Alexander Eiler

A Guide to the Natural History of Freshwater Lake Bacteria

SUMMARY Freshwater bacteria are at the hub of biogeochemical cycles and control water quality in lakes. Despite this, little is known about the identity and ecology of functionally significant lake bacteria. Molecular studies have identified many abundant lake bacteria, but there is a large variation in the taxonomic or phylogenetic breadths among the methods used for this exploration. Because of this, an inconsistent and overlapping naming structure has developed for freshwater bacteria, creating a significant obstacle to identifying coherent ecological traits among these groups. A discourse that unites the field is sorely needed. Here we present a new freshwater lake phylogeny constructed from all published 16S rRNA gene sequences from lake epilimnia and propose a unifying vocabulary to discuss freshwater taxa. With this new vocabulary in place, we review the current information on the ecology, ecophysiology, and distribution of lake bacteria and highlight newly identified phylotypes. In the second part of our review, we conduct meta-analyses on the compiled data, identifying distribution patterns for bacterial phylotypes among biomes and across environmental gradients in lakes. We conclude by emphasizing the role that this review can play in providing a coherent framework for future studies.

Heterotrophic bacterial growth efficiency and community structure at different natural organic carbon concentrations.

ABSTRACT Batch cultures of aquatic bacteria and dissolved organic matter were used to examine the impact of carbon source concentration on bacterial growth, biomass, growth efficiency, and community composition. An aged concentrate of dissolved organic matter from a humic lake was diluted with organic compound-free artificial lake water to obtain concentrations of dissolved organic carbon (DOC) ranging from 0.04 to 2.53 mM. The bacterial biomass produced in the cultures increased linearly with the DOC concentration, indicating that bacterial biomass production was limited by the supply of carbon. The bacterial growth rate in the exponential growth phase exhibited a hyperbolic response to the DOC concentration, suggesting that the maximum growth rate was constrained by the substrate concentration at low DOC concentrations. Likewise, the bacterial growth efficiency calculated from the production of biomass and CO2 increased asymptotically from 0.4 to 10.4% with increasing DOC concentration. The compositions of the microbial communities that emerged in the cultures were assessed by separation of PCR-amplified 16S rRNA fragments by denaturing gradient gel electrophoresis. Nonmetric multidimensional scaling of the gel profiles showed that there was a gradual change in the community composition along the DOC gradient; members of the β subclass of the class Proteobacteria and members of the Cytophaga-Flavobacterium group were well represented at all concentrations, whereas members of the α subclass of the Proteobacteria were found exclusively at the lowest carbon concentration. The shift in community composition along the DOC gradient was similar to the patterns of growth efficiency and growth rate. The results suggest that the bacterial growth efficiencies, the rates of bacterial growth, and the compositions of bacterial communities are not constrained by substrate concentrations in most natural waters, with the possible exception of the most oligotrophic environments.

Coherent dynamics and association networks among lake bacterioplankton taxa.

Bacteria have important roles in freshwater food webs and in the cycling of elements in the ecosystem. Yet specific ecological features of individual phylogenetic groups and interactions among these are largely unknown. We used 454 pyrosequencing of 16S rRNA genes to study associations of different bacterioplankton groups to environmental characteristics and their co-occurrence patterns over an annual cycle in a dimictic lake. Clear seasonal succession of the bacterioplankton community was observed. After binning of sequences into previously described and highly resolved phylogenetic groups (tribes), their temporal dynamics revealed extensive synchrony and associations with seasonal events such as ice coverage, ice-off, mixing and phytoplankton blooms. Coupling between closely and distantly related tribes was resolved by time-dependent rank correlations, suggesting ecological coherence that was often dependent on taxonomic relatedness. Association networks with the abundant freshwater Actinobacteria and Proteobacteria in focus revealed complex interdependencies within bacterioplankton communities and contrasting linkages to environmental conditions. Accordingly, unique ecological features can be inferred for each tribe and reveal the natural history of abundant cultured and uncultured freshwater bacteria.

