Denaturing Gradient Gel Electrophoresis Gels Research Articles

Tropical Soil Bacterial Diversity in Sabah, Malaysia

Bacteria are an essential biological component of soil function that plays fundamental roles in biogeochemical cycling, soil quality improvement, habitat-shaping, and ecosystem conservation. It is therefore important to have a good record of soil bacteria in the tropics in order to monitor future changes that may occur due to global warming and other factors. However, extremely limited data are available on the diversity of bacteria in soils in some tropical Borneo regions such as Sabah, Malaysia. This research, therefore, was undertaken to determine the bacterial diversity of soils from various locations in Sabah, Malaysia. Ten soil samples (n=10) were collected around Sabah. 16S rDNA of bacterial DNA extracted from soils were amplified and analysed using the Denaturing Gradient Gel Electrophoresis (DGGE). A total of 100 dominant and well-defined DNA fragments observed in the DGGE gel were extracted, sequenced, and aligned. The results indicated that 93 different bacterial operational taxonomic units (OTUs) representing bacteria from 8 different phyla were present. The most abundant phyla in the analysed Sabah soils were Proteobacteria followed by Acidobacteria, Firmicutes, Actinobacteria, Planctomycetes, Verrucomicrobia, Chloroflexi, and Bacteroidetes. The examined soils of Sabah and Peninsular Malaysia had similar dominant phyla in general, except that the most dominant phylum in Peninsular Malaysia soils is the Acidobacteria instead of Proteobacteria. These baseline data generated from this work are important and can be used to track bacterial diversity shifts due to soil or environmental changes in the future.

Metagenomic Analysis of Bacteria Phylum Firmicutes and Bacteroidetes in Women with Type 2 Diabetes

Diabetes mellitus (DM) is a serious health problem in Indonesia. Type 2 diabetes is a metabolic disease which primarily caused by obesity-linked insulin resistance. There is a link between insulin resistance and bacterial populations in the gut. Therefore, the aims of this study was to analyze composition of bacteria belong to the phylum Firmicutes and Bacteroidetes in women with type 2 diabetes and non-diabetic persons as control. The study included five female adults type 2 diabetes patients and five healthy controls. The bacteria composition was analyzed for abundance by denaturing gradient gel electrophoresis (DGGE). Metagenomic analysis based on 16S rRNA gene was represented by 12 DGGE bands. The twelve respective bands showed the similarity ranging from 77 up to 98%. Diversity of microbial composition in women type 2 diabetes was represented by five DGGE bands are Bacteroides fragilis, Bacteroides vulgatus, uncultured Bacteroides sp. clone Lb4eF4, uncultured bacterium clone 16sms90-5g05, and uncultured bacterium clone Malaga 1F14. Four DGGE bands in women healthy controls are closely related to uncultured bacterium isolate DGGE gel band Eub42, uncultured bacterium isolate DGGE gel band K115, uncultured bacterium clone HFV04255, and Enterococcus sp. the finding shows bacterial species belong to the phylum Bacteroidetes was mostly found in women diabetic group, while those belong to the phylum Firmicutes was mostly found in healthy controls.

Effect of Silene vulgaris and Heavy Metal Pollution on Soil Microbial Diversity in Long-Term Contaminated Soil

In this study, we analysed the impact of heavy metals and plant rhizodeposition on the structure of indigenous microbial communities in rhizosphere and bulk soil that had been exposed to heavy metals for more than 150 years. Samples of the rhizosphere of Silene vulgaris and non-rhizosphere soils 250 and 450 m from the source of emission that had different metal concentrations were collected for analyses. The results showed that soils were collected 250 m from the smelter had a higher number of Cd-resistant CFU compared with the samples that were collected from 450 m, but no significant differences were observed in the number of total and oligotrophic CFU or the equivalent cell numbers between rhizosphere and non-rhizosphere soils that were taken 250 and 450 m from the emitter. Unweighted pair group method with arithmetic mean (UPGMA) cluster analysis of the denaturing gradient gel electrophoresis (DGGE) profiles, as well as a cluster analysis that was generated on the phospholipid fatty acid (PLFA) profiles, showed that the bacterial community structure of rhizosphere soils depended more on the plant than on the distance and metal concentrations. The sequencing of the 16S rDNA fragments that were excised from the DGGE gel revealed representatives of the phyla Bacteroidetes, Acidobacteria, Gemmatimonadetes, Actinobacteria and Betaproteobacteria in the analysed soil with a predominance of the first three groups. The obtained results demonstrated that the presence of S. vulgaris did not affect the number of CFUs, except for those of Cd-resistant bacteria. However, the presence of S. vulgaris altered the soil bacterial community structure, regardless of the sampling site, which supported the thesis that plants have a higher impact on soil microbial community than metal contamination.

