Laura G. Leff

Diversity of Fungi, Bacteria, and Actinomycetes on Leaves Decomposing in a Stream

ABSTRACT Although fungi, bacteria, and specific bacterial taxa, such as the actinomycetes, have been studied extensively in various habitats, few studies have examined them simultaneously, especially on decomposing leaves in streams. In this study, sugar maple and white oak leaves were incubated in a stream in northeastern Ohio for 181 days during which samples were collected at regular intervals. Following DNA extraction, PCR-denaturing gradient gel electrophoresis (DGGE) was performed using fungus-, bacterium-, and actinomycete-specific primers. In addition, fungal and bacterial biomass was estimated. Fungal biomass differed on different days but not between leaves of the two species and was always greater than bacterial biomass. There were significant differences in bacterial biomass through time and between leaf types on some days. Generally, on the basis of DGGE, few differences in community structure were found for different leaf types. However, the ribotype richness of fungi was significantly greater than those of the bacteria and actinomycetes, which were similar to each other. Ribotype richness decreased toward the end of the study for each group except bacteria. Lack of differences between the two leaf types suggests that the microorganisms colonizing the leaf biofilm were primarily generalists that could exploit the resources of the leaves of either species equally well. Thus, we conclude that factors, such as the ecological role of the taxa (generalists versus specialists), stage of decay, and time of exposure, appeared to be more important determinants of microbial community structure than leaf quality.

Direct observations of aluminosilicate weathering in the hyporheic zone of an Antarctic Dry Valley stream

Abstract This study focused on chemical weathering and bacterial ecology in the hyporheic zone of Green Creek, a McMurdo Dry Valley (Antarctica) stream. An in situ microcosm approach was used to observe dissolution features on the basal-plane surface of muscovite mica. Four mica chips were buried in December 1999 and dug up 39 d later. Atomic force microscopy (AFM) of the basal-plane surfaces revealed small, anhedral ∼10-A-deep etch pits covering ∼4% of the surfaces, from which an approximate basal-plane dissolution rate of 8.3 × 10−18 mol muscovite cm−2 s−1 was calculated (on the basis of the geometric surface area) for the study period. This is an integrated initial dissolution rate on a fresh surface exposed for a relatively brief period over the austral summer and should not be compared directly to other long-term field rates. The observation of weathering features on mica agrees with previous stream- and watershed-scale studies in the Dry Valleys, which have demonstrated that weathering occurs where liquid water is present, despite the cold temperatures. AFM imaging of mica surfaces revealed biofilms including numerous small (

Seasonal Response of Stream Biofilm Communities to Dissolved Organic Matter and Nutrient Enrichments

ABSTRACT Dissolved organic matter (DOM) and inorganic nutrients may affect microbial communities in streams, but little is known about the impact of these factors on specific taxa within bacterial assemblages in biofilms. In this study, nutrient diffusing artificial substrates were used to examine bacterial responses to DOM (i.e., glucose, leaf leachate, and algal exudates) and inorganic nutrients (nitrate and phosphate singly and in combination). Artificial substrates were deployed for five seasons, from summer 2002 to summer 2003, in a northeastern Ohio stream. Differences were observed in the responses of bacterial taxa examined to various DOM and inorganic nutrient treatments, and the response patterns varied seasonally, indicating that resources that limit the bacterial communities change over time. Overall, the greatest responses were to labile, low-molecular-weight DOM (i.e., glucose) at times when chlorophyll a concentrations were low due to scouring during significant storm events. Different types of DOM and inorganic nutrients induced various responses among bacterial taxa in the biofilms examined, and these responses would not have been apparent if they were examined at the community level or if seasonal changes were not taken into account.

