Qinglong L. Wu

Environmental issues of Lake Taihu, China

Lake Taihu is characterized by its shallowness (mean depth = 1.9 m) and large surface area (2,338 km2). Runoff sources are mostly from the mountainous west and southwest, and outflows are located throughout East Taihu. This causes shorter retention times in the south. In contrast, urban pollutants discharge into northern Taihu and result in poor water quality. Non-point pollution from rural areas and sewage wastewater is the primary pollution source. Water current velocity ranges from 10–30 cm s−1, and surface currents normally follow wind direction. Bottom currents appear to be a compensation flow. Most wave heights are less than 40 cm, and underwater irradiance correlates to seston in the water column. Lacustrine sediment is distributed in littoral zones, mostly along the western shoreline, with almost no accumulation in the lake center. Intensive aquaculture in East Taihu caused eutrophication and hampered water supply in surrounding areas. In addition, development of marshiness in the eastern littoral zones and East Taihu has occurred. The function of flood discharging of East Taihu has been limited by flourishing macrophytes. The problems facing in Lake Taihu will be alleviated by improving the management of nutrient sources into the lake.

Isolation of Novel Ultramicrobacteria Classified as Actinobacteria from Five Freshwater Habitats in Europe and Asia

ABSTRACT We describe the first freshwater members of the class Actinobacteria that have been isolated. Nine ultramicro-size (<0.1 μm3) strains were isolated from five freshwater habitats in Europe and Asia. These habitats represent a broad spectrum of ecosystems, ranging from deep oligotrophic lakes to shallow hypertrophic lakes. Even when the isolated strains were grown in very rich media, the cell size was <0.1 μm3 and was indistinguishable from the cell sizes of bacteria belonging to the smaller size classes of natural lake bacterioplankton. Hybridization of the isolates with oligonucleotide probes and phylogenetic analysis of the 16S rRNA gene sequences of the isolated strains revealed that they are affiliated with the class Actinobacteria and the family Microbacteriaceae. The previously described species with the highest levels of sequence similarity are Clavibacter michiganensis and Rathayibacter tritici, two phytopathogens of terrestrial plants. The 16S rRNA gene sequences of the nine isolates examined are more closely related to cloned sequences from uncultured freshwater bacteria than to the sequences of any previously isolated bacteria. The nine ultramicrobacteria isolated form, together with several uncultured bacteria, a diverse phylogenetic cluster (Luna cluster) consisting exclusively of freshwater bacteria. Isolates obtained from lakes that are ecologically different and geographically separated by great distances possess identical 16S rRNA gene sequences but have clearly different ecophysiological and phenotypic traits. Predator-prey experiments demonstrated that at least one of the ultramicro-size isolates is protected against predation by the bacterivorous nanoflagellate Ochromonas sp. strain DS.

Bacterioplankton community composition along a salinity gradient of sixteen high-mountain lakes located on the tibetan plateau, China

ABSTRACT The influence of altitude and salinity on bacterioplankton community composition (BCC) in 16 high-mountain lakes located at altitudes of 2,817 to 5,134 m on the Eastern Qinghai-Xizang (Tibetan) Plateau, China, spanning a salinity gradient from 0.02% (freshwater) to 22.3% (hypersaline), was investigated. Three different methods, fluorescent in situ hybridization, denaturing gradient gel electrophoresis (DGGE) with subsequent band sequencing, and reverse line blot hybridization (RLB) with probes targeting 17 freshwater bacterial groups, were used for analysis of BCC. Furthermore, the salt tolerances of 47 strains affiliated with groups detected in or isolated from the Tibetan habitats were investigated. Altitude was not found to influence BCC significantly within the investigated range. Several groups of typical freshwater bacteria, e.g., the ACK-M1 cluster and the Polynucleobacter group, were detected in habitats located above 4,400 m. Salinity was found to be the dominating environmental factor controlling BCC in the investigated lakes, resulting in only small overlaps in the BCCs of freshwater and hypersaline lakes. The relative abundances of different classes of Proteobacteria showed a sharp succession along the salinity gradient. Both DGGE and RLB demonstrated that a few freshwater bacterial groups, e.g., GKS98 and LD2, appeared over wide salinity ranges. Six freshwater isolates affiliated with the GKS98 cluster grew in ecophysiological experiments at maximum salinities of 0.3% to 0.7% (oligosaline), while this group was detected in habitats with salinities up to 6.7% (hypersaline). This observation indicated ecologically significant differences in ecophysiological adaptations among members of this narrow phylogenetic group and suggested ecological significance of microdiversity.

