Seasonal Variation in Sedimentary Microbial Community Structure as

Chapter 4

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Seasonal Variation in Sedimentary Microbial Community Structure as a Backdrop for the Detection of Anthropogenic Stress 1

2

Robert H. Findlay and Les Watting 1

Department of Microbiology, Miami University, Oxford, OH 45056 Darling Marine Center, University of Maine, Walpole, ME 04573 2

Benthic microbial communities exhibit natural seasonal variations in community structure. Analysis of microbial community structure by phospholipid fatty acid profiles and principal component analysis revealed similar seasonal patterns of change for two marine sites. One site was considered pristine while the other experienced anthropogenic organic enrichment (the waste food and feces from a salmon net-pen facility). Seasonal patterns of change were found to dominate at both sites, but the changes induced by organic enrichment could be detected after variation due to the seasonal patterns were removed. The seasonal patterns of change were best described as increased importance of microeukaryotes and aerobes (20:5ω3, 16:4ω1, 18:1ω7) during cold-water months and increased importance of bacteria and anaerobes(i16:0,a15:0,16:1ω7t)during warm-water months. Organic enrichment induced increased importance of a chemolithotrophic community within the sediments that is best characterized by the microbial assemblage found in Beggiatoa-type mats. These results once again demonstrate the utility of the phospholipid fatty acid profiles for the quantitative description of microbial community structure and suggest that care must be taken to include seasonal factors into experimental designs attempting to determine the effects of anthropogenic stress. Introduction All natural biological systems are characterized by two features; they are spatially heterogeneous, and they are dynamic in that community structure changes with time. Hence, the greatest challenge to determining ecosystem health by investigating changes in populations is to be able to discriminate between changes induced by natural versus anthropogenic causes. In marine and estuarine environments the most common pollutants are organic. Duursma and Marchand (7) list sewage, pulp mill effluent, detergents, © 1997 American Chemical Society

Eganhouse; Molecular Markers in Environmental Geochemistry ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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MOLECULAR MARKERS IN ENVIRONMENTAL GEOCHEMISTRY

agriculture runoff, hydrocarbons, and xenobiotics as common marine and estuarine organic pollutants. All, with exception of xenobiotics, cause, to a lesser or greater extent, a similar pattern of change in marine and estuarine benthic communities. Most commonly utilized for monitoring purposes is the pattern of change observed in the macrobenthic community (2). Changes in the benthic microbial community, however, can also serve effectively as an indicator of organic enrichment in estuarine environments and allow for the early detection of possible ecosystem change due to anthropogenic stress. The microbial component of the benthos offers many advantages over using patterns of change in the macrobenthic community for monitoring purposes. These are: 1. As living members of the sediment community they must adapt to environmental stress or perish. 2. Their response to sediment contamination facilitates the spatial definition of impacts. 3. Benthic microorganisms are effective indicators of impacts at higher levels of organization because of their central role in overall ecosystem structure and function. 4. Not only do bacteria have the potential to mediate transfer of toxic substances to higher trophic levels, they can transform toxic substances sometimes increasing their toxicity or their mobility within the food web. 5. Benthic microorganisms mediate nutrient recycling from the sediments into the water column. 6. The biomass of benthic microorganisms is generally controlled by sediment grain size and organic content and, as such, is sensitive to organic enrichment. 7. They are numerous, allowing small sample sizes and frequent replication. 8. They have short generation times, thus, offering the opportunity for rapid response. Coupled with the fact that bacteria and bacterial products comprise between 2-10% of sedimentary organic matter, the above points form a strong rationale for the need to understand bacterial biomass and community structure in environmental geochemical studies. Most of the bacteria in sediments are heterotrophic and, therefore, deperident on the decomposition of organic matter (5). Catabolic processes in sediments generally proceed following a definite succession of oxidizing agents. The reason why this succession occurs is generally explained in terms of the metabolic free energy yield of the reactions (4). It is assumed that the greater the theoretical energy yield of a bacterial reaction, the greater the probability that the reaction will predominate over other competing reactions. Hence, diagenetic reactions within sediments can be modeled in terms of the availability of organic carbon and terminal electron acceptors. The prevalence of oxygen and sulfate in marine waters leads to their importance as electron acceptors within sediment systems, although nitrate, iron, manganese and carbon can also play a role. This model of early diagenesis is most often observed in the zonation of prevalent metabolic types within a sediment (Figure 1). Microbial degradation will proceed aerobically as long as molecular oxygen is available. If the supply of oxygen exceeds the demand generated by the availability of carbon, the sediments remain aerobic. When demand exceeds availability, oxygen is depleted and anaerobic processes will predominate, including the reduction of sulfate to hydrogen sulfide. The hydrogen sulfide produced can then, in turn, serve as an energy source for chemoautotrophic bacteria (e.g. Beggiatoa-type organisms) that reside at the sediment horizon where molecular oxygen and hydrogen sulfide coexist. If the amount of metabolizable organic matter is extreme, most of the available O2 is consumed as an oxidant for sulfides and not



FINDLAY & WATLING

Seasonal Variation in Microbial Communities

LOW CARBON INPUT (0.1 kg C/m2/y)

120 cm I

HIGH CARBON INPUT (1.8 kgC/m2/y)

i.

Figure 1. Diagrammatic representation of the distribution of functional groups of microorganisms within sediments and its relationship to organic matter input. (Drawn, in part, using data published in ref. 28-29).



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for organic carbon (5). Under these conditions visible Beggiatoa mats form at the sediment-water interface. If sufficient organic material is present, sulfate will also be depleted in the sediments and diagenesis will proceed via production of biogenic methane. Findlay et al. (6) have demonstrated that organic enrichment does indeed induce such changes in community structure and Findlay and Watling (in press, Mar.Ecol.Prog. Ser.) have shown that these changes are not only dependent on benthic carbon flux but also dependent on delivery of O2 to the benthos. Combined, these two studies demonstrate that changes caused by variation in the balance of electron acceptors and metabolizable organic matter induce characteristic changes in microbial community structure detectable using phospholipid fatty acids (PLFA) profiles. What remains is to separate changes in microbial community structure induced by anthropogenic stresses from those that occur due to natural processes. It is simplistic to assume that carbon content and terminal electron acceptor availability are the only factors to affect microbial community structure. Factors likely to influence the structure of microbial communities in sediments are: 1) season (Findlay and Watling submitted, Micro.Ecol.),2) sediment grain size and shape (7-5), 3) organic carbon content, 4) fluid flux over the bed (Findlay and Watling in press, Mar. Ecol. Prog. Ser.), 5) disturbance or disruption of the sediments (P-72) and 6) animal-microbe interactions (13-14). In this paper, we compare and contrast the patterns of change found in microbial community structure for two shallow-water sites from coastal Maine. Both sites were sampled approximately monthly during 1991, and microbial community structure was determined by phospholipid fatty acid profiles (75). The sites differ in that one is considered pristine (Damariscotta River estuary) while the other is impacted by organic waste (Toothacher Cove). Data from both sites were generated independently and were used to examine two separate research problems. The Toothacher Cove site was sampled to determine the effects of increased organic flux to the benthos generated by salmon aquaculture on the benthos. These results have been previously published (6). The Damariscotta River site was used to explore the natural seasonal variation in sedimentary microbial community structure and the manuscript relating these changes is currently in review (Findlay and Watling submitted, Micro. Ecol.). Because both sites were sampled approximately monthly for a year and microbial community structure was determined by the same method (PLFA and principal component analysis), a comparison of the findings of these two independent studies affords a unique opportunity to evaluate the relative strength of two important structuring processes; seasonality and organic enrichment. This paper evaluates interactions between seasonal changes and organic enrichment with the goal of separating those changes in microbial community structure induced by anthropogenic stresses from those that occur due to natural processes. Materials and Methods. Experimental Design. Permanent benthic study sites were established within two Maine coastal embayments. These study sites differed in that one embayment was pristine and the other anthropogenically impacted. Sampling stations were marked