Molecular Study of Indigenous Bacterial Community Composition on Exposure to Soil Arsenic Concentration Gradient.

Community structure of bacteria present in arsenic contaminated agricultural soil was studied with qPCR (quantitative PCR) and DGGE (Denaturing Gradient Gel Electrophoresis) as an indicator of extreme stresses. Copy number of six common bacterial taxa (Acidobacteria, Actinobacteria, α-, β- and γ-Proteobacteria, Firmicutes) was calculated using group specific primers of 16S rDNA. It revealed that soil contaminated with low concentration of arsenic was dominated by both Actinobacteria and Proteobacteria but a shift towards Proteobacteria was observed with increasing arsenic concentration, and number of Actinobacteria eventually decreases. PCA (Principle Component Analysis) plot of bacterial community composition indicated a distinct resemblance among high arsenic content samples, while low arsenic content samples remained separated from others. Cluster analysis of soil parameters identifies three clusters, each of them was related to the arsenic content. Further, cluster analysis of 16S rDNA based DGGE fingerprint markedly distributed the soil bacterial populations into low (< 10 ppm) and high (> 10 ppm) arsenic content subgroups. Following analysis of diversity indices shows significant variation in bacterial community structure. MDS (Multi Dimensional Scaling) plot revealed distinction in the distribution of each sample denoting variation in bacterial diversity. Phylogenetic sequence analysis of fragments excised from DGGE gel revealed the presence of γ-Proteobacteria group across the study sites. Collectively, our experiments indicated that gradient of arsenic contamination affected the shape of the soil bacterial population by significant structural shift.

Seasonal changes in bacterial communities associated with healthy and diseased Porites coral in southern Taiwan.

We compared the bacterial communities associated with healthy scleractinian coral Porites sp. with those associated with coral infected with pink spot syndrome harvested during summer and winter from waters off the coast of southern Taiwan. Members of the bacterial community associated with the coral were characterized by means of denaturing gradient gel electrophoresis (DGGE) of a short region of the 16S rRNA gene and clone library analysis. Of 5 different areas of the 16S rRNA gene, we demonstrated that the V3 hypervariable region is most suited to represent the coral-associated bacterial community. The DNA sequences of 26 distinct bands extracted from DGGE gels and 269 sequences of the 16S rRNA gene from clone libraries were determined. We found that the communities present in diseased coral were more heterogeneous than the bacterial communities of uninfected coral. In addition, bacterial communities associated with coral harvested in the summer were more diverse than those associated with coral collected in winter, regardless of the health status of the coral. Our study suggested that the compositions of coral-associated bacteria communities are complex, and the population of bacteria varies greatly between seasons and in coral of differing health status.

Invasion by Conyza sumatrensis alters soil microbial community structure in urban ecosystems

Whether invasive plants stimulate or inhibit the soil microbial diversity is still an open question. Despite large-scale invasion by Conyza sumatrensis (Retz.) E. Walker in the urban ecosystems of the Srinagar city of the Kashmir Himalayan region, limited information exists on its impact, particularly, on the belowground microbial diversity. The present study was thus conducted to compare the soil microbial (bacterial and ascomycetous fungal) diversity between the sites invaded by C. sumatrensis and un-invaded (control) sites. Soil metagenome was extracted from C. sumatrensis invaded and un-invaded plots at the three study sites. A total of six plots (5 × 5 m each in size), including three invaded by C. sumatrensis and three un-invaded plots were nested within each study site. DNA after amplification was subject to denaturing gradient gel electrophoresis (DGGE); the bands were extracted from the DGGE gel, re-amplified, and sequenced for identification of the species. The number of bacterial species was reduced in the invaded plots at two out of the three sites while as it was relatively higher in the un-invaded plots with many species exclusively found in these plots. Fungal species richness was higher in the invaded plots compared to the un-invaded plots at all the three sites. Also, more fungal species were found to occur exclusively in the invaded plots without being represented in the un-invaded plots. Invasion by C. sumatrensis alters soil microbial community structure in the urban ecosystems in the Kashmir Himalaya. How this species does so and what benefits does it draw from such alteration promise to be an interesting future discourse.