Nutrients and Other Abiotic Factors Affecting Bacterial Communities in an Ohio River (USA)

Nitrogen and phosphorus additions from anthropogenic sources can alter the nutrient pool of aquatic systems, both through increased nutrient concentrations and changes in stoichiometry. Because bacteria are important in nutrient cycling and aquatic food webs, information about how nutrients affect bacterial communities enhances our understanding of how changes in nutrient concentrations and stoichiometry potentially affect aquatic ecosystems as a whole. In this study, bacterial communities were examined in biofilms from cobbles collected across seasons at three sites along the Mahoning River (Ohio) with differing levels of inorganic nutrient inputs. Members of the alpha-, beta-, and gamma-proteobacteria, the Cytophaga–Flavobacteria cluster, and the Domain Bacteria were enumerated using fluorescent in situ hybridization. Detrended canonical correspondence analysis (DCCA) revealed that stoichiometric ratios, especially the dissolved inorganic nitrogen (DIN):soluble reactive phosphorus (SRP) molar ratio (NO2/NO3 + NH4:soluble reactive phosphorus), were correlated with abundance of the various bacterial taxa. However, the patterns were complicated by correlations with single nutrient concentrations and seasonal changes in temperature. Seasonal cycles appeared to play an important role in structuring the community, as there were distinct winter communities and temperature was negatively correlated with abundance of both alpha-proteobacteria and Cytophaga–Flavobacteria. However, nutrients and stoichiometry also appeared to affect the community. Numbers of cells hybridizing the Domain Bacteria probe were correlated with the DOC:DIN ratio, the beta-proteobacteria had a negative correlation with soluble reactive phosphorus concentrations and a positive correlation with the DIN:SRP ratio, and the Cytophaga–Flavobacteria had a significant negative partial correlation with the DIN:SRP ratio. Abundances of the alpha- or gamma-proteobacteria were not directly correlated to nutrient concentrations or stoichiometry. It appears that nutrient stoichiometry may be an important factor structuring bacterial communities; however, it is one of many factors, such as temperature, that are interlinked and must be considered together when studying environmental bacteria.

Bacterial Populations in an Anthropogenically Disturbed Stream: Comparison of Different Seasons.

A bstractTo determine the effects of environmental changes on stream bacterial populations, assemblage- and population-level measurements were compared between an anthropogenically disturbed stream and an undisturbed reference stream during different seasons. Physical and chemical variables monitored at two disturbed sites from a stream affected by multiple environmental perturbations confirmed discernibly different water quality from three reference sites: two from an adjacent, undisturbed watershed and one from the headwaters of the polluted stream. Assemblage-level variables, including total number of bacteria, colony forming units, and number of Bacteria from in situ hybridization revealed only one statistically significant difference between disturbed and undisturbed sites. Population-level changes of three bacterial species, Burkholderia cepacia, Pseudomonas putida, and Acinetobacter calcoaceticus, were determined by colony hybridization with rDNA probes. Abundance of culturable A. calcoaceticus was higher at disturbed sites in November and February; B. cepacia and P. putida did not exhibit pollution-associated responses. In contrast, in situ hybridization indicated that there was more A. calcoaceticus at the reference sites in November and April, suggesting that culturability of the species increased at disturbed sites. To determine if differences among sites were attributable to changes in water quality among the streams, three bacterial strains isolated from the disturbed stream were grown for 64 h in flasks in water from disturbed and reference sites. As observed in the stream, A. calcoaceticus numbers increased in polluted stream water after an initial lag period of approximately 24 h. Our results indicate that although assemblage-level measurements of bacterial communities did not reflect environmental differences among sites, A. calcoaceticus population sizes differed between disturbed and reference sites, suggesting that anthropogenic disturbance can alter some bacterial populations and not others.

The Response of Three Bacterial Populations to Pollution in a Stream

A bstractPractical methods for biomonitoring of natural systems are still under development. Bacteria are potentially useful indicators of water quality because of their species diversity and ability to rapidly respond to changing environmental conditions. In this study, bacterial populations from unpolluted and polluted stream sites in two watersheds were compared to determine their suitability for use as environmental indicators. Upper Three Runs Creek and Four Mile Creek headwaters have had little anthropogenic disturbance, as opposed to lower Four Mile Creek which received thermal, radioactive, and chemical perturbations. Chemical and physical measurements provided evidence that seepage from holding ponds polluted Four Mile Creek. Polluted sites did not have altered total bacterial numbers but had decreased numbers of colony-forming units. Abundances of three bacterial species, Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida, were determined by colony hybridization with species-specific rDNA probes. Contribution of A. calcoaceticus to the assemblage was higher at polluted sites, which indicated either tolerance of polluted conditions or the ability to utilize compounds existing at these sites to reach larger populations. No differences in B. cepacia populations were detected, and differences in P. putida populations could not be attributed solely to disturbance. The pollution of Four Mile Creek induced differences in bacterial populations that could be monitored using the described approach.

Comparison of Methods for the Concentration of Bacterioplankton for in Situ Hybridization

Three approaches for concentrating stream bacterioplankton were compared for analysis by in situ hybridization: centrifugation; filtration onto a polymer membrane and transfer to a gel-coated slide; and filtration onto an inorganic filter disk. Stream bacterioplankton were hybridized with a species-specific oligonucleotide probe targeted for 23S rRNA of Burkholderia cepacia for analysis of variability among techniques. The inorganic filter technique yielded three to six times higher total cell counts and appeared to be effected less by interfering suspended particles and differences in site conditions than the other two concentration methods. Hybridization of samples with minimum cell loss yielded different, and presumably more accurate, estimates of the relative abundance of B. cepacia than the other methods.

Seasonal dynamics of bacterial assemblages in epilithic biofilms in a northeastern Ohio stream

Abstract Seasonal changes in the abundance of bacteria belonging to different phylogenetic groups in epilithic biofilms from a northeastern Ohio (USA) stream were examined using fluorescent in situ hybridization (FISH). Changes over a 13-mo period were observed in the biofilm assemblages, dominated by α- and β-proteobacteria. Numbers of β-proteobacteria and Cytophaga–Flavobacterium peaked during the winter months and coincided with increased NO3 concentration. Actinobacteria (Gram-Positive bacteria with high guanine and cytosine [GC] content) had no relationship with any measured environmental variable and accounted for <3% of the overall bacterial assemblage. Abundance of 3 bacterial species examined, Acinetobacter calcoaceticus, Burkholderia cepacia, and Pseudomonas putida, was similar, except in the summer when numbers of B. cepacia were higher than the other 2 species. Detrended correspondence analysis extracted 2 factors that explained 69.2% of the total variation. β-proteobacteria and Cytophaga–Flavobacterium clustered with conductivity and concentrations of NO3, dissolved organic C, and soluble reactive P in the 1st factor, while A. calcoaceticus, B. cepacia, and P. putida clustered with temperature and turbidity in the 2nd factor. Our study revealed large seasonal fluctuations in the abundance of the different bacterial taxa examined in biofilms, and also demonstrated the potential influences of various environmental variables on microbial community composition in aquatic systems.

Genetic Diversity of Bacteria along a Stream Continuum

Adaptation is a fundamental concept in ecology and evolution. Determining whether organisms are adapted requires genetic analysis of the organism. We characterized the genetic structure of isolates of two species of aquatic bacteria (Pseudomonas cepacia and P. pickettii) using starch gel electrophoresis to determine the relationship between genetic diversity and stream location. Genetic diversity in these species of bacteria did not change longitudinally. However, each location in the stream had genetically unique bacteria. Genetic distance among isolates was found to be related to geographical distance, i.e., isolates farther apart in the stream were more genetically dissimilar. The results suggest localized adaptation. Soil forms of P. cepacia collected from the same catchment were very different genetically from the aquatic forms of the same species. Analyzing patterns of allozymes may be of value in determining the source of bacteria in aquatic ecosystems.

The Effect of Leaf Pack Composition on Processing: A Comparison of Mixed and Single Species Packs

The effect of leaf species composition on decomposition patterns was examined in a coastal plain stream. Red maple leaves (Acer rubrum) decomposed at the same rate separately or when mixed with cypress leaves (Taxodium distichum). Cypress addition increased structural integrity but its effects differed between sites with different hydrologic regimes. Invertebrate communities varied slightly between mixed and single species packs, however invertebrates did not appear to be the primary agent of decomposition. Mixed species packs may be an alternative method to fine mesh bags for studying processing of small, narrow leaves in a more realistic manner.