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FINDLAY & WATLING

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with moored buoys. Three replicate benthic sediment samples were collected monthly (or as closely as possible given the rigors of diving in coastal waters) by SCUBA-assisted divers using push cores from each sampling station and subsamples of approximately 1.1 cc were removed using 5 cc plastic syringes with the canula end removed. This effectively sampled the 0-1 cm sediment horizon with minimal disturbance to the sediment. Benthic microbial community structure was determined by phospholipid fatty acid (PLFA) analysis (75). Patterns of seasonal variation in benthic microbial community structure were determined using Principal Components Analysis (PCA). Study Sites. The first study site was located approximately 100 m north of the Darling Marine Center dock in the Damariscotta River estuary system and is approximately 7 river miles from the mouth of the estuary. A complete description of this site and the observed seasonal changes in microbial community structure can be found in Findlay and Watling (submitted, Microb.Ecol.).This site is in approximately 10 m of water (Mean Low Water). Currents range from 0-20 cm s' and flow parallel to the shore (Findlay and Watling submitted, Micro. Ecol.). The bottom consists of a fine muddy sand. Water temperatures annually range from 0 to 20 °C, and salinities typically range from 25 to 33 o / (16). Total fecal coliform counts at mid channel are typically less than 2 Colony Forming Units/100 ml. This is a pristine site with no evidence of anthropogenic stress. A single sampling station established at this site was sampled monthly from April 1991 - June 1992. The second site was a working salmon aquaculture farm in Toothacher Cove, Swans Island, Maine. Swans Island is approximately 15 miles offshore, and the cove is exposed to ocean waves to the south-southwest. Water depth at this site is 15.7 m at mean low water, annual temperatures ranged from -1.2 to 15.5 °C, the directions of prevailing currents were west-southwest and east-northeast and ranged from 0 to 10 cm s" at 1 m above the sediment surface (6). Sediments consisted of a muddy sand over glacial till. The study was conducted between February 1991 and June 1992 with the majority of sampling effort (monthly samplings ~ again as closely as possible given the challenges of working with small boats and diving fifteen miles offshore in cold-temperate waters) concentrated during the summer and fall of 1991. We intensively studied sediments beneath and adjacent to a single salmon net-pen and sediments located approximately 100 m to the north-northwest of the pen (located perpendicular to the predominant flow to minimize the chance of a pen influence). A total of six sampling stations were established at this site; three ambient stations 100 m from the net-pen and spaced 10 m apart and three pen stations placed 1, 10 and 20 m from the edge of the net-pen. The rationale for this placement was that the ambient stations would be unimpacted by the aquaculture activity while the pen stations would experience a gradient of increasing benthic carbon flux. During thistimethe pen contained approximately 8,000 Atlantic salmon which were hand-fed approximately 0.012 kg semi-moist feed kg'* fish biomass daily (2 daily feedings). During the approximately 150 days that sediment traps were successfully deployed during the summer of 1991 ambient sediments received ca. 250 g C m" (including sediment resuspension), sediments below the net-pen received 500-750 g C nr and sediment 10 m downcurrent of the net-pen 1

00

1

2

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received ca. 325 g C rrr (6). Detailed descriptions of this site and the nature and extent of the benthic impact (from geochemical to epibenthic) associated with the increased carbon flux can be found in Findlay et al. (6). Determination of Microbial Community Structure. Microbial community structure was determined using PLFA analysis following the protocols of Findlay (75). Briefly, total lipids were extracted using a modified Bligh and Dyer extraction (77), and phospholipid fatty acids were purified using silicic acid column chromatography. Fatty acid methyl esters were formed by transmethylation (0.2 M methanolic KOH) and purified by Cis reverse-phase column chromatography. Fatty acid methyl esters were identified and quantified by gas chromatography. Individual fatty acids were expressed as weight percent, that is, (grams individual fatty acid x grams total fatty acids' ) x 100. A total of 42 individual PLFAs were identified and quantified. Fatty acids are named using the following convention — chain length,:, number of double bonds, co, the position offirstdouble bond from the omega or aliphatic end of the molecule. For example the fatty acid, 16:10)7, is 16 carbons long, has one double bond, and it occurs at the seventh carbon from the omega end of the molecule. All bonds are assumed to be of the cis configuration unless noted by a t. Terminal branching patterns are designated by i (ios) and a (anteiso)\ internal branching patterns are designated by the number of the carbon (with respect to the acyl end) at which the branch occurs and the abbreviation Me. 1