Relationships between ruminal Gram-positive bacteria and methane from lactating dairy cows supplemented with monensin and tallow, alone or in combination

The aim of this study was to explore the relationships between a Gram-positive bacterial population and methane production in the rumen of dairy cows supplemented with monensin and tallow, alone or in combination. Under each treatment condition, the following ruminal fermentation parameters were measured: pH, oxidation-reduction potential (ORP), VFA, and methane production. Quantification of Gram-positive bacteria present in the rumen was performed by amplifying the 16S rDNA gene using PCR. Diversity analysis of Gram-positive bacteria was performed by PCR–denaturing gradient gel electrophoresis (DGGE), and bacterial taxonomy data were obtained by bioinformatics tools. Neither additive affected the pH or ORP, but they both reduced the total quantity of VFA. Supplementation with monensin, tallow, or their combination caused a decrease in methane production compared to the control diet. The best treatment to reduce methane production was monensin, with a reduction of 7.2% compared to the control diet. Supplementation with monensin or tallow did not affect the bacterial population. The Shannon diversity index was higher with monensin supplementation than with the other treatments. The analysis of each DGGE microbial community by the unweighted pair group method with averaging (UPGMA) revealed two clusters, one group with the control and monensin diets and the other with the tallow and combination diets. Taxonomic analysis of the dominant bands from the DGGE gel with the use of the Ribosomal Database Project (RBP) revealed that the Ruminococcaceae family was predominant, followed by the Lachnospiraceae family. Correspondence analysis (CA) suggested that both were negatively correlated with methane production in all treatments.

Different substrates and starter inocula govern microbial community structures in biogas reactors

ABSTRACTThe influence of different starter inocula on the microbial communities in biogas batch reactors fed with fresh maize and maize silage as substrates was investigated. Molecular biological analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rRNA gene fragments showed that each inoculum bore specific microbial communities with varying predominant phylotypes. Both, bacterial and archaeal DGGE profiles displayed three distinct communities that developed depending on the type of inoculum. Although maize and silage are similar substrates, different communities dominated the lactate-rich silage compared to lactate-free fresh maize. Cluster analysis of DGGE gels showed the communities of the same substrates to be stable with their respective inoculum. Bacteria-specific DGGE analysis revealed a rich diversity with Firmicutes being predominant. The other abundant phylotypes were Bacteroidetes and Synergistetes. Archaea-specific DGGE analysis displayed less diverse community structures, identifying members of the Methanosarcinales as the dominant methanogens present in all the three biogas digesters. In general, the source of inoculum played a significant role in shaping microbial communities. Adaptability of the inoculum to the substrates fed also influenced community compositions which further impacted the rates of biogas production.

Variations in bacterial communities during foliar litter decomposition in the winter and growing seasons in an alpine forest of the eastern Tibetan Plateau.

