Culture-dependent Study Research Articles

A Culture-Independent and Culture-Dependent Study of the Bacterial Community from the Bedrock Soil Interface

In nutrient limited soils, minerals constitute a major reservoir of bio-essential elements. Consequently, the release of nutritive elements during weathering is crucial. Bacteria have been shown to enhance weathering rates; however, there has been limited work that has focused on the bacterial weathering of bedrock or parent rock, which are the major sources of minerals, in nutrient limiting soils. In this study, both a culture-independent and culture-dependent approach was used to study the bacterial community at the interface between basaltic bedrock and nutrient limiting soil in Cadiar Idris region of Snowdonia National Park, United Kingdom. High throughput sequencing method, Ion Torrent, was used to characterise the bacterial community, which generated over 250,000 sequences. Taxonomical assignment demonstrated that approximately 50% (125,000 sequences) of the community consisted of the orders Actinomycetales, Burkholderiales, Clostridales, Bacillales, Rhizobiales and Acidobacterium, with unclassified sequences representing 44% ± 1.46% (110,000 ± 3650). Bacteria belonging to the genera Serratia, Pseudomonas, Bacillus, Paenibacillus, Chromobacterium, Janthinobacterium, Burkholderia and Arthrobacter, were isolated from the sample site. All of the isolates were able to grow in a minimal growth medium, which contained glucose, ammonium chloride with basalt as the sole source of bio-essential elements. Seventy percent of the isolates significantly enhanced basalt dissolution (p < 0.05). The rate of dissolution correlated to the production of oxalic acid and acidification of the growth medium. The findings of this work suggest that at the interface between bedrock and soil heterotrophic members of the bacterial community can enhance weathering, an essential part of biogeochemical cycling in nutrient limiting soil.

Correlation between microbial community structure and biofouling in a laboratory scale membrane bioreactor with synthetic wastewater

Membrane biofouling was investigated in a laboratory-scale membrane bioreactor (MBR) to determine how microbial community structure influences the fouling process and the potential for quorum sensing. Differences in microbial communities of the mixed liquor and the membrane fouling layer were evaluated using pyrosequencing. During continuous MBR operation, macroscopic engineering parameters such as total cell concentration and organic removal rate were maintained at constant levels, however, microbial community structure was greatly changed showing a dynamic shift in the dominant bacterial species. The microbial composition in the membrane-biocake was very different from that in the mixed liquor and specific microbial groups such as the genera Enterobacter and Dyella were found to be closely associated with the initial and late biofouling stages, respectively. In parallel, in a culture-dependent study, a total of 61 isolates were obtained from the MBR. Both approaches indicate that Enterobacter cancerogenus is a dominant community member and that it is likely a contributor to membrane fouling in this particular MBR system. Furthermore, a bioassay with Agrobacterium tumefaciens A136 (Ti−)(pCF218)(pCF372) reporter strain demonstrated that this E. cancerogenus-like isolate can produce N-acyl homoserine lactone-type quorum sensing autoinducers, although an in-situ experiment would be needed to prove that QS mechanism is operative under actual operational conditions.

Detection and phylogenetic analysis of coastal bioaerosols using culture dependent and independent techniques

Abstract. Bioaerosols are emerging as important yet poorly understood players in atmospheric processes. Microorganisms can impact atmospheric chemistry through metabolic reactions and can potentially influence physical processes by participating in ice nucleation and cloud droplet formation. Microbial roles in atmospheric processes are thought to be species-specific and potentially dependent on cell viability. Using a coastal pier monitoring site as a sampling platform, culture-dependent (i.e. agar plates) and culture-independent (i.e. DNA clone libraries from filters) approaches were combined with 18S rRNA and 16S rRNA gene targeting to obtain insight into the local atmospheric microbial composition. From 13 microbial isolates and 42 DNA library clones, a total of 55 sequences were obtained representing four independent sampling events. Sequence analysis revealed that in these coastal samples two fungal phyla, Ascomycota and Basidiomycota, predominate among eukaryotes while Firmicutes and Proteobacteria predominate among bacteria. Furthermore, our culture-dependent study verifies the viability of microbes from all four phyla detected through our culture-independent study. Contrary to our expectations and despite oceanic air mass sources, common marine planktonic bacteria and phytoplankton were not typically found. The abundance of terrestrial and marine sediment-associated microorganisms suggests a potential importance for bioaerosols derived from beaches and/or coastal erosion processes.

Culture independent characterization of bacteria associated with the mucus of the coral Acropora digitifera from the Gulf of Mannar

Corals are sessile eukaryotic hosts which provide a unique surface for microbial colonization. Culture independent studies show that the coral mucus and tissue harbour diverse and abundant prokaryotic communities. However, little is known about the diversity of bacteria associated with the corals of Gulf of Mannar. The present study characterised the bacterial diversity associated with the mucus of the coral Acropora digitifera from the Gulf of Mannar by 16S rRNA gene clone library construction. The bacterial communities of the mucus of A. digitifera were diverse, with representatives within the Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes and several unclassified bacteria. The culture independent bacterial population was totally different from our previous culture dependent study of the mucus and tissue of the same coral. 36% of the bacteria in the clone library of A. digitifera were found to be novel after full length sequencing of the 16S rRNA gene wherein several clones were found to be novel at the Genus and species level. The current study further supports the findings that Actinobacteria amount to a certain proportion among bacterial communities associated with corals.