Environmental Influences on Vibrio Populations in Northern Temperate and Boreal Coastal Waters (Baltic and Skagerrak Seas)

ABSTRACT Even if many Vibrio spp. are endemic to coastal waters, their distribution in northern temperate and boreal waters is poorly studied. To identify environmental factors regulating Vibrio populations in a salinity gradient along the Swedish coastline, we combined Vibrio-specific quantitative competitive PCR with denaturant gradient gel electrophoresis-based genotyping. The total Vibrio abundance ranged from 4 × 103 to 9.6 × 104 cells liter−1, with the highest abundances in the more saline waters of the Skagerrak Sea. Several Vibrio populations were present throughout the salinity gradient, with abundances of single populations ranging from 5 × 102 to 7 × 104 cells liter−1. Clear differences were observed along the salinity gradient, where three populations dominated the more saline waters of the Skagerrak Sea and two populations containing mainly representatives of V. anguillarum and V. aestuarianus genotypes were abundant in the brackish waters of the Baltic Sea. Our results suggest that this apparent niche separation within the genus Vibrio may also be influenced by alternate factors such as nutrient levels and high abundances of dinoflagellates. A V. cholerae/V. mimicus population was detected in more than 50% of the samples, with abundances exceeding 103 cells liter−1, even in the cold (annual average water temperature of around 5°C) and low-salinity (2 to 4‰) samples from the Bothnian Bay (latitude, 65°N). The unsuspected and widespread occurrence of this population in temperate and boreal coastal waters suggests that potential Vibrio pathogens may also be endemic to cold and brackish waters and hence may represent a previously overlooked health hazard.

Microbial Community Composition and Diversity via 16S rRNA Gene Amplicons: Evaluating the Illumina Platform

As new sequencing technologies become cheaper and older ones disappear, laboratories switch vendors and platforms. Validating the new setups is a crucial part of conducting rigorous scientific research. Here we report on the reliability and biases of performing bacterial 16S rRNA gene amplicon paired-end sequencing on the MiSeq Illumina platform. We designed a protocol using 50 barcode pairs to run samples in parallel and coded a pipeline to process the data. Sequencing the same sediment sample in 248 replicates as well as 70 samples from alkaline soda lakes, we evaluated the performance of the method with regards to estimates of alpha and beta diversity. Using different purification and DNA quantification procedures we always found up to 5-fold differences in the yield of sequences between individually barcodes samples. Using either a one-step or a two-step PCR preparation resulted in significantly different estimates in both alpha and beta diversity. Comparing with a previous method based on 454 pyrosequencing, we found that our Illumina protocol performed in a similar manner – with the exception for evenness estimates where correspondence between the methods was low. We further quantified the data loss at every processing step eventually accumulating to 50% of the raw reads. When evaluating different OTU clustering methods, we observed a stark contrast between the results of QIIME with default settings and the more recent UPARSE algorithm when it comes to the number of OTUs generated. Still, overall trends in alpha and beta diversity corresponded highly using both clustering methods. Our procedure performed well considering the precisions of alpha and beta diversity estimates, with insignificant effects of individual barcodes. Comparative analyses suggest that 454 and Illumina sequence data can be combined if the same PCR protocol and bioinformatic workflows are used for describing patterns in richness, beta-diversity and taxonomic composition.

Flavobacteria blooms in four eutrophic lakes : Linking population dynamics of freshwater bacterioplankton to resource availability

