Populations of Methanogenic Bacteria in a Georgia Salt Marsh

Methanogens represented about 0.5% of the total bacteria in sediments from a Georgia salt marsh in which Spartina alterniflora is the predominant vegetation. The population of methanogens was composed of at least two groups of nearly equal size. One group was represented by cocci which were able to utilize trimethylamine and were unable to use H₂ or acetate. The second group was composed of two subgroups which were able to utilize H₂ but were unable to use trimethylamine or acetate. The more common subgroup included rod- or plate-shaped methanogens which could utilize isopropanol in addition to H₂ and formate. The second subgroup included Methanococcus maripaludis, which utilized only H₂ and formate. Other groups of methanogens were also present, including Methanosarcina sp. which utilized acetate, H₂, and methylamines. In addition to the overall variability in the types of methanogens, the numbers of methanogens in sediments also exhibited significant spatial variability both within and between tall- and short-Spartina zones.     

Quantification of Ammonia-Oxidizing Bacteria and Factors Controlling Nitrification in Salt Marsh Sediments

To elucidate the geomicrobiological factors controlling nitrification in salt marsh sediments, a comprehensive approach involving sediment geochemistry, process rate measurements, and quantification of the genetic potential for nitrification was applied to three contrasting salt marsh habitats: areas colonized by the tall (TS) or short (SS) form of Spartina alterniflora and unvegetated creek banks (CBs). Nitrification and denitrification potential rates were strongly correlated with one another and with macrofaunal burrow abundance, indicating that coupled nitrification-denitrification was enhanced by macrofaunal burrowing activity. Ammonia monooxygenase (amoA) gene copy numbers were used to estimate the ammonia-oxidizing bacterial population size (5.6 × 10⁴ to 1.3 × 10⁶ g of wet sediment⁻¹), which correlated with nitrification potentials and was 1 order of magnitude higher for TS and CB than for SS. TS and CB sediments also had higher Fe(III) content, higher Fe(III)-to-total reduced sulfur ratios, higher Fe(III) reduction rates, and lower dissolved sulfides than SS sediments. Iron(III) content and reduction rates were positively correlated with nitrification and denitrification potential and amoA gene copy number. Laboratory slurry incubations supported field data, confirming that increased amounts of Fe(III) relieved sulfide inhibition of nitrification. We propose that macrofaunal burrowing and high concentrations of Fe(III) stimulate nitrifying bacterial populations, and thus may increase nitrogen removal through coupled nitrification-denitrification in salt marsh sediments.     

Populations of Anaerobic Phototrophic Bacteria in a Spartina alterniflora Salt Marsh

Habitat-simulating media were used with the Hungate anaerobic roll tube technique to enumerate culturable anaerobic photosynthetic bacteria in sediment, tidal waters, and Spartina alterniflora plant samples collected from the salt marsh at Sapelo Island, Ga. No phototrophs were detected in samples of creekside (low marsh) sediment or in tidal waters in creekside regions. In the high marsh region, 90% of anaerobic phototrophic bacteria occurred in the top 5 mm of sediment and none were detected below 6 mm. There was a seasonal variation, with maximal populations occurring in summer and fall (mean, 4.4 × 10⁵ phototrophs g of dry sediment⁻¹) and minimal numbers occurring in winter (mean, 3.9 × 10³ phototrophs g of dry sediment⁻¹). During winter and late spring, phototrophs had a patchy distribution over the high marsh sediment surface. In contrast, during late summer they had a random uniform distribution. Tidal water collected over high marsh sediment contained an average of 8.7 × 10² phototrophs ml⁻¹, with no significant seasonal variation. Anaerobic phototrophic bacteria were also cultured from the lower stem tissue of S. alterniflora growing in both the high (4.3 × 10⁴ phototrophs g of dry tissue⁻¹) and creekside (4.9 × 10⁴ phototrophs g of dry tissue⁻¹) marsh regions. Chromatium buderi, Chromatium vinosum, Thiospirillum sanguineum, Rhodospirillum molischianum, and Chlorobium phaeobacteroides were the predominant anaerobic phototrophic species cultured from high marsh sediment. The two Chromatium species were dominant.     