Diversity of Culturable Bacteria, from the Anaerobic Zone of the Meromictic Lake Pavin, Able to Perform Dissimilatory-Iron Reduction in Different in Vitro Conditions

A culture-dependent study was performed with the aim of assessing the carbon, electron and Fe(III) sources used for the dissimilatory Fe(III) reduction pathway and the diversity of culturable Fe(III)-reducers in the anoxic zone of the meromictic Lake Pavin. This metabolic pathway was investigated in enrichment cultures inoculated with water samples collected at 70 m depth in the anoxic zone of Lake Pavin. Combinations of different media, organic acids, and incubation gas phases were performed. The potential for Fe(III) reduction in the different growth conditions was assessed by measuring the accumulation of Fe(II) overtime. Bacterial community structure was determined in each growth conditions by Temporal Temperature gradient Gel Electrophoresis (TTGE) profiles of 16S rDNA genes and bands of interest in positive enrichments were sequenced. Comparisons of bacterial community structure between growth conditions revealed that the electron donor, the basal media as well as the Fe(III) source yielded to the selection of different bacterial populations, suggesting that Fe(III) reducers occupy different ecological niches in the anoxic zone of Lake Pavin. Facultative Fe(III) reducers, such as fermentative (e.g., Pseudomonas, Clostridium) and sulphate-reducing (e.g., Desulfovibrio sp.) bacteria, were retrieved in enrichments but well-known obligatory Fe(III) reducers (e.g., Geobacter) were not detected. A greater Fe(III) reduction was noted under H2:CO2 gas phase, suggesting that H2 is used as an electron donor for Fe(III) reduction. Acetate was not used as a precursor for this terminal electron-accepting process, and a high Fe(III) reduction was observed with fumarate provided as the electron donor and carbon sources suggesting that this metabolite may be energetically more beneficial for Fe(III)-reducers.

Culture-dependent and culture-independent microbial investigation of pine litters and soil in subtropical Australia

Background, aim, and scope Forest plantations, widely grown for wood production, involve the selective promotion of single-tree species or replacement of natural species by exotic tree species. Slash pine (Pinus elliottii) has been chosen for reforestation of infertile sandy soils in southeast Queensland, Australia. These exotic pine plantations minimize soil and water losses and are important scientific study sites. The soil environment of these plantations, though devoid of sufficient nutrients, organic carbon and other factors, harbors innumerable bacteria that may play a crucial role in maintaining soil quality and ecosystem functions. These soil microorganisms also have the potential for use as sensitive biological indicators to reflect environmental changes. It is therefore essential to understand the interrelationships among bacterial communities and their environment by assessing their structural and functional diversity and their responses to disturbances. The main aim of our investigation was to determine the diversity of bacterial communities in forest litters and soil during the forest leaf litter decomposition using culturedependent and culture-independent techniques. Materials and methods A 25-cm (diameter)×40-cm core sample was collected and fractionated into three subsamples designated E1 (L leaf litter layer), E2 (F leaf litter layer), and E5 (0–10 cm soil layer). Both culture-dependent and culture-independent methods were applied in this study. In the culture-independent study, a strategy of wholecommunity DNA extraction, polymerase chain reaction (PCR) amplification followed by cloning and 16S rDNA sequence analysis was used; for culture-dependent study, the strategy included sample plating and bacteria isolating, DNA extraction, PCR amplification, and 16S rDNA sequence analysis. The diversity similarities between two bacterial communities and two methods are quantified using Jensen–Shannon divergence. Results From culture-dependent study, 336 colonies in total were isolated and grouped from the three subsamples, and the 16S rRNA sequence analysis from a representative isolate from each morphogroup (21 isolates) indicated that they belonged to the phyla Actinobacteria, Firmicutes, and Proteobacteria. Culture-independent assessment based on 16S rRNA gene library comprising 194 clones revealed that members of the phylum Actinobacteria were absent in the culture-independent studies. Clones in libraries from E1 consisted exclusively of members of the Firmicutes. The majority of clones from E2 were related to Firmicutes (79%) and Proteobacteria (21%). Clones derived from E5 were mostly affiliated with Acidobacterium (42%), followed by unclassified bacteria (27%), Verrucomicrobiales (12%), Proteobacteria (11%), and Planctomycetes (8%). Discussion This study showed that bacterial culturabilities in different fractions of leaf litters were similar, and both of them were higher than the bacterial culturability in the soil. Unculturable bacterial diversity in the soil, however, was much higher than the leaf litter bacterial diversity. The bacterial diversity on the top layer of leaf litters was slightly