ABSTRACT Heterotrophic bacteria are major contributors to biogeochemical cycles and influence water quality. Still, the lack of representative isolates and the few quantitative surveys leave the ecological role and significance of single bacterial populations to be revealed. Here we analyzed the diversity and dynamics of freshwater Flavobacteria populations in four eutrophic temperate lakes. From each lake, clone libraries were constructed using primers specific for either the class Flavobacteria or Bacteria. Sequencing of 194 Flavobacteria clones from 8 libraries revealed a diverse freshwater Flavobacteria community and distinct differences among lakes. Abundance and seasonal dynamics of Flavobacteria were assessed by quantitative PCR with class-specific primers. In parallel, the dynamics of individual populations within the Flavobacteria community were assessed with terminal restriction fragment length polymorphism analysis using identical primers. The contribution of Flavobacteria to the total bacterioplankton community ranged from 0.4 to almost 100% (average, 24%). Blooms where Flavobacteria represented more than 30% of the bacterioplankton were observed at different times in the four lakes. In general, high proportions of Flavobacteria appeared during episodes of high bacterial production. Phylogenetic analyses combined with Flavobacteria community fingerprints suggested dominance of two Flavobacteria lineages. Both drastic alterations in total Flavobacteria and in community composition of this class significantly correlated with bacterial production, emphasizing that resource availability is an important driver of heterotrophic bacterial succession in eutrophic lakes.

Evidence for the ubiquity of mixotrophic bacteria in the upper ocean: implications and consequences.

Microorganisms are usually grouped into those relying solely on harvesting light (phototrophy) or those relying solely on the assimilation of organic or inorganic compounds (chemotrophy) to meet their requirements for energy. As a carbon source for biomass production, they can use either inorganic carbon (autotrophy) or organic substances (heterotrophy) (Table ​(Table1).1). Most biogeochemical studies of marine environments use the dichotomy of grouping microorganisms into photo(auto)trophs (primary producers like algae and cyanobacteria) and (organo)heterotrophs (secondary producers, like most heterotrophic bacteria) (see, for example, references 2 and 74). TABLE 1. Definitions of metabolic strategies to obtain carbon and energya It is well known that metabolic modes of aquatic microorganisms are more diverse than that, and modes such as mixotrophy, chemoautotrophy, chemoheterotrophy, or photoheterotrophy do exist. For microeukaryotes, mixotrophy is a widespread phenomenon in aquatic habitats and is observed in many organisms (see, for example, references 16, 62, 82, and 83). Some Bacteria, like purple nonsulfur bacteria (anoxygenic photosynthetic bacteria), are also able to alter between photo-, hetero-, auto-, litho-, and organotrophy, depending on the environmental conditions (93). Although mixotrophic bacteria, which invest in both phototrophic and heterotrophic enzymatic apparatuses and combine them with autotrophic and/or organotrophic strategies, have been isolated from various aquatic environments (see, for example, references 66, 75, and 93), their contribution to total biomass and their importance for biogeochemical processes in aquatic systems have been ignored. However, recent genome data of two Prochlorococcus strains (67) and one Synechococcus strain (56), together with results from isotope tracer uptake experiments (45, 97, 98), suggest that at least certain strains of these ubiquitous picocyanobacteria are not pure photoautotrophs and can take up organic compounds. Cyanobacteria are not the only bacteria that can harvest light energy. Aerobic anoxygenic photosynthetic bacteria (AAnPB) use mainly bacteriochlorophyll a (Bchl-a) for photosynthesis (for example, see references 75 and 93), whereas other bacteria can use proteorhodopsin, a light-driven proton pump (3, 13, 17, 23). Isolated representatives of these bacteria usually use organic carbon for cell synthesis and are characterized as photo(organo)heterotrophs (22-24, 63, 93). Genetic community surveys suggest that AAnPB and proteorhodopsin-containing bacteria (PRB) are common in surface waters throughout the oceans of the world and can make up a substantial fraction of the bacterial community in oligotrophic marine environments (see, for example, references 3-5, 10, 34, 35, 69-72, 80, and 89). Furthermore, it has been shown that phylogenetic clades, including organisms with photoreceptors, can be significant in degradation processes of organic compounds (43-45). These observations suggest that mixotrophic bacteria may be major players in photo-, hetero-, auto-, and organotrophic processes in the upper ocean, which may demand a thorough revision of our understanding of how microorganisms contribute to biogeochemical cycles in the marine environment.

Distinct and diverse anaerobic bacterial communities in boreal lakes dominated by candidate division OD1

Lakes have a central role in the carbon cycle of the boreal landscape. These systems typically stratify in summer and their hypolimnetic microbial communities influence burial of biogenic organic matter in sediments. The composition of bacterial communities in these suboxic habitats was studied by pyrosequencing of 16S rRNA amplicons from five lakes with variable dissolved organic carbon (DOC) concentrations. Bacterioplankton communities in the hypolimnetic waters were clearly different from the surface layer with candidate division OD1, Chlorobi and Bacteroidetes as dominant community members. Several operational taxonomic units (OTUs) affiliated with candidate division OD1 were abundant and consistently present in the suboxic hypolimnion in these boreal lakes. The overall representation of this group was positively correlated with DOC and methane concentrations. Network analysis of time-series data revealed contrasting temporal patterns but suggested similar ecological roles among the abundant OTUs affiliated with candidate division OD1. Together, stable isotope data and taxonomic classification point to methane oxidation and autotrophic denitrification as important processes in the suboxic zone of boreal lakes. Our data revealed that while hypolimnetic bacterial communities are less dynamic, they appear to be more diverse than communities from the oxic surface layer. An appreciable proportion of the hypolimnetic bacteria belong to poorly described phyla.

Determining indicator taxa across spatial and seasonal gradients in the Columbia River coastal margin.

Bacterioplankton communities are deeply diverse and highly variable across space and time, but several recent studies demonstrate repeatable and predictable patterns in this diversity. We expanded on previous studies by determining patterns of variability in both individual taxa and bacterial communities across coastal environmental gradients. We surveyed bacterioplankton diversity across the Columbia River coastal margin, USA, using amplicon pyrosequencing of 16S rRNA genes from 596 water samples collected from 2007 to 2010. Our results showed seasonal shifts and annual reassembly of bacterioplankton communities in the freshwater-influenced Columbia River, estuary, and plume, and identified indicator taxa, including species from freshwater SAR11, Oceanospirillales, and Flavobacteria groups, that characterize the changing seasonal conditions in these environments. In the river and estuary, Actinobacteria and Betaproteobacteria indicator taxa correlated strongly with seasonal fluctuations in particulate organic carbon (ρ=−0.664) and residence time (ρ=0.512), respectively. In contrast, seasonal change in communities was not detected in the coastal ocean and varied more with the spatial variability of environmental factors including temperature and dissolved oxygen. Indicator taxa of coastal ocean environments included SAR406 and SUP05 taxa from the deep ocean, and Prochlorococcus and SAR11 taxa from the upper water column. We found that in the Columbia River coastal margin, freshwater-influenced environments were consistent and predictable, whereas coastal ocean community variability was difficult to interpret due to complex physical conditions. This study moves beyond beta-diversity patterns to focus on the occurrence of specific taxa and lends insight into the potential ecological roles these taxa have in coastal ocean environments.

Bacterial diversity along a 2600 km river continuum

Summary The bacterioplankton diversity in large rivers has thus far been under‐sampled despite the importance of streams and rivers as components of continental landscapes. Here, we present a comprehensive dataset detailing the bacterioplankton diversity along the midstream of the Danube River and its tributaries. Using 16S rRNA‐gene amplicon sequencing, our analysis revealed that bacterial richness and evenness gradually declined downriver in both the free‐living and particle‐associated bacterial communities. These shifts were also supported by beta diversity analysis, where the effects of tributaries were negligible in regards to the overall variation. In addition, the river was largely dominated by bacteria that are commonly observed in freshwaters. Dominated by the acI lineage, the freshwater SAR11 (LD12) and the P olynucleobacter group, typical freshwater taxa increased in proportion downriver and were accompanied by a decrease in soil and groundwater‐affiliated bacteria. Based on views of the meta‐community and River Continuum Concept, we interpret the observed taxonomic patterns and accompanying changes in alpha and beta diversity with the intention of laying the foundation for a unified concept for river bacterioplankton diversity.