Bacterial communities are the primary engineers during litter decomposition and related material cycling, and they can be strongly controlled by seasonal changes in temperature and other environmental factors. However, limited information is available on changes in the bacterial community from winter to the growing season as litter decomposition proceeds in cold climates. Here, we investigated the abundance and structure of bacterial communities using real-time quantitative PCR and denaturing gradient gel electrophoresis (DGGE) during a 2-year field study of the decomposition of litter of 4 species in the winter and growing seasons of an alpine forest of the eastern Tibetan Plateau. The abundance of the bacterial 16S rRNA gene was relatively high during decomposition of cypress and birch litter in the first winter, but for the other litters 16S rRNA abundance during both winters was significantly lower than during the following growing season. A large number of bands were observed on the DGGE gels, and their intensities and number from the winter samples were lower than those from the growing season during the 2-year decomposition experiment. Eighty-nine sequences from the bands of bacteria that had been cut from the DGGE gels were affiliated with 10 distinct classes of bacteria and an unknown group. A redundancy analysis indicated that the moisture, mass loss, and elemental content (e.g., C, N, and P) of the litter significantly affected the bacterial communities. Collectively, the results suggest that uneven seasonal changes in climate regulate bacterial communities and other decomposers, thus affecting their contribution to litter decomposition processes in the alpine forest.

MICROBIAL COMMUNITY ANALYSIS IN ANAEROBIC PALM OIL MILL EFFLUENT (POME) WASTEWATER BY DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE)

Microorganisms play a key role in wastewater bio-treatment processes and understanding the microbial community structure is of great importance to improve treatment performance. Denaturing gradient gel electrophoresis (DGGE) was used to monitor succession of the microbial community and thus predominant bands were sequenced to reveal the microbial community composition inside palm oil mill effluent (POME) wastewater.DNA bands from DGGE gels were excised with a sterile blade and placed in 1.5 ml eppendorf tube containing 50 μl deionized water (ddH2O). Tubes were incubated overnight at 4C to elute the DNA. Eluted DNA was purified using QIAquick gel extraction kits (QIAGEN, Inc., Valencia, CA) and was frozen and thawed three times.Microbial DNA successfully excised and purified from DGGE was amplified using polymerase chain reaction (PCR).Five micro liters of the supernatant were used as a template to re-amplify the DNA using 16s rDNA primers,341f (with no GC-clamp) (5'- cct-acg-gga-ggc-agc-ag-3') and reverse(r) primers 907r (5'-ccc-cgt-caa-ttc-att-tga-gtt-t-3'). Amplification was repeated referring to the steps in ‘PCR amplification of 16s rDNA’. PCR products from agarose gels were cut and purified using QIAquick Gel Extraction Kit (QIAGEN, Inc., Valencia, CA), which were similar to the purification steps after recovery of DNA from DGGE, and sequenced in both directions with the same primers (with no GC-clamp) as used in PCR. Moreover, start-up is an important step in establishing proper community structure in all kinds of biological treatment processes. In anaerobic POME wastewater, 6 sequences of Firmicutes, 5 sequence of Proteobacterium and 2 sequences of Bacteroidetes were found through denaturing gradient gel electrophoresis (DGGE) results. Sequence closely related to Rummeliibacillus suwonensis strain G20 was detected grows at band BE10, BE11, BE12, BE15, BE16, BE17 and BE18. Meanwhile BE25 and BE26 were found at ban d after treatment process done.Rummeliibacillus suwonensis is an aerobic, Gram-positive, rod shaped, round-spore-forming bacteria which were isolated from aerobic condition.

Identification of predominant yeasts associated with artisan Mexican cocoa fermentations using culture-dependent and culture-independent approaches.

The process of cocoa fermentation is a very important step for the generation or aromatic compounds, which are attributable to the metabolism of the microorganisms involved. There are some reports about this process and the identification of microorganisms; however, there are no reports identifying the yeasts involved in a Mexican cocoa fermentation process using molecular biology techniques, including restricted fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE). The aim of this study was to identify the main yeast species associated with Mexican cocoa fermentations employing culture-dependent and -independent techniques achieving two samplings with a 1 year time difference at the same site. Isolation of the microorganisms was performed in situ. Molecular identification of yeast isolates was achieved by RFLP analysis and rDNA sequencing. Total DNA from the microorganisms on the cocoa beans was utilized for the DGGE analysis. Bands from the DGGE gels were excised and sequenced. Nineteen isolated yeasts were identified (al specie level), three of which had never before been associated with cocoa fermentations worldwide. The detected predominant yeast varied from one technique to another. Hanseniaspora sp. resulted dominant in DGGE however Saccharomyces cerevisiae was the principal isolated species. In conclusion, the culture-dependent and -independent techniques complement each other showing differences in the main yeasts involved in spontaneous cocoa fermentation, probably due to the physiological states of the viable but non culturable yeasts. Furthermore important differences between the species detected in the two samplings were detected.

