Biofilm Community Structure and Resource Availability in Experimental Flow Cells China A. Hanson Microbial Diversity 2008 MBL, Woods Hole Contact Info: Department of Ecology and Evolutionary Biology University of California, Irvine cahanson@uci.edu Abstract Artificial one- and two-species biofilm communities were constructed in a flow cell (continuous flow biofilm chambers) experiment in order to investigate the effects of nutrient availability on biofilm community structure. Flow cells were inoculated with Vibrio natriegens (a relatively fast grower), Hyphomonas neptunium (a slower grower, which prefers low nutrient conditions, and is a well-characterized biofilm former), or a mixed culture consisting of approximately equal cell numbers of each of the two species. Cells were allowed to attach to surfaces, and then were amended with either a high nutrient or low nutrient medium. After 18 hours, relative abundance of the two strains were enumerated by FISH. The results did not support the hypothesis that V. natriegens would outcompete H. neptunium under high nutrients. Instead, H neptunium was present in all biofilm communities, indicating that its ability to attach to surfaces readily may facilitate its coexistence with competitors. 1
Introduction Community development and structure among microbes is vastly complex, and understanding the biotic and abiotic factors that influence microbial community assembly, structure, and diversity remains a central goal in microbial ecology. One of the most common small-scale community patterns observed among microbes is the development of biofilms (Battin, T. J., et al., 2007). Biofilm development is an effective ecological strategy for microbes and has been implicated in antibiotic resistance, water quality concerns, and structural damage, or biofouling. However, biofilms also represent a complex assemblage of microbial species, harboring much micro-scale temporal and spatial habitat heterogeneity. As such, biofilms can be used as an interesting model system for investigations of the effect of environmental factors, spatial complexity, and competition on microbial community succession, species coexistence, and community structure. In order to examine the effect of nutrient availability on the development and community structure of simple one- and two-species biofilms, I established artificial biofilm communities in a flow-cell experiment. The secondary goals of this pilot study were to: 1) develop and optimize an experimental, home-made flow-cell system; 2) develop and optimize a procedure for fluorescent in situ hybridization (FISH) on biofilms formed within flow cells (glass microscope slides). Background and Hypotheses For this experiment, I chose two relatively fast growing marine aerobic heterotrophic bacterial isolates: Vibrio natriegens (ATCC 14048) and Hyphomonas neptunium (ATCC 15444), both of which have been found colonizing the surfaces of sea water purification systems. However, both strains are also readily found as pelagic, non-sessile cells. These isolates were chosen from a number of possibilities available through the Marine Resources Center at the MBL on the basis of similarity in optimal growth conditions (26 C, sea water media), difference in potential growth rate and rrna copy numbers (V. natriegens is relatively fast, contains 13 rrna gene copy numbers per genome, while H. neptunium has slower growth rates and contains 1 rrna gene copy number per genome) and ability to distinguish them using FISH probes for broad taxonomic groups (V. natriegens is a γ proteobacterium, while H. neptunium is a α- proteobacterium). V. natriegens is a ubiquitous marine bacterium, while H. neptunium is more 2
rare, typically found in oligotrophic environments, and grows well on a defined minimal medium consisting of 30% seawater supplemented with low concentrations of just four amino acids (Havenner, J. A., et al., 1979; Poindexter, J.S., 2006). H. neptunium is also a well-characterized biofilm former, having an unusual reproductive morphology that is thought to supplement the ability to attach to surfaces. Bacteria with this morphology are known as dimorphic prosthecate bacteria, and form an asymmetrical protrusion of the cell wall and cytoplasm called the prosthecate. This structure eventually buds into a daughter cell, and may also aid in surface attachment (Poindexter, J. S., 2006). Based on the potential growth rates and optimal nutrient requirements of the two species, I developed two hypotheses: if inoculated in equal initial abundances and growth phases, 1) V. natriegens would out-compete H. neptunium in biofilms under high nutrient availability; 2) H. neptunium would coexist with V. natriegens in biofilms under low nutrient availability. These hypotheses would be supported by a reduced abundance of H. neptunium relative to V. natriegens under high