Tracer Analysis of Methanogenesis in Salt Marsh Soils

Differences in paths of carbon flow have been found in soils of the tall (TS) and short (SS) Spartina alterniflora marshes of Sapelo Island, Ga. Gaseous end products of [U-¹⁴C]glucose metabolism were ¹⁴CO₂ and ¹⁴CH₄ in the SS region and primarily ¹⁴CO₂ in the TS region. Sulfate concentration did not demonstrably affect glucose catabolism or the distribution of end products in either zone. [U-¹⁴C]acetate was converted to ¹⁴CO₂ and ¹⁴CH₄ in the SS soils and almost exclusively to ¹⁴CO₂ in the TS soils. Sulfate concentration did not affect acetate metabolism in the SS soils; however, a noticeable effect of sulfate dilution was seen in TS soils. Sulfate dilution in TS samples resulted in increased methane formation. Total glucose and acetate metabolism were similar in TS and SS soils despite differences in end products. A microbial community characterized by fermentative/sulfate-reducing processes has developed in TS soils as opposed to the fermentative/methanogenic/sulfate-reducing community found in SS soils.     

Methanogenesis and Sulfate Reduction: Competitive and Noncompetitive Substrates in Estuarine Sediments

Sulfate ions did not inhibit methanogenesis in estuarine sediments supplemented with methanol, trimethylamine, or methionine. However, sulfate greatly retarded methanogenesis when hydrogen or acetate was the substrate. Sulfate reduction was stimulated by acetate, hydrogen, and acetate plus hydrogen, but not by methanol or trimethylamine. These results indicate that sulfate-reducing bacteria will outcompete methanogens for hydrogen, acetate, or both, but will not compete with methanogens for compounds like methanol, trimethylamine, or methionine, thereby allowing methanogenesis and sulfate reduction to operate simultaneously within anoxic, sulfate-containing sediments.     

End Products of Anaerobic Chitin Degradation by Salt Marsh Bacteria as Substrates for Dissimilatory Sulfate Reduction and Methanogenesis

The anaerobic pathway of chitin decomposition by chitinoclastic bacteria was examined with an emphasis on end product coupling to other salt marsh bacteria. Actively growing chitinoclastic bacterial isolates produced primarily acetate, H₂, and CO₂ in broth culture. No sulfate-reducing or methanogenic isolates grew on chitin as sole carbon source or produced any measurable degradation products. Mixed cultures of chitin degraders with sulfate reducers resulted in positive sulfide production. Mixed cultures of chitin-degrading isolates with methanogens resulted in the production of CH₄ with reductions in headspace CO₂ and H₂. The combination of all three metabolic types resulted in the simultaneous production of methane and sulfide, with more methane being produced in mixed cultures containing CO₂-reducing methanogens and acetoclastic sulfate reducers because of less interspecific H₂ competition.     

Effects of Metals on Methanogenesis, Sulfate Reduction, Carbon Dioxide Evolution, and Microbial Biomass in Anoxic Salt Marsh Sediments

The effects of several metals on microbial methane, carbon dioxide, and sulfide production and microbial ATP were examined in sediments from Spartina alterniflora communities. Anaerobically homogenized sediments were amended with 1,000 ppm (ratio of weight of metal to dry weight of sediment) of various metals. Time courses in controls were similar for CH₄, H₂S, and CO₂, with short initial lags (0 to 4 h) followed by periods of constant gas production (1 to 2 days) and declining rates thereafter. Comparisons were made between control and experimental assays with respect to initial rates of production (after lag) and overall production. Methane evolution was inhibited both initially and overall by CH₃HgCl, HgS, and NaAsO₂. A period of initial inhibition was followed by a period of overall stimulation with Hg, Pb, Ni, Cd, and Cu, all as chlorides, and with ZnSO₄, K₂CrO₄, and K₂Cr₂O₇. Production of CO₂ was generally less affected by the addition of metals. Inhibition was noted with NaAsO₂, CH₃HgCl, and Na₂MoO₄. Minor stimulation of CO₂ production occurred over the long term with chlorides of Hg, Pb, and Fe. Sulfate reduction was inhibited in the short term by all metals tested and over the long term by all but FeCl₂ and NiCl₂. Microbial biomass was decreased by FeCl₂, K₂Cr₂O₇, ZnSO₄, CdCl₂, and CuCl₂ but remained generally unaffected by PbCl₂, HgCl₂, and NiCl₂. Although the majority of metals produced an immediate inhibition of methanogenesis, for several metals this was only a transient phenomenon followed by an overall stimulation. The initial suppression of methanogenesis may be relieved by precipitation, complexation, or transformation of the metal (possibly by methylation), with the subsequent stimulation resulting from a sustained inhibition of competing organisms (e.g., sulfate-reducing bacteria). For several environmentally significant metals, severe metal pollution may substantially alter the flow of carbon in sediments.     

