacterial community patterns, but have not fully explained the potential causes for those patterns (Field et al. 1997, Acinas et al. 1999). With this study, I plan to assess the bacterial diversity in Hood Canal, part of Puget Sound, WA, along both the vertical and North-South oxygen gradients. The deep, southern portions of Hood Canal have shown the lowest oxygen concentrations. I hypothesize that there are varying bacterial distributions in Hood Canal, and that oxygen availability could shape that pattern. Bacterial samples collected along the Canal will be analyzed for diversity using terminal restriction fragment length polymorphism (t-rflp) and clone libraries. Data analysis includes comparing diversity (number of types) to oxygen availability, and quantifying trflp peak similarity (proxy for type similarity) between stations. Types of especial interest (e.g. a commonly recurring peak throughout the Canal) will be inserted into a clone library and sequenced for identification. I expect to see a change in the numbers of prevalent types with a change in oxygen availability, and (or at least) a shift in the bacterial community based on that change in oxygen availability.

3 3 Introduction Bacteria are now recognized to be an integral part of the marine ecosystem, processing up to 50% of primary production (Acinas et al. 1997). Estimating the diversity of bacteria is a start toward understanding bacterial community dynamics, though that itself can be a challenging task. The use of the bacterial 16S rrna gene sequences to identify bacterial types provides a way to analyze the community without culturing each type (Field et al. 1997). Terminal restriction fragment length polymorphism is a rapid and effective way to characterize bacterial communities by defining the number of operational taxonomic units (OTUs) present in a given environment and sample (Moeseneder et al. 1999). As we apply such techniques to differing environments, we can attempt to understand the relationships between bacterial community patterns and environmental characteristics such as oxygen. Hood Canal, part of Puget Sound, Washington, has recognizable oxygen depletion patterns, with the least oxygen in the deepest, southernmost areas (Figure 1).Estuarine circulation creates layering of water masses with different ages, and therefore oxygen concentrations (Figure 2). Respiration patterns and marine community diversity contribute to and are affected by this oxygen distribution. Below the surface mixed layer, bacterial respiration may account for anywhere between % of the oxygen consumption in a stratified system (Tuomi et al. 1999). Previous studies of bacterial diversity elsewhere have described depth-specific bacterial community patterns, but have not fully explained the potential causes for those patterns (Field et al. 1997, Acinas et al. 1999). I hypothesize that there is depth distribution of bacteria in Hood Canal, and that oxygen availability could shape that depth-related distribution.

4 4 The influence of oxygen on diversity could be reflected in a changing North-South pattern as well. Observed patterns might be differing numbers of bacterial types, or different community composition, or both, between stations. There does not seem to be a strict cut-off between anaerobic/aerobic, and anaerobes could be present and thriving even in low-oxygen waters (Baughn and Malamy 2004), implying that many bacterial types could be present in both oxygenated and hypoxic areas. However, the abundances may shift due to increased activity of anaerobic or aerobic types, and that shift would be apparent in trflp analysis (Moeseneder et al. 1999). Shifts in the bacterial community could reflect, parallel, or even induce shifts in community ecology at higher food web levels. The current, and very relevant, concern regarding oxygen availability in Hood Canal calls for analysis at many levels. Understanding the bacterial dynamics, or at least recognizing pattern shifts related to oxygen, gives us insight into one more aspect of this complex system. Proposed Work Data Collection In March 2005, samples will be collected at four stations (Table 1) along the Canal, using the R/V Thomas G. Thompson. For two of the stations (2 and 3), water will be collected from the surface, depth of the chlorophyll maximum layer, mid-water column (80m), and near bottom. At the two endpoint stations (1 and 4) the southernmost and the northernmost, I will sample in greater detail to increase the resolution along the oxygen gradient. Water here will be taken from the very surface (using a hand-held

5 5 Niskin), 2m, 5m or depth of the chlorophyll maximum layer, 15m, 30m (at Northern station; 50m at Southern station), and near bottom. At each sampling depth, the salinity, chlorophyll concentration (proxy for phytoplankton biomass), oxygen concentration, and nutrient (PO 4, Si(OH) 4, NO 3, NO 2, NH 3 ) concentrations will be measured. Salinity and chlorophyll fluorescence will also be determined with the CTD profiler, calibrated to hand-analyzed samples. Sample Analysis Chlorophyll samples will be analyzed on board using acetone extraction and fluorometric detection. Oxygen samples will be analyzed on board, using a modification of the Winkler method (Codispoti 1988, unpublished). Salinity and nutrient samples will be sent to the UW Marine Chemistry Lab for processing. Bacterial water samples (1 liter) will be pre-ilowhuhgzlwk PPHVKVFUHHQWR exclude zooplankton, large diatoms, and large particulate debris. Samples will then be run through P GF/F filters (2.5 cm diameter); 500mL per filter, thereby obtaining two filters (replicates) per sampling depth. Filters will be frozen in liquid nitrogen aboard ship to preserve until analysis. Post-cruise analysis of bacterial samples will be completed at the Marine Molecular Biotechnology Lab (MMBL), using protocols I have already developed along with other previously developed molecular techniques for t-rflp and clone libraries. Bacterial DNA will be extracted from the filters, using the Qiagen DNeasy kit. PCR with labeled primers (27f FAM forward primer; unlabeled 1492r reverse primer) will be used to amplify the 16S rrna genes. Restriction enzymes (Hin6I and RsaI) added to the