Simultaneous detection and discrimination of Pratylenchus and Meloidogyne species in Japanese fields using group-specific primers and denaturing gradient gel electrophoresis

We developed a simultaneous detection and discrimination method for the main Pratylenchus and Meloidogyne species of nematodes inhabiting Japanese fields. The method consists of a single polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) process using group-specific primers (SNem 1–4) constructed from the 18S rDNA region with high specificity toward these nematodes. Optimal electrophoresis conditions to differentiate PCR products of each species were 60 °C at a constant voltage of 70 V for 17 h using DGGE gel, with a 6 % acrylamide concentration and a denaturant gradient ranging from 25 to 41 %. Differentiation of most species was achieved according to their main band positions. Only bands derived from the Pratylenchus and Meloidogyne species were present in the DGGE profiles of many nematode communities from various locations in Japan, so we concluded that these primers were sufficiently specific to the targeted nematodes. Detection sensitivity was so high that we could detect one second-stage juvenile of Pratylenchus species among 1,000 free-living nematodes using a single PCR step (35 cycles). Even when two or more target species coexisted in a community, we could usually detect and distinguish them accurately. Most target species formed some minor bands in addition to the primary band. Any interference of the minor bands with the main bands of other target species was overcome using optimal denaturing gradient conditions for the electrophoresis.

Methanogenic archaea diversity in hyporheic sediments of a small lowland stream

Abundance and diversity of methanogenic archaea were studied at five localities along a longitudinal profile of a Sitka stream (Czech Republic). Samples of hyporheic sediments were collected from two sediment depths (0–25 cm and 25–50 cm) by freeze-core method. Methanogen community was analyzed by fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and sequencing method. The proportion of methanogens to the DAPI-stained cells varied among all localities and depths with an average value 2.08 × 105 per g of dry sediment with the range from 0.37 to 4.96 × 105 cells per g of dry sediment. A total of 73 bands were detected at 19 different positions on the DGGE gel and the highest methanogen diversity was found at the downstream located sites. There was no relationship between methanogen diversity and sediment depth. Cluster analysis of DGGE image showed three main clusters consisting of localities that differed in the number and similarity of the DGGE bands. Sequencing analysis of representative DGGE bands revealed phylotypes affiliated with members belonging to the orders Methanosarcinales, Methanomicrobiales and Methanocellales. The knowledge about occurrence and diversity of methanogenic archaea in freshwater ecosystems are essential for methane dynamics in river sediments and can contribute to the understanding of global warming process.

Agarose gel purification of PCR products for denaturing gradient gel electrophoresis results in GC-clamp deletion.

The 16S ribosomal RNA (rRNA) gene of marine archaeal samples was amplified using a nested PCR approach, and the V3 region of 16S rRNA gene of crab gut microbiota (CGM) was amplified using the V3 universal primer pair with a guanine and cytosine (GC)-clamp. Unpurified PCR products (UPPs), products purified from reaction solution (PPFSs), and products purified from gel (PPFGs) of above two DNA samples were used for denaturing gradient gel electrophoresis (DGGE) analysis, respectively. In contrast to almost identical band patterns shared by both the UPP and PPFS, the PPFGs were barely observed on the DGGE gel for both the marine archaea and CGM samples. Both PPFS and PPFG of CGM V3 regions were subjected to cloning. A small amount of positive clones was obtained for PPFS, but no positive clones were observed for PPFG. The melt curve and direct sequencing analysis of PPFS and PPFG of E. coli V3 region indicated that the Tm value of PPFG (82.35 ± 0.19 °C) was less than that of PPFS (83.81 ± 0.11 °C), and the number of shorter GC-clamps was significant higher in PPFG than in PPFS. The ultraviolet exposure experiment indicated that the ultraviolet was not responsible for the deletion of the GC-clamps. We conclude that the gel purification method is not suitable for DGGE PCR products or even other GC-rich DNA samples.