Microbiological and Physical Properties of Salt Marsh and Microecosystem Sediments

Sediments from natural marsh and four microecosystem (MES) tanks were compared with reference to ATP, carbon content, and physical properties. Mean values for bacterial numbers and ATP were about twice as high in the MES, carbon content was slightly higher in the artificial systems, and the sediment of the MES was enriched for larger grain sizes and depleted of silt and clay. In the natural marsh sediment, ATP was strongly related (Pearson's correlation coefficient) to granulometric (or physical) factors, whereas ATP was related to biological factors in the MES. Approximately 61% of the variation in ATP concentrations in the marsh sediments could be accounted for by the parameters measured. Only half that amount could be explained on the basis of those same variables in the MES. Furthermore, one of the four tanks was significantly different from the other three in terms of ATP concentration, and an anomaly in respect to silt-clay content was demonstrated in another. Careful control and some type of equilibrating procedure will be necessary before the MES can be employed as replicate units.     

Detection and Enumeration of Sulphate-Reducing Bacteria in Estuarine Sediments by Competitive PCR

The distribution of sulphate-reducing bacteria (SRB) in the sediments of the Colne River estuary, Essex, UK covering different saline concentrations of sediment porewater was investigated by the use of quantitative competitive PCR. Here, we show that a new PCR primer set and a new quantitative method using PCR are useful tools for the detection and the enumeration of SRB in natural environments. A PCR primer set selective for the dissimilatory sulphite reductase gene (dsr) of SRB was designed. PCR amplification using the single set of dsr-specific primers resulted in PCR products of the expected size from all 27 SRB strains tested, including Gram-negative and positive species. Sixty clones derived from sediment DNA using the primers were sequenced and all were closely related with the predicted dsr of SRB. These results indicate that PCR using the newly designed primer set are useful for the selective detection of SRB from a natural sample. This primer set was used to estimate cell numbers by dsr selective competitive PCR using a competitor, which was about 20% shorter than the targeted region of dsr. This procedure was applied to sediment samples from the River Colne estuary, Essex, UK together with simultaneous measurement of in situ rates of sulphate reduction. High densities of SRB ranging from 0.2 ? 5.7 × 10⁸ cells ml^{? 1} wet sediment were estimated by the competitive PCR assuming that all SRB have a single copy of dsr. Using these estimates cell specific sulphate reduction rates of 10^{? 17} to 10^{? 15} mol of SO₄ ^{2 ?} cell^{? 1} day^{? 1} were calculated, which is within the range of, or lower than, those previously reported for pure cultures of SRB. Our results show that the newly developed competitive PCR technique targeted to dsr is a powerful tool for rapid and reproducible estimation of SRB numbers in situ and is superior to the use of culture-dependent techniques.     

Microbial Community Composition and Denitrifying Enzyme Activities in Salt Marsh Sediments

Denitrifying microbial communities and denitrification in salt marsh sediments may be affected by many factors, including environmental conditions, nutrient availability, and levels of pollutants. The objective of this study was to examine how microbial community composition and denitrification enzyme activities (DEA) at a California salt marsh with high nutrient loading vary with such factors. Sediments were sampled from three elevations, each with different inundation and vegetation patterns, across 12 stations representing various salinity and nutrient conditions. Analyses included determination of cell abundance, total and denitrifier community compositions (by terminal restriction fragment length polymorphism), DEA, nutrients, and eluted metals. Total bacterial (16S rRNA) and denitrifier (nirS) community compositions and DEA were analyzed for their relationships to environmental variables and metal concentrations via multivariate direct gradient and regression analyses, respectively. Community composition and DEA were highly variable within the dynamic salt marsh system, but each was strongly affected by elevation (i.e., degree of inundation) and carbon content as well as by selected metals. Carbon content was highly related to elevation, and the relationships between DEA and carbon content were found to be elevation specific when evaluated across the entire marsh. There were also lateral gradients in the marsh, as evidenced by an even stronger association between community composition and elevation for a marsh subsystem. Lastly, though correlated with similar environmental factors and selected metals, denitrifier community composition and function appeared uncoupled in the marsh.     