Intragenomic Heterogeneity of 16S rRNA Genes Causes Overestimation of Prokaryotic Diversity

ABSTRACT Ever since Carl Woese introduced the use of 16S rRNA genes for determining the phylogenetic relationships of prokaryotes, this method has been regarded as the “gold standard” in both microbial phylogeny and ecology studies. However, intragenomic heterogeneity within 16S rRNA genes has been reported in many investigations and is believed to bias the estimation of prokaryotic diversity. In the current study, 2,013 completely sequenced genomes of bacteria and archaea were analyzed and intragenomic heterogeneity was found in 952 genomes (585 species), with 87.5% of the divergence detected being below the 1% level. In particular, some extremophiles (thermophiles and halophiles) were found to harbor highly divergent 16S rRNA genes. Overestimation caused by 16S rRNA gene intragenomic heterogeneity was evaluated at different levels using the full-length and partial 16S rRNA genes usually chosen as targets for pyrosequencing. The result indicates that, at the unique level, full-length 16S rRNA genes can produce an overestimation of as much as 123.7%, while at the 3% level, an overestimation of 12.9% for the V6 region may be introduced. Further analysis showed that intragenomic heterogeneity tends to concentrate in specific positions, with the V1 and V6 regions suffering the most intragenomic heterogeneity and the V4 and V5 regions suffering the least intragenomic heterogeneity in bacteria. This is the most up-to-date overview of the diversity of 16S rRNA genes within prokaryotic genomes. It not only provides general guidance on how much overestimation can be introduced when applying 16S rRNA gene-based methods, due to its intragenomic heterogeneity, but also recommends that, for bacteria, this overestimation be minimized using primers targeting the V4 and V5 regions.

Low Intraspecific Diversity in a Polynucleobacter Subcluster Population Numerically Dominating Bacterioplankton of a Freshwater Pond

ABSTRACT Cultivation-dependent and -independent methods were combined to investigate the microdiversity of a Polynucleobacter subcluster population (Betaproteobacteria) numerically dominating the bacterioplankton of a small, humic freshwater pond. Complete coverage of the population by cultivation allowed the analysis of microdiversity beyond the phylogenetic resolution of ribosomal markers. Fluorescent in situ hybridization with two probes specific for the narrow subcluster C (PnecC bacteria) of the Polynucleobacter cluster revealed that this population contributed up to 60% to the total number of bacterioplankton cells. Microdiversity was investigated for a date at which the highest relative numbers of PnecC were observed. A clone library of fragments of the ribosomal operon (16S rRNA genes, complete 16S-23S internal transcribed spacer 1 [ITS1], partial 23S rRNA genes) amplified with universal bacterial primers was constructed. The library was stepwise screened for fragments from PnecC bacteria and for different ITS genotypes of PnecC bacteria. The isolated PnecC strains were characterized by sequencing of the 16S rRNA genes and the ITS1. Both the clone library and the established culture collection contained only the same three ITS genotypes, and one of them contributed 46% to the entire number of clones. Genomic fingerprinting of the isolates with several methods always resulted in the detection of only one fingerprint per ITS genotype. We conclude that a Polynucleobacter population with an extremely low intraspecific diversity and an uneven structure numerically dominated the bacterioplankton community in the investigated habitat. This low intraspecific diversity is in strong contrast to the high intraspecific diversities found in marine bacterial populations.