Metabolic activity and genetic diversity of microbial communities inhabiting the rhizosphere of halophyton plants

A preliminary study was conducted to compare the community level physiological profile (CLPP) and genetic diversity of rhizosphere microbial communities of four plant species growing nearby Kiskunság soda ponds, namely Böddi-szék, Kelemen-szék and Zab-szék. CLPP was assessed by MicroResp method using 15 different substrates while Denaturing Gradient Gel Electrophoresis (DGGE) was used to analyse genetic diversity of bacterial communities. The soil physical and chemical properties were quite different at the three sampling sites. Multivariate statistics (PCA and UPGMA) revealed that Zab-szék samples could be separated according to their genetic profile from the two others which might be attributed to the geographical location and perhaps the differences in soil physical properties. Böddi-szék samples could be separated from the two others considering the metabolic activity which could be explained by their high salt and low humus contents. The number of bands in DGGE gels was related to the metabolic activity, and positively correlated with soil humus content, but negatively with soil salt content. The main finding was that geographical location, soil physical and chemical properties and the type of vegetation were all important factors influencing the metabolic activity and genetic diversity of rhizosphere microbial communities.

Bacterial community in the intestine of the sea urchin Strongylocentrotus intermedius during digestion of Macrocystis pyrifera

Denaturing gradient gel electrophoresis (DGGE) was used to study the bacterial community changes in the intestine of the sea urchin Strongylocentrotus intermedius during the digestion of Macrocystis pyrifera. The distinct bands in DGGE gels were sequenced, and the results indicated that the bacterial community in the large and small intestine varied at different periods of digestion. Samples from the large intestine included six specific bands belonging to the genus Psychromonas, whereas samples from the small intestine included eight specific bands representing Psychromonas, Shewanella, Saccharophagus degradans, and Nitrosomonas eutropha. The bacterial flora differed at different periods of digestion. The increase in the microbial community species in the large intestine was not obvious compared with that in the small intestinal microbial community. Several microbes involved in degradation of M. pyrifera were found in the intestine of sea urchin.

Suppression of Listeria monocytogenes by the Native Micro-Flora in Teewurst Sausage.

Modern consumers are interested in the use of non-chemical methods to control pathogens when heat sterilization is not an option. Such is the case with teewurst sausage, a raw spreadable sausage and a popular German commodity. Although Listeria was not found in teewurst, the optimal microbial growing conditions of teewurst coupled with the ubiquity of L. monocytogenes in nature, makes the possibility of contamination of products very possible. This pilot study was conducted to examine teewurst’s native micro-flora’s ability to suppress the outgrowth of L. monocytogenes at 10 °C using standard plate counts and PCR-DGGE. Traditional plating methods showed L. monocytogenes growth significantly decreased when in competition with the teewurst’s native micro-flora (p < 0.05). The native micro-flora of the teewurst suppressed the overall growth of L. monocytogenes by an average of two logs, under these conditions. Denaturing Gradient Gel Electrophoresis (DGGE) amplicons with unique banding patterns were extracted from DGGE gel for identification. Brochothrix thermosphacta and Lactobacillus curvatus were identified as a part of the teewurst’s native micro-flora. Although the native micro-flora did not decrease L. monocytogenes to below limits of detection, it was enough of a decrease to warrant further investigation.

A New Framework to Accurately Quantify Soil Bacterial Community Diversity from DGGE

Denaturing gradient gel electrophoresis (DGGE) has been and remains extensively used to assess and monitor the effects of various treatments on soil bacterial communities. Considering only abundant phylotypes, the diversity estimates produced by this technique have been proven to be uncorrelated to true community diversity. The aim of this paper was to develop a framework to estimate a community's true diversity from DGGE. Developed using in silico DGGE profiles generated from published pyrosequencing datasets, this framework elongates the rank-abundance distributions (RADs) drawn by band quantification using the peak-to-signal ratio (PSR) parameter, which was proven to be related to bacterial richness. The ability to compare DGGE-based diversity estimates to the true diversity of communities led to a unique opportunity to identify potential pitfalls when analyzing DGGE gels with commercial analysis software programs and gain insight into the process of DNA band clustering in the profiles. Bacterial diversity was compared through richness, Shannon, and Simpson's 1/D indices. Intermediate results demonstrated that, even though commercial gel analysis software programs were unable to produce consistent results throughout all samples, a newly developed Matlab-based framework unraveled the dominance profiles of communities from band quantification. Elongating these partial RADs using the PSRs extracted from the DGGE profiles chiefly made it possible to accurately estimate the true diversity of communities. For all the samples analyzed, the estimated Shannon and Simpson's 1/D were accurate at ±10 %. Richness estimations were less accurate, ranging from -11 to 31 % of the expected values. The framework showed great potential to study the structure and diversity of soil bacterial communities.