Naked Amoebas and Bacteria in an Oil-Impacted Salt Marsh Community

Populations of soil amoebas were monitored in two salt marshes in Staten Island, NY for 2 years. One site, Gulfport Reach on the Arthur Kill, has been highly impacted by numerous oil spills. In particular, in 1990 a massive no. 2 fuel oil spill from a ruptured pipe flooded the area; its sediments had total petroleum hydrocarbon (TPH) concentrations in the range 800-46,000 ppm. A reference site 11 km away, on the Atlantic coast, had low TPH levels. Amoeba population densities were in general higher in the impacted sediments. In laboratory microcosm experiments, sediment samples from unimpacted sites were treated with added fresh (unweathered) hydrocarbons (no. 2 fuel oil) and cultured; these also yielded higher amoeba numbers than untreated controls. Four distinct amoeba morphotypes were monitored. Changes in population levels of total amoebas were correlated in the two sites, particularly for morphotype 2 (r = 0.83). The ratios of total amoebas to total bacterial numbers were also correlated (r = 0.85) between the sites. This suggests the amoebas may function as generalists, and that their trophic relation to bacterial prey is not much affected by the presence of petroleum hydrocarbons, but rather may reflect regional parameters such as ambient temperature or other physical factors.     

Effect of Salinity on Mercury-Methylating Activity of Sulfate-Reducing Bacteria in Estuarine Sediments

The biomethylation of mercury was measured in anoxic estuarine sediments that ranged in salinity from 0.03 to 2.4% with or without added molybdate, an inhibitor of sulfate reducers. Mercury methylation was inhibited by molybdate by more than 95%, regardless of sediment salinity. In the absence of inhibitor, high-salinity sediments methylated mercury at only 40% of the level observed in low-salinity sediments. In response to molybdate inhibition of sulfate reducers, methanogenesis increased up to 258% in high-salinity sediments but only up to 25% in low-salinity sediments. In contrast to an earlier low-salinity isolate, a Desulfovibrio desulfuricans strain from high-salinity sediment required 0.5 M sodium for optimal growth and mercury methylation activity. The formation of negatively charged mercuric chloride complexes at high salinity did not noticeably interfere with the methylation process. Results of these studies demonstrate that sulfate reducers are responsible for mercury methylation in anoxic estuarine sediments, regardless of the prevailing salinity.     

Microbial Aldicarb Transformation in Aquifer, Lake, and Salt Marsh Sediments

The microbial transformation of [N-methyl-(sup14)C]aldicarb, a carbamate pesticide, occurred in aquifer, lake, and salt marsh sediments. Microbial degradation of aldicarb took place within 21 days in aquifer sediments from sites previously exposed to aldicarb (Jamesport, Long Island, N.Y.) but did not occur in sediments which were not previously exposed (Connetquot State Park, Long Island, N.Y.). At the Jamesport sites, higher aldicarb transformation rates occurred in deep, anoxic sediments than in shallow, oxic sediments. There was a significant negative relationship (P < 0.05) between transformation rates and ambient dissolved O(inf2) levels. Aldicarb hydrolysis rates in Jamesport sediments were 10- to 1,000-fold lower than rates previously reported for soils. In addition, aldicarb degradation rates were not significantly correlated with measurements of bacterial activity and density previously determined in the same sediments. Substantially higher aldicarb degradation rates were found in anoxic lake and salt marsh than in aquifer sediments. Furthermore, we investigated the anaerobic microbial processes involved in aldicarb transformation by adding organic substrates (acetate, glucose), an alternative electron acceptor (nitrate), and microbial inhibitors (molybdate, 2-bromoethanesulfonic acid) to anoxic aquifer, lake, and salt marsh sediments. The results suggest that a methanogenic consortium was important in aldicarb transformation or in the use of aldicarb-derived products such as methylamine. In addition, microbial aldicarb transformation proceeded via different pathways under oxic and anoxic conditions. In the presence of O(inf2), aldicarb transformation was mainly via an oxidation pathway, while in the absence of O(inf2), degradation took place through a hydrolytic pathway (including the formation of methylamine precursors). Under anoxic conditions, therefore, aldicarb can be transformed by microbial consortia to yield products which can be of direct benefit to natural populations of methanogens present in sediments.     

