Timothy F. Steppe

Microbial indicators of aquatic ecosystem change: current applications to eutrophication studies

Human encroachment on aquatic ecosystems is increasing at an unprecedented rate. The impacts of human pollution and habitat alteration are most evident and of greatest concern at the microbial level, where a bulk of production and nutrient cycling takes place. Aquatic ecosystems are additionally affected by natural perturbations, including droughts, storms, and floods, the frequency and extent of which may be increasing. Distinguishing and integrating the impacts of natural and human stressors is essential for understanding environmentally driven change of microbial diversity and function. Microbial bioindicators play a major role in detecting and characterizing these changes. Complementary use of analytical and molecular indicator tools shows great promise in helping us clarify the processes underlying microbial population, community, and ecosystem change in response to environmental perturbations. This is illustrated in phytoplankton (microalgal and cyanobacterial) and bacterial community changes in a range of US estuarine and coastal ecosystems experiencing increasing development in their water- and airsheds as well as climatic changes (e.g., increasing hurricane frequency). Microbial indicators can be adapted to a range of monitoring programs, including ferries, moored instrumentation, and remote sensing, in order to evaluate environmental controls on microbial community structure and function over ecosystem to global scales.

Consortial N2 fixation: a strategy for meeting nitrogen requirements of marine and terrestrial cyanobacterial mats

Abstract Four microbial mat-forming, non-axenic, strains of the non-heterocystous, filamentous, cyanobacterial genus Microcoleus were maintained in culture and examined for the ability to fix atmospheric nitrogen (N 2 ). Each was tested for nitrogenase activity using the acetylene reduction assay (ARA) and for the presence of the dinitrogenase reductase gene ( nifH ), an essential gene for N 2 fixation, using the polymerase chain reaction (PCR). The Microcoleus spp. cultures were incapable of growth without an exogenous nitrogen source and never exhibited nitrogenase activity. Attempts to amplify a 360-bp segment of the nifH gene using DNA purified from the cyanobacterial cultures did not produce any cyanobacteria-specific nifH sequences. However, several non-cyanobacterial homologous nifH sequences were obtained. Phylogenetic analysis showed these sequences to be most similar to sequences from heterotrophic bacteria isolated from a marine microbial mat in Tomales Bay (California, USA), and bulk DNA extracted from a cryptobiotic soil crust in Moab (Utah, USA). Microcoleus spp. dominated the biomass of both systems. Cyanobacteria-specific 16S rDNA sequences obtained from the cultured cyanobacterial strains demonstrate that the lack of cyanobacteria-specific nifH sequences was not due to inefficiency of extracting Microcoleus DNA. Hence, both the growth and genetic data indicate that, contrary to earlier reports, Microcoleus spp. appear incapable of fixing N 2 because they lack at least one of the requisite genes for this process. Furthermore, our study suggests epiphytic N 2 -fixing bacteria form a diazotrophic consortium with these Microcoleus spp. and are likely key sources of fixed N 2 generated within soil crusts and marine microbial mats.

Genetic variance in the composition of two functional groups (diazotrophs and cyanobacteria) from a hypersaline microbial mat

ABSTRACT Examination of variation in ecological communities can lead to an understanding of the forces that structure communities, the consequences of change at the ecosystem level, and the relevant scales involved. This study details spatial and seasonal variability in the composition of nitrogen-fixing and cyanobacterial (i.e., oxygenic photosynthetic) functional groups of a benthic, hypersaline microbial mat from Salt Pond, San Salvador Island, Bahamas. This system shows extreme annual variability in the salinity of the overlying water and the extent of water coverage. Analysis of molecular variance and FST tests of genetic differentiation of nifH and cyanobacterial 16S rRNA gene clone libraries allowed for changes at multiple taxonomic levels (i.e., above, below, and at the species level) to inform the conclusions regarding these functional groups. Composition of the nitrogen-fixing community showed significant seasonal changes related to salinity, while cyanobacterial composition showed no consistent seasonal pattern. Both functional groups exhibited significant spatial variation, changing with depth in the mat and horizontally with distance from the shoreline. The patterns of change suggest that cyanobacterial composition was more insensitive to water stress, and consequently, cyanobacteria dominated the nitrogen-fixing community during dry months but gave way to a more diverse community of diazotrophs in wet months. This seasonal pattern may allow the mat community to respond quickly to water-freshening events after prolonged dry conditions (system recovery) and maintain ecosystem function in the face of disturbance during the wet season (system resilience).

