Characterization of the indigenous PAH-degrading bacteria of <Emphasis Type="Italic">Spartina</Emphasis> dominated salt marshes in the New York/New Jersey Harbor

The aerobic polyaromatic hydrocarbon (PAH) degrading microbial communities of two petroleum-impacted Spartina-dominated salt marshes in the New York/New Jersey Harbor were examined using a combination of microbiological, molecular and chemical techniques. Microbial isolation studies resulted in the identification of 48 aromatic hydrocarbon-degrading bacterial strains from both vegetated and non-vegetated marsh sediments. The majority of the isolates were from the genera Paenibacillus and Pseudomonas. Radiotracer studies using ¹⁴C-phenanthrene and ¹⁴C-pyrene were used to measure the PAH-mineralization activity in salt marsh sediments. The results suggested a trend towards increased PAH mineralization in vegetated sediments relative to non-vegetated sediments. This trend was supported by the enumeration of PAH-degrading bacteria in non-vegetated and vegetated sediment using a Most Probable Numbers (MPN) technique, which demonstrated that PAH-degrading bacteria existed in non-vegetated and vegetated sediments at levels ranging from 10² to 10⁵ cells/g sediment respectively. No difference between microbial communities present in vegetated versus non-vegetated sediments was found using terminal restriction fragment length polymorphism (of the 16S rRNA gene) or phospholipid fatty acid analysis. These studies provide information on the specific members and activity of the PAH-degrading aerobic bacterial communities present in Spartina-dominated salt marshes in the New York/New Jersey Harbor estuary.     

Ectoenzymatic activity and glucose heterotrophic metabolism in a shallow estuary (Ria de Aveiro,Portugal): influence of bed sediments and salt marshes

Bacterioplankton abundance and activity were studied in the estuarine system of Ria de Aveiro (Portugal) to test if tidal resuspension of sediments and transport of particles from the salt marshes may act as factors of variability of bacterial communities. The total and attached cell abundance, ectoenzymatic activity and the heterotrophic metabolism of glucose, as well as seston, chlorophyll a and particulate organic carbon (POC) were monitored during four 10-h periods along the tidal cycle at four sampling sites across a transect. The variation of particulate materials (seston, POC and chlorophyll a ) along the transect was not significantly correlated with either distance to the margin or distance to the sediment surface. Nevertheless, proximity to the salt marsh or to the bottom sediment surface favoured glucose incorporation and aminopeptidase activity. A multiple stepwise linear regression analysis using temperature, salinity, seston, POC, chlorophyll a , distance to sediment surface and distance to the margin as independent variables explained 66.5% of the variability of the fraction of particle-attached bacteria and only a very small proportion (12–43%) of the observed variability of total bacterial abundance, ectoenzymatic activity and glucose utilization. The spatial patterns of variation of the concentration of particulate material (seston, POC and chlorophyll a) do not clearly indicate the occurrence of sediment resuspension and runoff from the margins. This, together with the poor contribution of these parameters to the transversal and tidal variability of bacterial activity, dismisses the importance of inputs of suspended material across the sediment/water interface and from neighbouring salt marshes in the control of bacterial density and activity.     

1973-2020年黄河三角洲滨海盐沼湿地景观格局演化模式和驱动因素

明晰滨海盐沼湿地景观格局演化模式和驱动因素,有助于制定合理的盐沼湿地修复策略、维护区域生态系统健康和可持续发展。以黄河三角洲滨海盐沼湿地为例,基于Landsat系列卫星影像获取1973—2020年共十个时期土地利用/覆被数据,得出盐沼湿地时空变化及其与周边土地利用/覆被的相互转化;利用改进的景观格局状态与演化识别模型(SEDMS),分析盐沼湿地景观格局演化模式,并利用地理探测器探究其空间分异驱动因素。结果表明：(1)1973—2020年,盐沼湿地面积减少了252.35 km²,空间范围总体向外海迁移且趋于集中。盐沼湿地转出类型主要为草地、养殖池/盐田和耕地,转入类型主要为滩涂未利用地和水体。(2)盐沼湿地景观格局演化模式呈明显的阶段性特征：1973—1995年为动荡期,演化模式以消失和破碎为主导;1995—2010为过渡期,格局演化模式逐渐由消失和破碎为主导转变为扩张为主导;2010年后为稳定期,格局发生演化的区域较少,总体以新增和扩张为主。(3)36%的盐沼湿地出现了多次格局演变模式的转变,滩涂未利用地、耕地对于景观格局演化频数的影响最为显著,人工表面、养殖池/...     

A Most Probable Number method (MPN) for the estimation of cell numbers of heterotrophic nitrifying bacteria in soil

A Most Probable Number (MPN) method was developed allowing for the first time estimation of populations of bacteria capable of heterotrophic nitrification. The method was applied to an acidic soil of a coniferous forest exhibiting nitrate production. In this soil nitrate production was unlikely to be catalyzed by autotrophic nitrifiers, since autotrophic ammonia oxidizers never could be detected, and autotrophic nitrite oxidizers were usually not found in appreciable cell numbers. The developed MPN method is based on the demonstration of the presence/absence of nitrite/nitrate produced by heterotrophic nitrifying bacteria during growth in a complex medium (peptone-meat-extract softagar medium) containing low concentrations of agar (0.1%). Both the supply of the growing cultures in MPN test tubes with sufficient oxygen and the presence of low agar concentrations in the medium were found to be favourable for sustainable nitrite/nitrate production. The results demonstrate that in the acidic forest soil the microbial population capable of heterotrophic nitrifcation represents a significant part of the total aerobic heterotrophic population. By applying the developed MPN method, several bacterial strains of different genera not previously described to perform heterotrophic nitrification have been isolated from the soil and have been identified by bacterio-diagnostic tests.     

Statistical approach for comparison between methods of bacterial enumeration in drinking water

A general statistical procedure based on the likelihood ratio test is presented for the purpose of comparing estimates of mean bacterial density derived from different sets of data. This approach is much more appropriate than the conventional ways of analyzing bacteriological results (e.g., analysis of variance) which usually require previous transformation of the data. An illustrative application of the method compares three distinct titration techniques for enumerating heterotrophic bacteria in drinking water at 20°C incubation temperature. It was shown that both the standard plate count (SPC) and the membrane filter (MF) procedures supplied substantially the same information, whereas the microplate technique using the most probable number (MPN) for total bacterial enumeration could yield considerably different estimates: MPN values were significantly lower in three cases and significantly higher in one case out of a total of five experiments. The results consistently indicate a strong interaction between the technique used and the sample analyzed. Three different media (nutrient agar, R-2A low nutrient agar and m-SPC agar) were then evaluated for enumerating heterotrophic bacteria, using the MF technique at 48, 72 and 96 h of incubation time at 20°C. Although the media recovered approximately the same numbers of bacteria after 96 h of incubation, statistically significant discrepancies occurred after intermediate periods of incubation, perhaps because the relative rates of bacterial growth differed among media.     

Influence of nutrients addition and bioaugmentation on the hydrocarbon biodegradation of a chronically contaminated Antarctic soil

Complexity involved in the transport of soils and the restrictive legislation for the area makes on-site bioremediation the strategy of choice to reduce hydrocarbons contamination in Antarctica. The effect of biostimulation (with N and P) and bioaugmentation (with two bacterial consortia and a mix of bacterial strains) was analysed by using microcosms set up on metal trays containing 2·5 kg of contaminated soil from Marambio Station. At the end of the assay (45 days), all biostimulated systems showed significant increases in total heterotrophic aerobic and hydrocarbon-degrading bacterial counts. However, no differences were detected between bioaugmented and nonbioaugmented systems, except for J13 system which seemed to exert a negative effect on the natural bacterial flora. Hydrocarbons removal efficiencies agreed with changes in bacterial counts reaching 86 and 81% in M10 (bioaugmented) and CC (biostimulated only) systems. Results confirmed the feasibility of the application of bioremediation strategies to reduce hydrocarbon contamination in Antarctic soils and showed that, when soils are chronically contaminated, biostimulation is the best option. Bioaugmentation with hydrocarbon-degrading bacteria at numbers comparable to the total heterotrophic aerobic counts showed by the natural microflora did not improve the process and showed that they would turn the procedure unnecessarily more complex.     

Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination

We investigated the changes in the community structure of ammonia-oxidizing bacteria (AOB) in activated sludge during incubation of the sludge in a medium selective for AOB. The number of AOB present in the activated sludge sample was enumerated by the most-probable-number (MPN) method. Both the activated sludge sample and the incubated samples for MPN determination were analyzed by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Universal PCR-DGGE indicated that even after 40-d incubation in a medium selected for AOB, the MPN samples were predominantly composed of heterotrophic bacteria and not AOB. Denitrification by heterotrophic bacteria might lead to the underestimation of the MPN count of AOB. Not dominated in whole bacteria, one species of AOB was detected in both original activated sludge and samples after MPN incubation by PCR-DGGE targeting AOB. Furthermore, two new species of AOB were detected only after incubation. Therefore, the community structure of AOB in the MPN samples partially resembled that in the original activated sludge.     

Most-probable-number technique for the enumeration of aromatic degraders in natural environments

A most-probable-number (MPN) method is described for the enumeration of heterotrophic populations capable of utilizing chlorinated and nonchlorinated benzoates and phenols as sole carbon sources. A correlation coefficient of 0.91 was obtained between the numbers determined by the MPN technique and the standard plate count. The MPN method gave realistic cell counts when population densities were low, and the presence of oligocarbophiles did not give spurious results.     

Influence of salt marsh on bacterial activity in two estuaries with different hydrodynamic characteristics (Ria de Aveiro and Tagus Estuary)

The influence of salt marsh on estuarine bacterioplankton was investigated in two estuaries with different hydrodynamic characteristics (Ria de Aveiro and Tagus Estuary). In the Ria de Aveiro, bacteria in the flood water overlying the marsh were two times more abundant and five to six times more active than in the main channel. In the Tagus Estuary, bacterial abundance was similar in flooding and channel water, but bacterial activity was up to two times higher in the main channel. The two salt marshes have distinct influences on estuarine bacterioplankton abundance and activity. In the Ria de Aveiro, salt marsh enhanced estuarine bacterial communities, increasing their size and stimulating their activity. By contrast, the salt marsh in the Tagus Estuary does not seem to increase the bacterial abundance and production in the channel water. These distinct influences may be explained by the hydrodynamic characteristics of the salt marshes, which were confirmed by the hydrodynamic model implemented for both systems.     

Bacterial Activity in a Reservoir Determined by Autoradiography and its Relationships to Phyto- and Zooplankton

In the drinking water reservoir Římov (Southern Bohemia) bacterioplankton was studied during 1983. Special attention was given to the relationships between parameters of bacterial abundance, total and individual activity. Bacterial counts and biomass was assessed and autoradiographic determinations of the proportion of active bacteria incorporating thymidine (Th) and mixture of amino acids (AA) and total uptake rate of AA were made over a year in the surface layer and during summer stratification from the thermocline and 15 m depth. Specific activity of metabolically active bacteria (SAMAB) and specific activity per unit of biomass (SAUB) were negatively correlated with counts of metabolizing cells and with bacterial biomass, respectively. Total and individual heterotrophic activity and counts of bacteria coincided with the changes of phytoplankton biomass, whereas bacteria incorporating Th were more tightly correlated with primary production. The most significant relation of metabolically active bacteria was found to cladoceran biomass. Thus, this part of heterotrophic bacterial activity seems to be stimulated by leakage of dissolved organic matter from phytoplankton being disrupted and incompletely digested by cladocerans rather than from healthy photosynthetizing cells.     

Microbial community analysis of a coastal salt marsh affected by the Deepwater Horizon oil spill

Coastal salt marshes are highly sensitive wetland ecosystems that can sustain long-term impacts from anthropogenic events such as oil spills. In this study, we examined the microbial communities of a Gulf of Mexico coastal salt marsh during and after the influx of petroleum hydrocarbons following the Deepwater Horizon oil spill. Total hydrocarbon concentrations in salt marsh sediments were highest in June and July 2010 and decreased in September 2010. Coupled PhyloChip and GeoChip microarray analyses demonstrated that the microbial community structure and function of the extant salt marsh hydrocarbon-degrading microbial populations changed significantly during the study. The relative richness and abundance of phyla containing previously described hydrocarbon-degrading bacteria (Proteobacteria, Bacteroidetes, and Actinobacteria) increased in hydrocarbon-contaminated sediments and then decreased once hydrocarbons were below detection. Firmicutes, however, continued to increase in relative richness and abundance after hydrocarbon concentrations were below detection. Functional genes involved in hydrocarbon degradation were enriched in hydrocarbon-contaminated sediments then declined significantly (p<0.05) once hydrocarbon concentrations decreased. A greater decrease in hydrocarbon concentrations among marsh grass sediments compared to inlet sediments (lacking marsh grass) suggests that the marsh rhizosphere microbial communities could also be contributing to hydrocarbon degradation. The results of this study provide a comprehensive view of microbial community structural and functional dynamics within perturbed salt marsh ecosystems.     

Factors Stimulating Propagation of Legionellae in Cooling Tower Water

Our survey of cooling tower water demonstrated that the highest density of legionellae, ≥10⁴ CFU/100 ml, appeared in water containing protozoa, ≥10² MPN/100 ml, and heterotrophic bacteria, ≥10⁶ CFU/100 ml, at water temperatures between 25 and 35°C. Viable counts of legionellae were detected even in the winter samples, and propagation, up to 10⁵ CFU/100 ml, occurs in summer. The counts of legionellae correlated positively with increases in water temperature, pH, and protozoan counts, but not with heterotrophic bacterial counts. The water temperature of cooling towers may promote increases in the viable counts of legionellae, and certain microbes, e.g., protozoa or some heterotrophic bacteria, may be a factor stimulating the propagation of legionellae.     

Effects of fish farming on microbial enzyme activities and densities: comparison between three Mediterranean sites

AIM: The effects of fish farming on microbial enzyme activities and heterotrophic bacterial density were investigated in three Mediterranean sites before and after the start of mariculture. METHODS AND RESULTS: Microbial activities were measured on water and sediment samples by using fluorogenic substrates specific for leucine aminopeptidase, beta-glucosidase and alkaline phosphatase (AP); bacterial counts were determined by Marine agar plates. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison of activity and abundance values obtained before and after the experiment showed that fish farming mainly affected the levels of microbial activities; they were significantly enhanced both in water and sediments, reaching an increase of 183.66 times for AP in Castellammare Gulf. After mariculture, no significant variations were recorded in heterotrophic bacterial density in the waters, while significant changes were observed in the sediments. Effects induced appeared to be extended not only to stations in which cages were located, but also to control sites far from the direct influence of fish farming.     

Effects of abiotic factors on the life span of the invasive cordgrass <Emphasis Type="Italic">Spartina densiflora</Emphasis> and the native <Emphasis Type="Italic">Spartina maritima</Emphasis> at low salt marshes

We analyzed variations in the life span of the invasive cordgrass Spartina densiflora at low marshes of SW Iberian Peninsula, and identified the abiotic factors limiting the plant in the absence of competition. With these objectives, clump survivorship, flowering, and growth of S. densiflora were studied in two natural populations at different low marsh elevations during more than three years, and at a transplant experiment in comparison with the native Spartina maritima. The life spans of both cordgrasses changed depending on small variations of a few centimeters in elevation. S. maritima, which tolerates better than S. densiflora the stressful abiotic environment of lower marshes, showed a significant lower distribution limit for its perennial habit, with survivorship longer than three years (from 1997 to 2000), than the neophyte (+1.57 m SHZ vs. +2.00 m SHZ). S. densiflora clumps flowered before dying at mostly all elevations, showing low relative growth rates. In contrast, clumps of S. maritima, with non-viable seeds, only flowered when they were three years old at higher elevations in the low marsh. Our results have applications for salt marshes bioengineering projects and to prevent S. densiflora from invading European marshes since our data improve the knowledge of its colonization mechanisms through salt marsh zonation and so identify those portions of restored and native marshes most susceptible to invasion due to the establishment of perennial populations.     