The Passive Yet Successful Way of Planktonic Life: Genomic and Experimental Analysis of the Ecology of a Free-Living Polynucleobacter Population

Background The bacterial taxon Polynucleobacter necessarius subspecies asymbioticus represents a group of planktonic freshwater bacteria with cosmopolitan and ubiquitous distribution in standing freshwater habitats. These bacteria comprise <1% to 70% (on average about 20%) of total bacterioplankton cells in various freshwater habitats. The ubiquity of this taxon was recently explained by intra-taxon ecological diversification, i.e. specialization of lineages to specific environmental conditions; however, details on specific adaptations are not known. Here we investigated by means of genomic and experimental analyses the ecological adaptation of a persistent population dwelling in a small acidic pond. Findings The investigated population (F10 lineage) contributed on average 11% to total bacterioplankton in the pond during the vegetation periods (ice-free period, usually May to November). Only a low degree of genetic diversification of the population could be revealed. These bacteria are characterized by a small genome size (2.1 Mb), a relatively small number of genes involved in transduction of environmental signals, and the lack of motility and quorum sensing. Experiments indicated that these bacteria live as chemoorganotrophs by mainly utilizing low-molecular-weight substrates derived from photooxidation of humic substances. Conclusions Evolutionary genome streamlining resulted in a highly passive lifestyle so far only known among free-living bacteria from pelagic marine taxa dwelling in environmentally stable nutrient-poor off-shore systems. Surprisingly, such a lifestyle is also successful in a highly dynamic and nutrient-richer environment such as the water column of the investigated pond, which was undergoing complete mixis and pronounced stratification in diurnal cycles. Obviously, metabolic and ecological versatility is not a prerequisite for long-lasting establishment of abundant bacterial populations under highly dynamic environmental conditions. Caution should be exercised when generalizing the obtained insights into the ecology and adaptation of the investigated lineage to other Polynucleobacter lineages.

Do Patterns of Bacterial Diversity along Salinity Gradients Differ from Those Observed for Macroorganisms

It is widely accepted that biodiversity is lower in more extreme environments. In this study, we sought to determine whether this trend, well documented for macroorganisms, also holds at the microbial level for bacteria. We used denaturing gradient gel electrophoresis (DGGE) with phylum-specific primers to quantify the taxon richness (i.e., the DGGE band numbers) of the bacterioplankton communities of 32 pristine Tibetan lakes that represent a broad salinity range (freshwater to hypersaline). For the lakes investigated, salinity was found to be the environmental variable with the strongest influence on the bacterial community composition. We found that the bacterial taxon richness in freshwater habitats increased with increasing salinity up to a value of 1‰. In saline systems (systems with >1‰ salinity), the expected decrease of taxon richness along a gradient of further increasing salinity was not observed. These patterns were consistently observed for two sets of samples taken in two different years. A comparison of 16S rRNA gene clone libraries revealed that the bacterial community of the lake with the highest salinity was characterized by a higher recent accelerated diversification than the community of a freshwater lake, whereas the phylogenetic diversity in the hypersaline lake was lower than that in the freshwater lake. These results suggest that different evolutionary forces may act on bacterial populations in freshwater and hypersaline lakes on the Tibetan Plateau, potentially resulting in different community structures and diversity patterns.