6 6 cleaned PCR product will cut the DNA into the fragments for trflp analysis. The final restriction digests will be analyzed using a MegaBACE1000 DNA Analyzer (Molecular Dynamics) to measure trflp. Data Processing The number of bacterial types (proxied by the number of t-rflp peaks) will be compared between stations. The main analysis includes comparing diversity (number of types) to oxygen availability, and quantifying trflp peak similarity (proxy for type similarity) between stations and again, relating to oxygen concentrations. The presence or absence of fragment peaks can be used to give a preliminary assessment of community shifts between stations, and a calculation of percent similarity will be completed. Bacterial types of especial interest (e.g. a commonly recurring peak throughout the Canal) will be inserted into a clone library (using an E. coli kit) and sequenced for identification. Since chlorophyll and salinity could also affect the distribution of bacteria, these data will be checked for correlation with bacterial type shifts (Gordon and Giovannoni 1996). Again, the data will be analyzed for clustering based on percent similarity for a given salinity or chlorophyll concentration.

7 7 Draft Budget For 20 samples; 5 day cruise Item Supplier Cost R/V T. G. Thompson UW Oceanography Class Budget Single Niskin, with messenger UW Ocean Pooled Equipment??.?? Dewar (for liquid N 2 ) Keil lab (?) Liquid nitrogen Filtration rig ($6/day) Pooled Equipment Filters (GF/F; $0.40 each) Biochemistry Stores Cryovials ($0.30 each) Keil lab (pay back) 6.00 Forceps, Sharpies, etc. Keil lab Chlorophyll analysis (incl. tubes) Chemicals, etc., from MCL Centrifuge ($6/day) Pooled Equipment Chlorophyll bottles Keil lab/mcl Fluorometer ($15/day) Pooled equipment Nutrient filters ($2.25 each) Marine Chemistry Lab Salinity bottles ($3/day; 24 per case) Pooled Equipment Salinity analysis ($9.50 ea; 3 samples) Marine Chemistry Lab Nutrient analysis ($10.50 each) Marine Chemistry Lab Oxygen bottles ($6/day; 24 per case) Pooled Equipment Chemicals for Winkler O 2 titration Marine Chemistry Lab Dosimat (for O 2 ; $15/day (1 day paid Pooled Equipment for Hudson)) Sampling collection bottles Keil lab Acetone (for chlorophyll analysis) Marine Chemistry Lab Qiagen DNA extraction kit Fisher Scientific Tips, tubes, other plastics MegaBACE runs MMBL Taq polymerase (estimated price) Promega Restriction enzymes Fermentas, Inc. Cloning kit Total $834.45

8 8 Station No. Region Position Latitude (ºN) Longitude (ºW) Bottom Depth (m) 1 Hood Canal sill Under Dabob Bay Mid-canal Great Bend (south) Table 1. Station data

9 Figure 1. A typical oxygen concentration plot for Hood Canal (North is on the right). 9

10 Figure 2. Schematic diagram of Hood Canal circulation. 10

11 11 References Acinas, S. G., J. Antón, and F. Rodriguez-Valera Diversity of free-living and attached bacteria in offshore western Mediterranean waters as depicted by analysis of genes encoding 16S rrna. Appl. Environ. Microbiol. 65: Acinas, S. G., F. Rodriguez-Valera, and C. Pedrós-Alió Spatial and temporal variation in marine bacterioplankton diversity as shown by RFLP fingerprinting of PCR amplified 16S rdna. FEMS Microbiol. Ecol. 24: Baughn, A. D., and M. H. Malamy The strict anaerobe Bacteroides fragilis grows in and benefits from nanomolar concentrations of oxygen. Nature 427: Codispoti, L One man s advice on the determination of dissolved oxygen in seawater. Unpublished. Field, K. G., D. Gordon, T. Wright, M. Rappé, E. Urbach, K. Vergin, and S. J. Giovannoni Diversity and depth-specific distribution of SAR11 cluster rrna genes from marine planktonic bacteria. Appl. Environ. Microbiol. 63: Gordon, D. A, and S. J. Giovannoni Detection of stratified microbial populations related to Chlorobium and Fibrobacter species in the Atlantic and Pacific Oceans. Appl. Environ. Microbiol. 62: Moeseneder, M. M., J. M. Arrieta, G. Muyzer, C. Winter, and G. Herndl Optimization of Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis for complex marine bacterioplankton communities and comparison with Denaturing Gradient Gel Electrophoresis (DGGE). Appl. Environ. Microbiol. 65: Tuomi, P., K. Suominen, and R. Autio Phytoplankton and bacterioplankton production and bacterial biomass in a fjord-like bay open sea gradient. Hydrobiologia 393: