Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern

Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined.     

Characterization of Microbial Diversity in Hypersaline Environments by Melting Profiles and Reassociation Kinetics in Combination with Terminal Restriction Fragment Length Polymorphism (T-RFLP)

The diversity of prokaryotes inhabiting solar saltern ponds was determined by thermal melting and reassociation of community DNA. These measurements were compared with fingerprinting techniques such as terminal restriction fragment length polymorphisms (T-RFLP) analysis, denaturant gradient gel electrophoresis (DGGE), and cloning and sequencing approaches. Three ponds with salinities of 22, 32, and 37% (NaCl saturation) were studied. The combination of independent molecular techniques to estimate the total genetic diversity provided a realistic assessment to reveal the microbial diversity in these environments. The changes in the prokaryotic communities at different salinity (22, 32, and 37% salt) were significant and revealed that the total genetic diversity increased from 22% to 32% salinity. At 37% salinity the diversity was reduced again to nearly half that at 22% salinity. Our results revealed that the community genome had a DNA complexity that was 7 (in 22% salinity pond), 13 (in 32% salinity pond), and 4 (in 37% salinity pond) times the complexity of an Escherichia coli genome. The base composition profiles showed two abundant populations, which changed in relative amount between the three ponds. They indicated an uneven taxon distribution at 22% and 37% salinity and a more even distribution at 32% salinity. The results indicated a large predominating population at 37% salinity, which might correspond to the abundance of square archaea (SPhT) observed by transmission electron microscopy (TEM) and also indicated by the same T-RFLP fragment as the SPhT. The SPhT phylotype has also been reported to be the most frequently retrieved phylotype from this environment by culture independent techniques. In addition, two different operational taxonomic units (OTU) were detected at 37% salinity based on PCR with bacterial specific primers and T-RFLP. One of these predominant phylotypes is the extreme halophilic bacterium belonging to the bacteroidetes group, Salinibacter ruber.     

Spatial and temporal changes in microbial community structure associated with recharge-influenced chemical gradients in a contaminated aquifer

In a contaminated water-table aquifer, we related microbial community structure on aquifer sediments to gradients in 24 geochemical and contaminant variables at five depths, under three recharge conditions. Community amplified ribsosomal DNA restriction analysis (ARDRA) using universal 16S rDNA primers and denaturing gradient gel electrophoresis (DGGE) using bacterial 16S rDNA primers indicated: (i). communities in the anoxic, contaminated central zone were similar regardless of recharge; (ii). after recharge, communities at greatest depth were similar to those in uncontaminated zones; and (iii). after extended lack of recharge, communities at upper and lower aquifer margins differed from communities at the same depths on other dates. General aquifer geochemistry was as important as contaminant or terminal electron accepting process (TEAP) chemistry in discriminant analysis of community groups. The Shannon index of diversity (H) and the evenness index (E), based on DGGE operational taxonomic units (OTUs), were statistically different across community groups and aquifer depths. Archaea or sulphate-reducing bacteria 16S rRNA abundance was not clearly correlated with TEAP chemistry indicative of methanogenesis or sulphate reduction. Eukarya rRNA abundance varied by depth and date from 0 to 13% of the microbial community. This contaminated aquifer is a dynamic ecosystem, with complex interactions between physical, chemical and biotic components, which should be considered in the interpretation of aquifer geochemistry and in the development of conceptual or predictive models for natural attenuation or remediation.     

Semi-automated genetic analyses of soil microbial communities: comparison of T-RFLP and RISA based on descriptive and discriminative statistical approaches

Cultivation independent analyses of soil microbial community structures are frequently used to describe microbiological soil characteristics. This approach is based on direct extraction of total soil DNA followed by PCR amplification of selected marker genes and subsequent genetic fingerprint analyses. Semi-automated genetic fingerprinting techniques such as terminal restriction fragment length polymorphism (T-RFLP) and ribosomal intergenic spacer analysis (RISA) yield high-resolution patterns of highly diverse soil microbial communities and hold great potential for use in routine soil quality monitoring, when rapid high throughput screening for differences or changes is more important than phylogenetic identification of organisms affected. Our objective was to perform profound statistical analysis to evaluate the cultivation independent approach and the consistency of results from T-RFLP and RISA. As a model system, we used two different heavy metal treated soils from an open top chamber experiment. Bacterial T-RFLP and RISA profiles of 16S rDNA were converted into numeric data matrices in order to allow for detailed statistical analyses with cluster analysis, Mantel test statistics, Monte Carlo permutation tests and ANOVA. Analyses revealed that soil DNA-contents were significantly correlated with soil microbial biomass in our system. T-RFLP and RISA yielded highly consistent and correlating results and both allowed to distinguish the four treatments with equal significance. While RISA represents a fast and general fingerprinting method of moderate cost and labor intensity, T-RFLP is technically more demanding but offers the advantage of phylogenetic identification of detected soil microorganisms. Therefore, selection of either of these methods should be based on the specific research question under investigation.     

Comparison of bacterioplankton communities in three mariculture ponds farming different commercial animals in subtropical Chinese coast

In order to explore the responses of the bacterioplankton community to different types of aquaculture environments, three mariculture ponds comprised of groupers (Epinephelus diacanthus, ED), prawns (Penaeus vannamei, PV), and abalone (Haliotis diversicolor supertexta, HDS) in southeast, coastal China were investigated. The free-living bacterial diversity was analyzed through the construction of 16S rDNA clone library. A total of 203 16S rDNA sequences from three clone libraries were classified into 118 operational taxonomic units (OTUs), of which 51, 31, and 42 OTUs were distributed in the ED, PV, and HDS pond, respectively, with Bacteroidetes (30.6%), Actinobacteria (55.2%), and Cyanobacteria (32.8%) as the dominant division in the respective ponds. Meanwhile, each pond occupied some unique OTUs that were affiliated with uncommon (sub-)phyla, such as candidate OP11 division, Acidobacteria, Deltaproteobacteria, Planctomycetes, and Verrucomicrobia. Bacterial diversity in the ED pond was the richest, followed by the HDS and the PV pond. OTUs of 61.9% and 94.9% have less than 90% and 97% similarity to their nearest neighbors in public databases, respectively. All OTUs were grouped into 67 clusters, covering 11 (sub-)phyla. The OTUs only from single pond distributed in 53 clusters (79.1%), the OTUs shared by two ponds were affiliated with 14 clusters (20.9%), and none of clusters was formed by the OTUs which commonly originated from the three pond libraries, suggesting that the composition of bacterial populations in these ponds were significantly different. These results indicate that the aquatic environment created by different mariculture animals may foster very special and complex bacterial communities. Handling editor: David Philip Hamilton     

Litter Breakdown and Microbial Succession on Two Submerged Leaf Species in a Small Forested Stream

Microbial succession during leaf breakdown was investigated in a small forested stream in west-central Georgia, USA, using multiple culture-independent techniques. Red maple (Acer rubrum) and water oak (Quercus nigra) leaf litter were incubated in situ for 128 days, and litter breakdown was quantified by ash-free dry mass (AFDM) method and microbial assemblage composition using phospholipid fatty acid analysis (PLFA), ribosomal intergenic spacer analysis (RISA), denaturing gradient gel electrophoresis (DGGE), and bar-coded next-generation sequencing of 16S rRNA gene amplicons. Leaf breakdown was faster for red maple than water oak. PLFA revealed a significant time effect on microbial lipid profiles for both leaf species. Microbial assemblages on maple contained a higher relative abundance of bacterial lipids than oak, and oak microbial assemblages contained higher relative abundance of fungal lipids than maple. RISA showed that incubation time was more important in structuring bacterial assemblages than leaf physicochemistry. DGGE profiles revealed high variability in bacterial assemblages over time, and sequencing of DGGE-resolved amplicons indicated several taxa present on degrading litter. Next-generation sequencing revealed temporal shifts in dominant taxa within the phylum Proteobacteria, whereas γ-Proteobacteria dominated pre-immersion and α- and β-Proteobacteria dominated after 1 month of instream incubation; the latter groups contain taxa that are predicted to be capable of using organic material to fuel further breakdown. Our results suggest that incubation time is more important than leaf species physicochemistry in influencing leaf litter microbial assemblage composition, and indicate the need for investigation into seasonal and temporal dynamics of leaf litter microbial assemblage succession.     

Microbial diversity and composition in acidic sediments of freshwater finfish culture ponds fed with two types of feed: a metagenomic approach

Microbial community profile associated with acidic pond sediments (APS) (pH = 3·0–4·5) of freshwater finfish aquaculture ponds (n = 8) was investigated. Sediment DNA extracted from the eight APS were subjected to high-throughput sequencing of V3 and V4 regions which yielded 7236 operational taxonomic units (OTUs) at a similarity of 97%. Overall results showed higher proportion of bacterial OTUs than archaeal OTUs in all the APS. Euryarchaeota (23%), Proteobacteria (19%), Chloroflexi (17%), Crenarchaeota (5·3%), Bacteroidetes (4·8%), Nitrospirae (3·2%), Nanoarchaeaeota (3%) which together constituted 75% of the microbial diversity. At the genus level, there was high preponderance of methanogens namely Methanolinea (5·4%), Methanosaeta (4·5%) and methanotrops, Bathyarchaeota (5%) in APS. Moreover, the abundant phyla in the APS were not drastically affected by the administration of chicken slaughter waste (R-group ponds) and commercial fish feed (C-group ponds), since 67% of the OTUs generated remained common in the APS of both the groups of ponds. There was a minimal difference of 24–26% of OTUs between C-group and R-group ponds, suggesting the existence of a core microbial community in these ponds driven by acidic pH over the years. This study concludes that microbial diversity in pond sediment was influenced to a lesser extent by the addition of chicken slaughter waste but was majorly driven by acidic nature of the pond.     

Characterization of the methanogenic Archaea within two-phase biogas reactor systems operated with plant biomass

The two-phase leach-bed system is a biogas reactor system optimized for the utilization of energy crop silages at maximized loading rates under maintenance of an optimal microbial activity. In this study, a characterization of the methanogenic microbial community within this reactor system was conducted for the first time. Accordingly, effluent samples from the anaerobic filter and the silage digesting leach-bed reactors of both a laboratory-scale two-phase biogas reactor system and a scaled-up commercial on-farm pilot plant were investigated. In total, five Archaea-specific 16S rDNA libraries were constructed and analyzed by amplified rDNA restriction analysis (ARDRA), with subsequent phylogenetic analysis of nucleotide sequences for individual ARDRA patterns. A quantification of major methanogenic Archaea groups was conducted by real-time PCR. A total of 663 clones were analyzed and 45 operational taxonomic units (OTUs) related to methanogenic Archaea were detected. These OTUs were related to the orders Methanosarcinales, Methanomicrobiales and Methanobacteriales, as well as the hitherto uncultured CA-11 and ARC-I groups, and most of them occurred throughout all the compartments of both two-phase biogas reactors. The proportion of acetotrophic to hydrogenotrophic methanogens differed between the laboratory and the pilot scale system. A total of 56% of the clones from the 16S rDNA library derived from the laboratory biogas system were assigned to presumably acetotrophic members of Methanosarcinales. In contrast, these OTUs were less abundant in the 16S rDNA library derived from samples of the pilot plant. Therein, the most dominant OTUs were Methanoculleus-related OTUs, which presumably indicated the predominant presence of hydrogenotrophic methanogens. These findings were confirmed by group-specific quantitative real-time PCR assays. The results indicated that the fraction of acetotrophic and hydrogenotrophic methanogens within a biogas reactor caused certain variations, which may reflect varying substrate utilization during methanogenesis.     