Heterogeneity of archaeal and bacterial ammonia‐oxidizing communities in Lake Taihu, China

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) communities within the surface sediments of Lake Taihu, a large eutrophic freshwater lake in China, were investigated using molecular approaches targeting the ammonia monooxygenase subunit A (amoA) gene. Large intra-lake variability in the composition and the relative abundance of both groups of ammonia-oxidizing prokaryotes was observed. Archaeal amoA far outnumbered bacterial amoA at most sites except those located in the Eastern Taihu Bay. This bay, which is used for intensive pen aquaculture, harboured the most unique AOA communities but was dominated by AOB in terms of relative abundance. Accumulation of organic substances rather than presence of submersed macrophytes significantly influenced the relative abundance of AOA. In contrast, shifts in the abundance of AOB were not found to be significantly related to the investigated environmental parameters. Phylogenetic analysis showed that all archaeal amoA sequences fell within either the Crenarchaeotal Group (CG) I.1b or the CG I.1a subgroup, and all AOB clustered exclusively with the genus Nitrosomonas. These findings represent the first detailed survey of AOA in eutrophic freshwater lake sediments by demonstrating that AOA dominate the ammonia-oxidizing communities, and are negatively correlated with the accumulation of organic substances.

Successful predation of filamentous bacteria by a nanoflagellate challenges current models of flagellate bacterivory

ABSTRACT Current models suggest that (i) filamentous bacteria are protected against predation by nanoflagellates, (ii) prey size is positively correlated with prey-predator contact probability, and (iii) contact probability is mainly responsible for size-selective predation by interception-feeding flagellates. We used five strains of filamentous bacteria and one bacterivorous nanoflagellate, Ochromonas sp. strain DS, to test these assumptions. The five strains, including one spirochete and four Betaproteobacteria strains, were isolated by the filtration-acclimatization method. All five strains possess flexible cells, but they differ in average cell length, which ranged from 4.5 to 13.7 μm. High-resolution video microscopy was used to measure contact, capture, and ingestion rates, as well as selectivity of the flagellate feeding. Growth and feeding experiments with satiating and nonsatiating food conditions, as well as experiments including alternative well-edible prey, were performed. In contrast to predictions by current models, the flagellate successfully consumed all the tested filamentous strains. The ingestion rate was negatively correlated with bacterial length. On the other hand, the lengths of the filamentous bacteria were not positively correlated to the contact rate and capture rate but were negatively correlated to ingestion efficiency. In experiments including alternative nonfilamentous prey, the flagellates showed negative selection for filamentous bacteria, which was independent of food concentration and is interpreted as a passive selection. Our observations indicate that (i) size alone is not sufficient to define a refuge for filamentous bacteria from nanoflagellate predation and (ii) for the investigated filamentous bacteria, prey-predator contact probability could be more influenced by factors other than the prey size.

Short-term bacterial community composition dynamics in response to accumulation and breakdown of Microcystis blooms

Short-term bacterial community composition (BCC) dynamics in response to accumulation and breakdown of Microcystis blooms were examined by conducting in situ mesocosm experiments with varying levels of Microcystis sp. biomass, ranging from 15 to 3217 μg/L as measured by chlorophyll-a concentration in the freshwater water column. The BCC was assessed by means of terminal restriction fragment length polymorphism (T-RFLP) of 16S ribosomal RNA genes followed by cloning and sequencing of selected samples. The results showed that the composition of both free-living and particle-attached bacterial communities changed during the accumulation and breakdown phases of a Microcystis bloom, and differences were also evident with different levels of Microcystis biomass. The relative abundance of bacteria affiliated with Micrococcineae and Legionellales increased in general after amendment with Microcystis. Significant correlation between the relative abundance of Micrococcineae and breakdown of Microcystis biomass was also observed. Canonical correspondence analysis (CCA) showed that the changes in the free-living and particle-attached bacterial community were mostly related to the changes in the concentrations of chlorophyll-a, dissolved organic carbon (DOC), dissolved oxygen (DO) and pH, which were mainly induced by the breakdown of Microcystis biomass. Overall, our study revealed the following: i) accumulation of Microcystis blooms and their breakdown have strong impacts on bacterial community composition; ii) there might be saprophytic association between Micrococcineae and decomposition of Microcystis biomass; iii) it is necessary to reveal potential associations between Legionellales organisms and Microcystis blooms in eutrophic freshwater lakes.