Spatial heterogeneity of cyanobacteria-inoculated sand dunes significantly influences artificial biological soil crusts in the Hopq Desert (China)

Artificial biological soil crusts (ABSCs), formed by inoculating Microcoleus vaginatus Gom. and Scytonema javanicum Born. et Flah. onto the topsoil of desert dunes, proved to be effective tools for the stabilization of moving dunes and promotion of soil fertility. As dominant driving forces in arid habitats, the abiotic environmental conditions of undulating dunes produce a gradient of abiotic stresses on cyanobacteria. Cyanobacteria are considered pioneering phototrophs in early soil crust communities in deserts. In this study, the development of ABSCs under various environmental site conditions was investigated using 16S rRNA-based polymerase chain reaction, denaturing gradient gel electrophoresis (DGGE), and soil property measurements. After inoculation in 2002 and long-term development, patchy moss crusts were observed on the dunes. All of the available sequenced bands in the DGGE gels belonged to Oscillatoriales and Nostocales. The dominant Nostocales genus in the ABSCs was still Scytonema; however, more Oscillatoriales genera were identified, which belonged to Microcoleus and Phormidium. The cyanobacterial compositions of different slope types were significantly distinct (p < 0.05), particularly those from windward slopes. The crusts of the top-dune slopes were more heterogeneous. In addition, the soil physicochemical properties and richness indices of the windward slopes were significantly lower than those of the leeward and interdune slopes (p < 0.05). Compared with uninoculated control dunes, all of the inoculated dunes had far higher biodiversities.

Evaluating Primers for Profiling Anaerobic Ammonia Oxidizing Bacteria within Freshwater Environments

Anaerobic ammonia oxidizing (anammox) bacteria play an important role in transforming ammonium to nitrogen gas and contribute to fixed nitrogen losses in freshwater environments. Understanding the diversity and abundance of anammox bacteria requires reliable molecular tools, and these are not yet well established for these important Planctomycetes. To help validate PCR primers for the detection of anammox bacteria within freshwater ecosystems, we analyzed representative positive controls and selected samples from Grand River and groundwater sites, both from Ontario, Canada. The objectives of this study were to identify a suitable anammox denaturing gradient gel electrophoresis (DGGE) fingerprint method by using GC-clamp modifications to existing primers, and to verify the specificity of anammox-specific primers used for DGGE, cloning and qPCR methods. Six primer combinations were tested from four published primer sets (i.e. A438f/A684r, Amx368f/Amx820r, An7f/An1388r, and Pla46/1392r) for both direct and nested PCR amplifications. All PCR products were run subsequently on DGGE gels to compare the resulting patterns. Two anammox-specific primer combinations were also used to generate clone libraries and quantify anammox bacterial 16S rRNA genes with qPCR. The primer set A438f/A684r was highly specific to anammox bacteria, provided reliable DGGE fingerprints and generated a high proportion of anammox-related clones. A second primer set (Amx368f/Amx820r) was anammox specific, based on clone library analysis, but PCR products from different candidate species of anammox bacteria resolved poorly using DGGE analysis. Both DGGE and cloning results revealed that Ca. Brocadia and an uncharacterized anammox bacterial cluster represented the majority of anammox bacteria found in Grand River sediment and groundwater samples, respectively. Together, our results demonstrate that although Amx368f/Amx820r was useful for anammox-specific qPCR and clone library analysis, A438f/A684r was the most suitable primer set for multiple molecular assessments of anammox bacteria in freshwater environments.