Distribution and Diversity of Archaeal and Bacterial Ammonia Oxidizers in Salt Marsh Sediments

Diversity and abundance of ammonia-oxidizing Betaproteobacteria (β-AOB) and archaea (AOA) were investigated in a New England salt marsh at sites dominated by short or tall Spartina alterniflora (SAS and SAT sites, respectively) or Spartina patens (SP site). AOA amoA gene richness was higher than β-AOB amoA richness at SAT and SP, but AOA and β-AOB richness were similar at SAS. β-AOB amoA clone libraries were composed exclusively of Nitrosospira-like amoA genes. AOA amoA genes at SAT and SP were equally distributed between the water column/sediment and soil/sediment clades, while AOA amoA sequences at SAS were primarily affiliated with the water column/sediment clade. At all three site types, AOA were always more abundant than β-AOB based on quantitative PCR of amoA genes. At some sites, we detected 10⁹ AOA amoA gene copies g of sediment⁻¹. Ratios of AOA to β-AOB varied over 2 orders of magnitude among sites and sampling dates. Nevertheless, abundances of AOA and β-AOB amoA genes were highly correlated. Abundance of 16S rRNA genes affiliated with Nitrosopumilus maritimus, Crenarchaeota group I.1b, and pSL12 were positively correlated with AOA amoA abundance, but ratios of amoA to 16S rRNA genes varied among sites. We also observed a significant effect of pH on AOA abundance and a significant salinity effect on both AOA and β-ΑΟΒ abundance. Our results expand the distribution of AOA to salt marshes, and the high numbers of AOA at some sites suggest that salt marsh sediments serve as an important habitat for AOA.Nitrification, the sequential oxidation of ammonia to nitrite and nitrate, is a critical step in the nitrogen cycle and is mediated by a suite of phylogenetically and physiologically distinct microorganisms. The recent discovery of ammonia oxidation among Archaea (17, 38) has led to a dramatic shift in the current model of nitrification and to new questions of niche differentiation between putative ammonia-oxidizing Archaea (AOA) and the more-well-studied ammonia-oxidizing Betaproteobacteria (β-AOB). Based on surveys of 16S rRNA genes and archaeal amoA genes, it is evident that AOA occupy a wide range of niches (10), suggesting a physiologically diverse group of Archaea. Additionally, in studies where AOA and β-AOB were both targeted, AOA were typically more abundant than their bacterial counterparts (19, 21, 42). However, there are reports of β-AOB outnumbering AOA in estuarine systems (6, 33), suggesting a possible shift in competitive dominance under certain conditions.Patterns of β-AOB diversity in estuaries have been well characterized and appear to be regulated by similar mechanisms within geographically disparate systems (4, 11, 32). However, AOA distribution and their role in nitrification relative to β-AOB remain to be determined. A few studies have begun to address this question in different estuaries, but no unifying patterns or mechanisms have emerged. Although β-AOB have been well studied along estuarine salinity gradients (1, 3, 4, 7, 11, 13, 22, 33, 39) and recent studies have begun to address AOA in estuaries (1, 6, 22, 32, 33), few have investigated β-AOB in salt marshes (9), and none has included AOA.In this study, we investigated the distribution and abundance of AOA and β-AOB based on the distribution and abundance of amoA genes in salt marsh sediments dominated by different types of vegetation. Although we equate the presence of archaeal amoA genes with the genetic potential to oxidize ammonia, we acknowledge the possibility that all Archaea that have amoA genes may not all represent functional ammonia oxidizers. Vegetation patterns of New England salt marshes are strongly correlated with marsh elevation and are controlled by a combination of interspecific competition and tolerance to physico-chemical stress (28). The dominant grasses of New England salt marshes are Spartina alterniflora and Spartina patens, which typically grow as pure stands. S. alterniflora is found in two phenotypically distinct but genetically identical forms, a tall and a short growth form (34). The tall S. alterniflora grows to heights of 1 to 2 m and is typically found at the edges of the marsh and along creek banks (SAT sites), while the short-form S. alterniflora may reach heights of only 30 cm and is found in sites (SAS sites) slightly higher on the marsh where soil drainage is limited and conditions are more reduced compared to SAT sites (14). Conversely, S. patens, due to its lower tolerance of salt and more reduced conditions, is found in sites (SP sites) highest on the marsh, in areas that receive less flooding (5). Because the marsh is subjected to daily tidal fluctuations, most sites experience periods of anoxia, the degree of which depends on the marsh elevation. We hypothesized that ammonia-oxidizing communities in areas dominated by different marsh grasses would reflect the different edaphic conditions associated with each type of grass, due to differences in vertical zonation in the marsh.     