nutrient availability, and a more even relative abundance of both species under low nutrient conditions. Experimental Design, Flow-Cell Approach, FISH Flow-cell chambers were created by sealing together two glass slides separated by round capillary tubes. The flow cells and multi-channel flow-cell pump system were created as depicted in Fig. 1 and following a similar procedure as Palmer, R. J. and Caldwell, D. E., 1994 and Foster, J. S and Kolenbrander, P. E., 2004. Ten flow cells were run simultaneously each consisting of one of 4 inoculation treatments and one of 2 nutrient treatments: V. natriegens (single-species) + low nutrient medium (n=1); V natriegens (single-species) + high nutrient medium (n=1); H. neptunium (single-species) + low nutrient medium (n=1); H. neptunium (single-species) + high nutrient medium (n=1); H. neptunium and V. natriegens (two-species) + low nutrient medium (n=2); H. neptunium and V. natriegens (two-species) + high nutrient medium (n=2); control (no inoculum) + low nutrient medium; control (no inoculum) + high nutrient medium. Complete flow cell system was autoclaved, set up, and connected. To initiate the flow cells, each line (tubing+ bubble trap+ flowcell) was flushed simultaneously with 10% bleach at 5-20 rpm for 30 min; flushed with sterile seawater (SW) base at 20 rpm for 20 min, then at 8 rpm for 1 hour. Flow cells were then inoculated with a liquid 3
culture of the appropriate type by running the inoculum through each line (see discussion for caveats) for 30 min at 4 rpm. Liquid inoculum was prepared by transferring 50 ul of overnight cultures of either V. natriegens or H. neptunium into 50 ml of sterile SWC medium; these were grown up at room temperature with shaking over 1 day and continually transferred in the case of V. natriegens, or allowed to grow up at room temperature with shaking for 3 days (H. neptunium). Single species inoculum consisted of fresh overnight culture of V. natriegens or 3 day culture of H. neptunium, diluted to an OD at 600 nm of 0.67-0.68 in sterile SWC medium. Two-species inoculum consisted of a the same diluted single-species cultures combined in equal volume. Once flow cells looked visibly turbid, flow was halted by removing all lines from the pump and clamping the tubing down directly upstream and downstream of the flow cell chambers using binder clips (as shown in Fig. 1a). Inoculated flow cells were incubated for 2 hours, and then flushed with sterile SW base for 60 min at 2-4 rpm to remove unattached cells. Either high or low nutrient sterile medium was then pumped through the appropriate cells at 2 rpm for 18 hours. Both media types consisted of modified SWC medium with 30% final concentration of SW base. High nutrient medium: 300 ml of SW base, 700 ml water, 5 g Bactotryptone; 1 g yeast extract, 3 ml glycerol. Low nutrient medium: 300 ml of SW base, 700 ml water, 2.5 g Bacto-tryptone. Flow cell chambers were removed from the system, immediately split apart using a scalpel; and fixed for DAPI staining + FISH by immersing the bottom slide of each flow chamber in a sterile 50 ml tube containing 1X PBS with 1% formaldehyde. Slides were allowed to fix in this solution for 1 hour at room temperature, then were washed in sterile 1X PBS, sterile water, and finally dipped in ethanol (99%), and allowed to dry for several hours at room temperature. FISH: All silicone adhesive was removed from slides after fixation, and each slide was broken into 4 rectangular pieces, one for each of 4 FISH probe hybridizations: Non 338, Eub I- III, Alf968, Gam42a. Slide pieces were labeled on the back by scoring, and edge was sealed with PAP pen. Hybridization buffer was prepared as described in the Microbial Diversity Course manual: all hybridization buffers prepared with 35% formamide, except buffer for Alf968 was 30% formamide. 30 ul of probe + hybridization buffer solution (3 ul probe + 27 ul buffer) was pipetted onto each slide piece and allowed to incubate in hybridization chambers at 48 C for 90 min. Slide pieces were washed in 48 C wash buffer (recipe as in manual) for 10 min; washed in 4
sterile purified water for 2 min, then allowed to dry at room temperature in the dark for several hours until dry. Slide pieces were mounted onto a large slide using nail polish, and 2 ul of mounting media + DAPI was applied to the slide piece, covered with a small cover slip; and immediately counted by epifuorescence microscopy. At least 1,000 cells were counted per piece per fluor. Other Materials and Methods Isolates of Vibrio natriegens (ATCC 14048) and Hyphomonas neptunium (ATCC 15444) and data on growth time, optimal growth conditions, rrna gene copy numbers were obtained from the Marine Resources Center, MBL, Woods Hole, MA. Flow-cell chambers and a multi-channel flow-cell pump system were created as depicted in Fig. 1. Materials used to create each flow-cell