Statistical Analysis. Patterns of seasonal variation in microbial community structure were determined by PCA using Systat (Systat Inc., Chicago). For the Damariscotta site 46 samples were analyzed, and for the Toothacher Cove site 107 samples were analyzed resulting in two matrices (45 x 42 and 107 x 42) from which the major components of variation were determined. Data were transformed [ln(x=l)] prior to statistical analysis. PLFA patterns were interpreted using the algorithms outlined in Findlay (75). Functional group designations for the PLFAs and the citations supporting the designations are given in Table I. Safety. Several safety issues were addressed during the course of this work. They were SCUBA-assisted diving, the use of hydrogen as a carrier and flame gas during gas chromatography and use of organic solvents. Each of these carries a risk that is minimized by proper training and adherence to established safety procedures. Results and Discussion Seasonal Variation at a Pristine Site. The pattern of seasonal variation at the Damariscotta River site as determined by PLFA profiles and PCA was one of the predictable changes in PLFA profiles that correlated with months of the year (Figure 2). Samples taken from April and May had the most negative component scores for principal component 1 while samples from August, September and November (no October samples were collected) had the highest positive component scores for principal component 1. In general, sediments from cold-water months had negative component scores and sedimentsfromwarm-water months had



i

1

Table I. Phospholipid fatty acids that defined major components of variation at the Damarisocota River and Toothacher Cove study sites jFptfyacid Functional group assignment % 1. 14:0 Gram-positive and some Gram-negative anaerobic bacteria (18) RP 2. /15:0 Gram-positive and some Gram-negative anaerobic bacteria (18) 3. al5:0 Gram-positive and some Gram-negative anaerobic bacteria (18) 5 4. 15:0 Gram-positive and some Gram-negative anaerobic bacteria (18) 5. 16:4©1 Microeukaryotes (18) 6. 16:3 Microeukaryotes (18); Diatoms (19) i I. 16:1 ©7/ Gram-negative bacteria experiencing stress (20) 8. 16: l©13t Eukaryotic phototrophs (18); Diatoms (19) 9 ./16:0 Gram-positive and some Gram-negative anaerobic bacteria (18) 10. 10Mel6:0 Sulfate-reducing bacteria and other anaerobes (18); Desulfobacter (19) s II. 17: lco6 Aerobic bacteria and eukaryotes (18); Beggiatoa-type mat if 17: lco6,22:6(03,22:5oo3,22:1 col 1 are present (6). sr 12. al7:0 Sulfate-reducing bacteria and other anaerobes (18) =; 13. 18:2o6 Aerobic bacteria and eukaryotes (18); Beggiatoa-type mat if 17:1©6,22:6©3, 22:5o>3,22: 1©1 1 are present (6); Fungi (19) 14. 18:10)7 Aerobic bacteria and eukaryotes (18) 15. 20:5ofl3 Microeukaryotes (18); Diatoms or higher plants (19) s 16. 20:4co6 Heterotrophic microeukaryotes (18); Protozoa (19) s 17. 22:6a)3 Microeukaryotes (18); Beggiatoa-type mat if 17:10)6, 22:6©3, 22:5G)3, 22:1©11 are present (6) 18. 22:5a)3 Nficroeukaryotes (75); ^eggia/oa-type mat if 17:1©6,22:6©3,22:5©3,22:1©11 are present (6) 19. 22:10)11 Beggiatoa-type mat if 17:1©6,22:6©3,22:5©3,22:1©11 are present (6); Francisella tularensis (19)