Enrichment and Association of Bacteria and Particulates in Salt Marsh Surface Water

Elevated counts of bacteria were found during outgoing tides in surface microlayers (~300 μm) of Sippewissett salt marsh, Falmouth, Massachusetts, and Palo Alto salt marsh, Palo Alto, California. At both sampling sites, the degrees by which bacteria were concentrated into the surface microlayer were linearly dependent upon surface concentration of particulate material. A significant percentage of bacteria in the microlayer were found to be attached to particulate material, while bacterial populations in the subsurface water were largely planktonic. Proportions of the bacterial populations which could be grown on seawater nutrient agar were also greater in the microlayer than in the subsurface waters and were positively correlated with the fraction of bacteria attached to particulate matter. Data from these studies suggest that particulates in the microlayer waters of the salt marsh influenced the observed increase in both the readily grown and the total numbers of bacteria.     

Enumeration of Particle-Bound and Unattached Respiring Bacteria in the Salt Marsh Environment

Proportions of respiring bacteria determined with a 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride dye-epifluorescent technique were significantly elevated in the 300-μm surface layer of a salt marsh estuary. Almost all the detectably respiring bacteria in the particle-laden surface layer and a significant proportion in subsurface waters were attached to particles.     

A Note on the Recognition of Pollution Stress in Populations of Estuarine Bacteria

Water samples were collected from the Humber estuary at a site subject to pollution by metal refinery effluent. V _max/bacterium for glucose mineralization, the percentage of glucose uptake by bacteria which was mineralized (% R ) and dissolved copper concentrations were measured. V _max/bacterium was negatively correlated with copper concentration and might be of use as an indicator of pollution stress. % R was not related to copper concentration and was not suitable as an indicator of pollution stress at this site.     

Bioturbation of Burrowing Crabs Promotes Sediment Turnover and Carbon and Nitrogen Movements in an Estuarine Salt Marsh

Ecological functions of bioturbation in ecosystems have received increasing attention over the recent decades, and crab burrowing has been considered as one of the major bioturbations affecting the physical and chemical processes in salt marshes. This study assessed the integrated effects of crab excavating and burrow mimic trapping on sediment turnover and vertical C and N distributions in a Chinese salt marsh in the Yangtze River estuary. Crab burrowing increased soil water content and the turnover of carbon and nitrogen and decreased bulk soil density. Vertical movement of materials, nutrient cycling and reuse driven by crab burrowing might be obstructed by vegetation (Phragmites australis and Spartina alterniflora communities). The amount of soil excavated by crab burrowing was higher than that deposited into burrow mimics. In Phragmites marshes, Spartina marshes and unvegetated mudflats, net transport of soil to the marsh surface was 171.73, 109.54, and 374.95 g m⁻² d⁻¹, respectively; and the corresponding estimated soil turnover time was 2.89, 4.07 and 1.83 years, respectively. Crab burrowing in salt marshes can mix surface and deeper soil over a period of years, accelerating litter decomposition and promoting the efficient reuse of nutrients by plants. Therefore, bioturbation affects soil physical processes and functioning of ecosystems, and needs to be addressed in ecosystem management.