High 16S rDNA bacterial diversity in glacial meltwater lake sediment,Bratina Island,Antarctica

The microbial diversity in maritime meltwater pond sediments from Bratina Island, Ross Sea, Antarctica was investigated by 16S rDNA-dependent molecular phylogeny. Investigations of the vertical distribution, phylogenetic composition, and spatial variability of Bacteria and Archaea in the sediment were carried out. Results revealed the presence of a highly diverse bacterial population and a significantly depth-related composition. Assessment of 173 partial 16S rDNA clones analyzed by amplified rDNA restriction analysis (ARDRA) using tetrameric restriction enzymes (HinP1I 5'G/CGC3'and Msp I. 5'C/CGG3', BioLabs) revealed 153 different bacterial OTUs (operational taxonomic units). However, only seven archaeal OTUs were detected, indicating low archaeal diversity. Based on ARDRA results, 30 bacterial clones were selected for sequencing and the sequenced clones fell into seven major lineages of the domain Bacteria; the alpha, gamma, and delta subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides, the Spirochaetaceae, and the Actinobacteria. All of the archaeal clones sequenced belonged to the group Crenarchaeota and phylogenetic analysis revealed close relationships with members of the deep-branching Group 1 Marine Archaea.     

Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1,632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.     

Spatial and seasonal prokaryotic community dynamics in ponds of increasing salinity of Sfax solar saltern in Tunisia

The spatial and seasonal dynamics of the halophilic prokaryotic community was investigated in five ponds from Sfax solar saltern (Tunisia), covering a salinity gradient ranging from 20 to 36%. Fluorescence in situ hybridization indicated that, above 24% salinity, the prokaryotic community shifted from bacterial to archaeal dominance with a remarkable increase in the proportion of detected cells. Denaturing gradient gel electrophoresis (DGGE) profiles were rather similar in all the samples analyzed, except in the lowest salinity pond (around 20% salt) where several specific archaeal and bacterial phylotypes were detected. In spite of previous studies on these salterns, DGGE analysis unveiled the presence of microorganisms not previously described in these ponds, such as Archaea related to Natronomonas or bacteria related to Alkalimnicola, as well as many new sequences of Bacteroidetes. Some phylotypes, such as those related to Haloquadratum or to some Bacteroidetes, displayed a strong dependence of salinity and/or magnesium concentrations, which in the case of Haloquadratum could be related to the presence of ecotypes. Seasonal variability in the prokaryotic community composition was focused on two ponds with the lowest (20%) and the highest salinity (36%). In contrast to the crystallized pond, where comparable profiles between autumn 2007 and summer 2008 were obtained, the non-crystallized pond showed pronounced seasonal changes and a sharp succession of “species” during the year. Canonical correspondence analysis of biological and physicochemical parameters indicated that temperature was a strong factor structuring the prokaryotic community in the non-crystallizer pond, that had salinities ranging from 20 to 23.8% during the year.     

DGGE and T-RFLP Analysis of Bacterial Succession during Mushroom Compost Production and Sequence-aided T-RFLP Profile of Mature Compost

The amount of button mushroom (Agaricus bisporus) harvested from compost is largely affected by the microbial processes taking place during composting and the microbes inhabiting the mature compost. In this study, the microbial changes during the stages of this specific composting process were monitored, and the dominant bacteria of the mature compost were identified to reveal the microbiological background of the favorable properties of the heat-treated phase II mushroom compost. 16S ribosomal deoxyribonucleic acid (rDNA)-based denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) molecular fingerprinting methods were used to track the succession of microbial communities in summer and winter composting cycles. DNA from individual DGGE bands were reamplified and subjected to sequence analysis. Principal component analysis of fingerprints of the composting processes showed intensive changes in bacterial community during the 22-day procedure. Peak temperature samples grouped together and were dominated by Thermus thermophilus. Mature compost patterns were almost identical by both methods (DGGE, T-RFLP). To get an in-depth analysis of the mature compost bacterial community, the sequence data from cultivation of the bacteria and cloning of environmental 16S rDNA were uniquely coupled with the output of the environmental T-RFLP fingerprints (sequence-aided T-RFLP). This method revealed the dominance of a supposedly cellulose-degrading consortium composed of phylotypes related to Pseudoxanthomonas, Thermobifida, and Thermomonospora.     

Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis.

The potential of terminal-restriction fragment length polymorphism (T-RFLP) and the detection of operational taxonomic units (OTUs) by capillary electrophoresis (CE) to characterize marine bacterioplankton communities was compared with that of denaturing gradient gel electrophoresis (DGGE). A protocol has been developed to optimize the separation and detection of OTUs between 20 and 1, 632 bp by using CE and laser-induced fluorescence detection. Additionally, we compared T-RFLP fingerprinting to DGGE optimized for detection of less abundant OTUs. Similar results were obtained with both fingerprinting techniques, although the T-RFLP approach and CE detection of OTUs was more sensitive, as indicated by the higher number of OTUs detected. We tested the T-RFLP fingerprinting technique on complex marine bacterial communities by using the 16S rRNA gene and 16S rRNA as templates for PCR. Samples from the Northern and Middle Adriatic Sea and from the South and North Aegean Sea were compared. Distinct clusters were identifiable for different sampling sites. Thus, this technique is useful for rapid evaluation of the biogeographical distribution and relationships of bacterioplankton communities.     

Microalgal assemblages in a poikilohaline pond

Microalgal strains for algal biofuels production in outdoor ponds will need to have high net growth rates under diverse environmental conditions. A small, variable salinity pond in the San Elijo Lagoon estuary in southern California was chosen to serve as a model pond due to its routinely high chlorophyll content. Profiles of microalgal assemblages from water samples collected from April 2011 to January 2012 were obtained by constructing 18S rDNA environmental clone libraries. Pond assemblages were found to be dominated by green algae Picochlorum sp. and Picocystis sp. throughout the year. Pigment analysis suggested that the two species contributed most of the chlorophyll a of the pond, which ranged from 21.9 to 664.3 μg · L^?1 with the Picocystis contribution increasing at higher salinities. However, changes of temperature, salinity or irradiance may have enabled a bloom of the diatom Chaetoceros sp. in June 2011. Isolates of these microalgae were obtained and their growth rates characterized as a function of temperature and salinity. Chaetoceros sp. had the highest growth rate over the temperature test range while it showed the most sensitivity to high salinity. All three strains showed the presence of lipid bodies during nitrogen starvation, suggesting they have potential as future biofuels strains.     

Terminal-restriction fragment length polymorphism (T-RFLP) screening of a marine archaeal clone library to determine the different phylotypes

T-RFLP clone characterization (screening) was optimized for a fast and basepair-accurate characterization of clones from marine Archaea collected from the Eastern Mediterranean Sea. Because of the high sensitivity of T-RFLP fingerprinting, a protocol was developed where 10 initial PCR cycles gave detectable terminal fragments from clones. Additionally, forward and reverse primers for PCR were individually labeled and detected simultaneously to assess the suitability of the forward and reverse fragments for T-RFLP screening. Based on independent restriction digests with the tetrameric restriction enzymes HhaI, RsaI and HaeIII to characterize the 49 archaeal clones in our library, the clones were grouped into 13 T-RFLP operational taxonomic units (OTUs). Reverse fragments generally gave less heterogeneous fragments in size. The accuracy of T-RFLP screening was evaluated by sequencing representative clones. Closely related clones ( approximately 97% similarity) could only be resolved with multiple restriction digests where forward and reverse fragments were included in the analysis. All fragments from the clone library were detected in the T-RFLP fingerprint from the complex archaeal community. We found representatives of marine group I, II and III Archaea. Thus, the recently discovered low abundant marine group III Archaea could be clearly differentiated from the other clones in our library and comprised a considerable fraction of the clone library ( approximately 12%). Therefore, our T-RFLP screening approach proved successful in characterizing novel archaeal sequences from the marine environment.     

Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.     

Detection of Microcystis in Lake Sediment using Molecular Genetic Techniques

Summary Microcystis, which are toxic microcystin-producing cyanobacteria, normally bloom in summer and drop in numbers during the winter season in Senba Lake, Japan. Recently, this lake has been treated by ultrasonic radiation and jet circulation which were integrated with flushing with river water. This treatment was most likely sufficient for the destruction of cyanobacterial gas vacuoles. In order to confirm whether Microcystis viridis was still present, a molecular genetic monitoring technique on the basis of DNA direct extraction from the sediment was applied. Three primer sets were used for polymerase chain reaction (PCR) based on rRNA intergenic spacer analysis (RISA), the DNA dependent RNA polymerase (rpoC1) and a Microcystis sp.-specific rpoC1 fragment. The results from each primer were demonstrated on the basis of single strand conformation polymorphisms (SSCP). Using the RISA primer showed different results from the rpoC1 and Microcystis sp.-specific rpoC1 fragment; meanwhile, the rpoC1 Microcystis sp.-specific fragment was more specific than the RISA primer. Therefore, the Microcystis sp.-specific rpoC1 fragment was further analysed by denaturing gradient gel electrophoresis (DGGE). The DNA pattern representing M. viridis could not be detected in any of the sediment samples. However, the results were confirmed with another technique, terminal restriction fragment length polymorphisms (T-RFLP). Although T-RFLP patterns of 16S rDNA in sediment at 91 bp and 477 bp lengths were matched with the T-RFLP of M. viridis (HhaI and MspI endonuclease digestion, respectively), the T-RFLP pattern of 75 bp length was not matched with M. viridis (both of HhaI and MspI endonuclease digestion) which were the major T-RFLP pattern of M. viridis. Therefore, the results most likely indicated that M. viridis seems to have disappeared because of the addition of the ultrasonic radiation and jet circulation to the flushing treatment.     

Characterization of Humus Microbial Communities in Adjacent Forest Types That Differ in Nitrogen Availability

To address the link between soil microbial community composition and soil processes, we investigated the microbial communities in forest floors of two forest types that differ substantially in nitrogen availability. Cedar-hemlock (CH) and hemlock-amabilis fir (HA) forests are both common on northern Vancouver Island, B.C., occurring adjacently across the landscape. CH forest floors have low nitrogen availability and HA high nitrogen availability. Total microbial biomass was assessed using chloroform fumigation-extraction and community composition was assessed using several cultivation-independent approaches: denaturing gradient gel electrophoresis (DGGE) of the bacterial communities, ribosomal intergenic spacer analysis (RISA) of the bacterial and fungal communities, and phospholipid fatty acid (PLFA) profiles of the whole microbial community. We did not detect differences in the bacterial communities of each forest type using DGGE and RISA, but differences in the fungal communities were detected using RISA. PLFA analysis detected subtle differences in overall composition of the microbial community between the forest types, as well as in particular groups of organisms. Fungal PLFAs were more abundant in the nitrogen-poor CH forests. Bacteria were proportionally more abundant in HA forests than CH in the lower humus layer, and Gram-positive bacteria were proportionally more abundant in HA forests irrespective of layer. Bacterial and fungal communities were distinct in the F, upper humus, and lower humus layers of the forest floor and total biomass decreased in deeper layers. These results indicate that there are distinct patterns in forest floor microbial community composition at the landscape scale, which may be important for understanding nutrient availability to forest vegetation.     

分子生物学方法在水体微生物生态研究中的应用

微生物是生态系统的重要组成部分,研究水体中微生物的多样性和群落结构对于开发微生物资源、进行水体生物修复具有重要意义。现代分子生物学的发展为研究水体微生物提供了行之有效的方法。综述了16S rDNA文库构建、变性梯度凝胶电泳、限制性片段长度多态性、末端标记限制性片段长度多态性等技术的原理以及在水体微生物研究中的主要应用。