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I 1

B B B M

L

0

_ c

C

c

J

(P

L

1

1

J

J

D

-

H

(Jf

C

G

W C

K

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D

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E

-1

F

F B

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-2

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B -3

-

B -4

i

i -

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20:5w3 ^ltf* 22:6w3 16:lwl3t

-

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0

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Seasonal variation

3

116:0 il5:0 10Mel6:0 al5:0 16:lw7t

Figure 2. Plot of factor scores resulting from principal component analysis of weight percent PLFA data from Damariscotta River estuary sediments. Component 1 was designated seasonal variability. Scores are plotted by sample date (April 9, 1991 through June 15, 1992). Symbols are: B = April, C = May, D = June, E = July, F = August, G = September, H = November, J = December, K = January, L = February, M = March. Note that there were no samples taken during October. (Adapted from ref. Findlay and Watling submitted, Microb. Ecol.)



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FINDLAY & WATLING


positive component scores for principal component 1. Samples from periods of temperature transition (December and June) had component scores that bracketed zero. Four functional groups of microorganisms were represented by 10 fatty acids carrying the highest eigenvalues for principle component 1. These were: 1) prototrophic microeukaryotes [20:5co3, 16:4col, 16:3, 22:6co3 and 16:lo)13f], 2) Gram-positive bacteria and some Gram-negative anaerobic bacteria [/16:0, /15:0 and al5:0], 3) sulfate-reducing and other anaerobic bacteria [10mel6:0] and 4) aerobic Gram-negative bacteria exposed to environmental stress [16:lco7f] (known stressor include reduced O2 tension, starvation and exposure to phenol (18,20)). The annual variation in microbial community structure at this site appeared to be driven, at least in part, by changes in biomass of phototrophic eukaryotes (Findlay and Watling submitted, Micro. Ecol.). Phototrophic eukaryotic biomass varied 6- to 11-fold while total biomass and biomass of the two functional groups of anaerobic bacteria varied 2- to 3-fold. Periods of peak total biomass and phototrophic eukaryotic biomass occurred during transition or cold-water months. During these periods, phototrophic eukaryotes accounted for 70% to 85% of total microbial biomass expressed as carbon (total biomass determined as total PLFA; PLFA concentration converted to carbon using the algorithms given in Findlay and Dobbs (18)). During warm-water months phototrophic eukaryotic biomass decreased and accounted for 35% to 55% of total microbial biomass. The biomass of anaerobic bacteria also increased during periods of highest microbial biomass but was relatively constant during other months. As total biomass was lowest during the warm-water months, the contribution of anaerobic bacteria to the community increased during these periods. The increase in the fatty acid 16: lu)7f indicated that aerobic bacteria were experiencing some form of environmental stress ~ in this case it was most likely decreased O2 concentrations in the sediments (Findlay and Watling in press, Mar. Ecol. Prog. Ser.). These results indicated that the observed pattern in microbial community structure was related to both the balance between eukaryotic and prokaryotic organisms and the balance between aerobic and anaerobic microorganisms within the sediments. As total microbial biomass and the abundance of eukaryotic phototrophs were strongly linked, the changes in the abundance of the latter strongly influenced total sedimentary microbial biomass in these sediments. The relatively small range in abundance of the marker fatty acids for the two anaerobic functional groups of bacteria indicated that their biomass was relatively constant compared to that of phototrophic microeukaryotes and that their increased importance within the sedimentary microbial community during the warmwater months was in part due to the decrease in eukaryotic biomass. Again, the pattern exhibited strong seasonal trends with maximum contributions to microbial biomass by eukaryotes and aerobes occurring during cold-water months and maximum contributions by bacteria and anaerobes occurring during warm-water months. Seasonal Variation at an Organically Enriched Site. The pattern of seasonal variation at the Toothacher Cove site as determined by PLFA profiles and PCA was a predictable change in PLFA profiles that correlated with months of the year (Figure 3). Samples taken from April and May had the most negative component


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al5:0 20:4w6 10Mel6:0 16:lw7 al7:0. 2

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Seasonal Variation in Sedimentary Microbial Community Structure as

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