Diazotrophy in Modern Marine Bahamian Stromatolites

N2 fixation (nitrogenase activity), primary production, and diazotrophic community composition of stromatolite mats from Highborne Cay, Exuma, Bahamas, were examined over a 2-year period (1997-1998). The purpose of the study was to characterize the ecophysiology of N2 fixation in modern marine stromatolites. Microbial mats are an integral surface component of these stromatolites and are hypothesized to have a major role in stromatolite formation and growth. The stromatolite mats contained active photosynthetic and diazotrophic assemblages that exhibited temporal separation of nitrogenase activity (NA) and photosynthesis. Maximal NA was detected at night. Seasonal differences in NA and net O2 production were observed. Photosynthetic activity and the availability of reduced organic carbon appear to be the key determinants of NA. Additions of the de novo protein synthesis inhibitor chloramphenicol did not inhibit NA in March 1998, but greatly inhibited NA in August 1998. Partial sequence analysis of the nifH gene indicates that a broad diversity of diazotrophs may be responsible for NA in the stromatolites.

Hypersaline Cyanobacterial Mats as Indicators of Elevated Tropical Hurricane Activity and Associated Climate Change

Abstract The Atlantic hurricanes of 1999 caused widespread environmental damage throughout the Caribbean and US mid-Atlantic coastal regions. However, these storms also proved beneficial to certain microbial habitats; specifically, cyanobacteria-dominated mats. Modern mats represent the oldest known biological communities on earth, stromatolites. Contemporary mats are dominant biological communities in the hypersaline Bahamian lakes along the Atlantic hurricane track. We examined the impacts of varying levels of hypersalinity on 2 processes controlling mat growth, photosynthesis and nitrogen fixation, in Salt Pond, San Salvador Island, Bahamas. Hypersalinity (> 5 times sea-water salinity) proved highly inhibitory to these processes. Freshwater input from Hurricane Floyd and other large storms alleviated this salt-inhibition. A predicted 10 to 40 year increase in Atlantic hurricane activity accompanied by more frequent “freshening” events will enhance mat productivity, CO2 sequestration and nutrient cycling. Cyanobacterial mats are sensitive short- and long-term indicators of climatic and ecological changes impacting these and other waterstressed environments.

Nitrogenase activity and nifH expression in a marine intertidal microbial mat.

N2 fixation, diazotrophic community composition, and organisms actively expressing genes for N2 fixation were examined over at 3−year period (1997–1999) for intertidal microbial mats on a sand flat located in the Rachel Carson National Estuarine Research Reserve (RCNERR) (Beaufort, NC, USA). Specifically, diel variations of N2 fixation in the mats from the RCNERR were examined. Three distinct diel patterns of nitrogenase activity (NA) were observed. NA responses to short-term inhibitions of photosynthesis corresponded to one of the three patterns. High rates of NA were observed during peak O2 production periods for diel experiments during summer months. Different types of NA diel variations correspond to different stages of mat development. Chloramphenicol treatments indicated that the mechanism of protein synthesis supporting NA changed throughout the day. Analysis of mat DNA and RNA gave further evidence suggesting that in addition to cyanobacteria, other functional groups were responsible for the NA observed in the RCNERR mats. The role of microbial diversity in the N2 fixation dynamics of these mats is discussed.