Shifts in Symbiotic Endophyte Communities of a Foundational Salt Marsh Grass following Oil Exposure from the Deepwater Horizon Oil Spill

Symbiotic associations can be disrupted by disturbance or by changing environmental conditions. Endophytes are fungal and bacterial symbionts of plants that can affect performance. As in more widely known symbioses, acute or chronic stressor exposure might trigger disassociation of endophytes from host plants. We tested this hypothesis by examining the effects of oil exposure following the Deepwater Horizon (DWH) oil spill on endophyte diversity and abundance in Spartina alterniflora – the foundational plant in northern Gulf coast salt marshes affected by the spill. We compared bacterial and fungal endophytes isolated from plants in reference areas to isolates from plants collected in areas with residual oil that has persisted for more than three years after the DWH spill. DNA sequence-based estimates showed that oil exposure shifted endophyte diversity and community structure. Plants from oiled areas exhibited near total loss of leaf fungal endophytes. Root fungal endophytes exhibited a more modest decline and little change was observed in endophytic bacterial diversity or abundance, though a shift towards hydrocarbon metabolizers was found in plants from oiled sites. These results show that plant-endophyte symbioses can be disrupted by stressor exposure, and indicate that symbiont community disassembly in marsh plants is an enduring outcome of the DWH spill.     

Natural attenuation of diesel-oil contamination in a subantarctic soil (Crozet Island)

An accidental contamination occurred in subantarctic Crozet Island between July and November 1997 near the "Alfred Faure" scientific station (51°51'E-46°25'S). More than 20,000 l of diesel fuel was spilled in the soil in the vicinity of the power station. In order to evaluate the efficiency of the expected bioattenuation process, the contaminated area was sampled in December 1999 for bacterial and chemical analysis. All samples were analysed for total bacteria, heterotrophic viable assemblages and hydrocarbon-utilising microflora. The results of this first analytical survey clearly show a significant response of subantarctic microbial soil communities to the hydrocarbon contamination. Significant increases of total, heterotrophic and hydrocarbon-utilising micro-organisms occurred in the more contaminated zone (from less than 5᎒⁴ MPN g^-1 wet soil to more than 10⁸ MPN g^-1 wet soil for hydrocarbon-degrading micro-organisms). The very high numbers of hydrocarbon-degrading micro-organisms present in the more contaminated zone are clearly linked to early bioattenuation activities. Chemical results provided some clear indications that spilled fuel was still well preserved from chemical and biological weathering 1 year after the spill, as light aliphatics and aromatics were present in all oiled samples and little differences were usually observed between samples.     

Seasonality and spatial variation in abundance, biomass and activity of heterotrophic bacterioplankton in relation to some biotic and abiotic variables in an Ethiopian rift-valley lake (Awassa)

SUMMARY. I. Seasonality and spatial variation in abundance, biomass and activity of heterotrophic bacterioplankton was studied in a tropical freshwater lake. Variation in phytoplankton biomass and production, and several other limnological parameters, were also quantified.
2. Bacterial number and biomass fluctuated between 3.88–8.33×10⁶ ml⁻¹ and 68–125 mg C m⁻³, respectively. No spatial variation was observed in these bacterial variables, although other limnological parameters showed vertical variation.
3. Seasonal and vertical variation of heterotrophic bacterial activity were estimated from oxygen consumption, ¹⁴CO₂dark uptake and plate counts of colony forming units. All three methods showed vertical heterogeneity, with higher activity in the hypolimnion during the long stratification period (February May). Oxygen consumption rate and plate counts showed seasonality.
4. In spite of the low seasonality of climate in the tropics, several biological, chemical and physical variables were correlated with changes in bacterial variables. Wind and rainfall, and their effects on stratification and mixing, vertical distribution of nutrients and rate of primary production appeared to be the important parameters affecting heterotrophic bacterioplankton.     

Development and N2-Fixing Activity of the Benthic Microbial Community in Transplanted Spartina alterniflora Marshes in North Carolina

Growth and maturation of transplanted salt marshes is often limited by the availability of nitrogen (N). We examined the role of N₂-fixing benthic microbial assemblages (microalgae and associated bacteria) in two restored marshes (1-year-old and 6-year-old marsh) and a natural salt marsh in the Newport River Estuary, North Carolina. Benthic N₂ fixation (nitrogenase activity, NA), chlorophyll a (Chl a ) concentration, Spartina alterniflora (smooth cordgrass) stem counts, and sediment organic matter content were determined in the three marshes. Significant differences were observed between sites for both Chl a and NA. The 1-year-old marsh always exhibited the highest levels of NA and Chl a . Sediment organic matter content was lowest in the 1-year-old marsh (∼2%), intermediate in the 6-year-old marsh (∼5%), and highest in the natural marsh (∼10%). Carbon and nitrogen analyses were also performed on the 1-year-old marsh sediments, which were depleted in N. A positive correlation was observed between surface sediment N and Chl a . Remineralized, microbially derived N may provide growth-limiting inorganic N to Spartina transplants. N₂-fixing microbial assemblages in the 1-year-old marsh may also be an important food source for marsh infauna. Benthic N₂-fixing microbial assemblages play a key role in the N economy of restored salt marshes.     

Salt Marsh Restoration in Connecticut: 20 Years of Science and Management

In 1980 the State of Connecticut began a tidal marsh restoration program targeting systems degraded by tidal restrictions and impoundments. Such marshes become dominated by common reed grass (Phragmites australis) and cattail (Typha angustifolia and T. latifolia), with little ecological connection to Long Island Sound. The management and scientific hypothesis was that returning tidal action, reconnecting marshes to Long Island Sound, would set these systems on a recovery trajectory. Specific restoration targets (i.e., pre‐disturbance conditions or particular reference marshes) were considered unrealistic. However, it was expected that with time restored tides would return ecological functions and attributes characteristic of fully functioning tidal salt marshes. Here we report results of this program at nine separate sites within six marsh systems along 110 km of Long Island Sound shoreline, with restoration times of 5 to 21 years. Biotic parameters assessed include vegetation, macroinvertebrates, and use by fish and birds. Abiotic factors studied were soil salinity, elevation and tidal flooding, and soil water table depth. Sites fell into two categories of vegetation recovery: slow, ca. 0.5%, or fast, more than 5% of total area per year. Although total cover and frequency of salt marsh angiosperms was positively related to soil salinity, and reed grass stand parameters negatively so, fast versus slow recovery rates could not be attributed to salinity. Instead, rates appear to reflect differences in tidal flooding. Rapid recovery was characterized by lower elevations, greater hydroperiods, and higher soil water tables. Recovery of other biotic attributes and functions does not necessarily parallel those for vegetation. At the longest studied system (rapid vegetation recovery) the high marsh snail Melampus bidentatus took two decades to reach densities comparable with a nearby reference marsh, whereas the amphipod Orchestia grillus was well established on a slow‐recovery marsh, reed grass dominated after 9 years. Typical fish species assemblages were found in restoration site creeks and ditches within 5 years. Gut contents of fish in ditches and on the high marsh suggest that use of restored marsh as foraging areas may require up to 15 years to reach equivalence with reference sites. Bird species that specialize in salt marshes require appropriate vegetation; on the oldest restoration site, breeding populations comparable with reference marshland had become established after 15 years. Use of restoration sites by birds considered marsh generalists was initially high and was still nearly twice that of reference areas even after 20 years. Herons, egrets, and migratory shorebirds used restoration areas extensively. These results support our prediction that returning tides will set degraded marshes on trajectories that can bring essentially full restoration of ecological functions. This can occur within two decades, although reduced tidal action can delay restoration of some functions. With this success, Connecticut's Department of Environmental Protection established a dedicated Wetland Restoration Unit. As of 1999 tides have been restored at 57 separate sites along the Connecticut coast.     

Microplate MPN-enumeration of monocyclic- and dicyclic-aromatic hydrocarbon degraders via substrate phase-partitioning

The high aqueous solubility of monoaromatic and some diaromatic oil components may hinder classical growth-based MPN enumeration of bacterial mono- and di-aromatics degraders because these aromatics are toxic in high concentrations. We developed a microplate MPN method for the enumeration of toluene-, xylene-, naphthalene-, biphenyl- and benzothiophene-degraders on the basis of phase-partitioning of substrate between a biologically inert organic phase and an aqueous mineral salt medium. This way, it was possible to maintain non-toxic, aqueous concentrations in the microplate wells. Depletion of aqueous aromatics by growth of the degraders was prevented by the continuous solubilization of aromatics from the silicone phase. The method was validated by MPN enumerating degrader cultures both with phase-partitioned aromatics and with tryptic soy broth as carbon sources. The applicability of the method was demonstrated by MPN-enumerating mono- and di-aromatic degraders in soils of varying hydrocarbon pre-exposure. An erratum to this article can be found at