included: glass slides; glass hollow thin capillary tubes; silicone adhesive (RTV 118), silicone tubing (a variety of outer diameters, but uniform inner diameters); polyethylene tube fittings of the appropriate sizes to match tube diameters; 200 ul pipette tips (2 per cell; end of tip used for outflow cut off ~1mm to eliminate pressure build-up at outflow), sterile syringes and needles of various sizes; commercially manufactured bubble traps; various flasks and stoppers; tubing appropriate for peristaltic pump; and a multi-channel peristaltic pump. Results Both stains grew in batch culture (as determined by turbidity) within either high or low nutrient medium, although the growth of V. natriegens was measurably slower in low nutrient conditions (Fig. 2). During flow with initially sterile media, all media, tubing, and flow cells became contaminated. This was likely due to back contamination of the original biofilm inocula into the media input reservoirs through the tubing. Although I was unable to test the identity of the contaminants, I do not think it was outside contamination because I made certain to use standard sterile technique when setting up the flow cell system and during liquid exchanges. If so, then the contamination experienced by the flow cells is similar to cross-contamination. After 18 hours, the high nutrient medium was much more visibly turbid than the low nutrient medium, and for 5
this reason, I did not enumerate abundances by FISH for all of the high nutrient amended biofilm communities. Despite the contamination, spatially-structured biofilm communities did form after 18 hours in most of the flow cells, and cells could be enumerated reliably with DAPI and FISH (Fig. 5). No inoculum, low nutrient controls consisted of very few total cells (Fig. 3), indicating that the contamination did not result in much attachment of contaminant cells to the surfaces. V. natriegens single-species inoculations with low nutrients resulted in lower numbers of cells than the H neptunium or mixed species treatments (Fig. 3). Due to low replication, relative abundances of V. natriegens, indicated by hybridization to the γ-proteobacteria probe, and H. neptunium, indicated by hybridization to the α- proteobacteria probe did not change by nutrient treatment in the mixed species communities (Fig. 4). Despite contamination, single-species inoculated communities were dominated by the species from the original inoculation (Fig 4). Discussion and Conclusion The results do not support my initial hypotheses that V. natriegens would outcompete H. neptunium under high nutrients but not under low nutrients. Instead the results indicate that after 18 hours of biofilm growth, H. neptunium was present in all biofilm communities, and is a strong competitor with the fast-growing V. natriegens within biofilms. This may be a result of H. neptunium prosthecate morphology which may allow it to attach to surfaces more readily than V. natriegens. In the future, it would be interesting to test this hypothesis by performing a similar nutrient amendment experiment in batch or chemostats where surface attachment could be minimized. References Battin, T. J., Sloan, W. T., Kjelleberg, S., et al. 2007. Microbial landscapes: new paths to biofilm research. Nat. Rev. Microbiol. 77: 76-81. Foster, J. S. and P. E. Kolenbrander. 2004. Development of a multispecies oral bacterial community in a saliva-conditioned flow-cell. Appl. Env. Microbiol. 70: 4340-4348. 6
Havenner, J. A., McCardell, B. A., and R. M. Weiner. 1979. Development of a defined, minimal and complete media for the growth of Hyphomicrobium neptunium. Appl. Environ. Microbiol. 38: 18-23. Palmer, R. J. and D. E. Caldwell. 1995. A flowcell for the study of plaque removal and regrowth. J. Microbiol. Methods, 24: 171-182 Poindexter, J. S. 2006. Dimorphic Prosthecate Bacteria. Prokaryotes. 5: 72-90. Acknowledgements Tom Schmidt, Bill Metcaff, Microbial Diversity Class of 2008 TAs and students, the MBL, Lillie Scholarship Fund, Wheeler Family Scholarship Fund, Jennifer B. H. Martiny, Heather Reed 7
Fig. 1. Flow cell chambers and pump system design. Fig. 1a Fig. 1b 8
Fig. 2. Exponential phase growth curves for V. natriegens grown in batch culture in high nutrient medium (red) or low nutrient medium (orange). Doubling time under high nutrient conditions in 45.3 min; doubling time under low nutrients is 138.6 min. Fig. 3. Total abundance of cells on biofilms as determined by DAPI staining. 9
Fig. 4. Relative abundance of cells hybridized with alpha-proteobacteria and gammaproteobacteria FISH probes, normalized to number of cells hybridized with the Eub FISH probe. Fig 5. Epifluorescence micrographs of flow cell biofilm consisting of mixed (two-species) inoculum grown with low nutrient medium. DAPI stain (blue) or FISH (red) using Eub I-III probe; bottom image is an overlay. 10