Denaturing Gradient Gel Electrophoresis Analysis Research Articles

Use of PCR-DGGE-Based Molecular Methods to Analyze Nematode Community Diversity.

DGGE (denaturing gradient gel electrophoresis) is a nucleic acid separation technique applied to the evaluation of microbial biodiversity. This technique is quite rapid and cheap compared to other types of analysis. Here we describe the comparison of nematode communities inhabiting different ecosystems. After an ecologically representative sampling collection and the nematode extraction from soil, nematodes are centrifuged in Eppendorf tubes to facilitate DNA extraction. DNA from the whole community of each type of soil is extracted, amplified with primers for 18 S rDNA and used in DGGE analysis. The profiles of DGGE can be analyzed with appropriate software, and biodiversity indices can be estimated.

Mangroves: A hotspot for novel bacterial and archaeal diversity

Mangrove ecosystems are highly productive ecosystems that harbor diverse microbial lineage playing multiple roles in nutrient cycling. Though several novel mangrove rhizospheres associated with bacterial genera and species have been reported from these unexplored wetland ecosystems, but yet to be studied for their richness and diversity including actinobacterial and archaeal communities. Therefore, this study was undertaken to decode the bacterial and archaeal communities associated with the rhizosphere regions of true mangrove species-Avicennia marina and Rhizophora mucronata and their intersecting regions; and associated halophytic species -Suaeda maritima, and Salicornia brachiata of Pichavaram mangroves, Tamil Nadu, India. Amplified ribosomal DNA restriction analysis (ARDRA) and PCR denaturing gradient gel electrophoresis (PCR-DGGE) analysis techniques were adopted. About 567 culturable bacterial isolates with distinct morphology were purified, and nearly 88 distinct Operational Taxonomic Units (OTUs) were identified by ARDRA analysis. Phylogenetic analysis of representative groups indicated that the majority of the isolates belonged to Firmicutes (10 genera), followed by Proteobacteria- Alphaproteobacteria (10 genera) and Gammaproteobacteria (8 genera), Actinobacteria (8 genera), Bacteroidetes (1genera), and Halomonas (9 genera) representing 38 genera with 105 species. In addition, a few phylogenetically distinct novel groups belonging to the genera Mangrovibacter, Vibrio, Demequina, and Catenococcus were also isolated. PCR-DGGE with the RDP-Classifier program identified the prominent DGGE bands up to genus level namely Bacillus, Pseudomonas, Vibrio, Halomonas, etc. In addition, this study for the first time describes the genera Bhargavaea, Exiguobacterium, Hoeflea, Catenococcus, Albirhodobacter, Dietzia, Brachybacterium, Aerococcus, and Myroides association with mangrove rhizospheres. Likewise, DGGE analysis of archaea represented 2 major groups such as Crenarchaeota and Euryarchaeota, which were further classified into 7 different genera.

Bacterial and fungal community composition and community-level physiological profiles in forest soils.

We characterized the potential functioning and composition of the bacterial and fungal communities in the O and A horizons of forest soils using community-level physiological profile (CLPP) based on BIOLOG analysis, and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S and 18S rDNA fragments, respectively. In addition, relationships between the potential functioning and the community composition in each horizon, and between the O and A horizons, were assessed using Procrustes analysis. For the bacterial and fungal communities, the CLPP and DGGE profile were clearly separated between the O and A horizons in a principal coordinate analysis except for the fungal CLPP. No significant links for CLPP and DGGE profile between the O and A horizons were observed for either bacterial or fungal communities, suggesting that different factors had considerable influence on the microbial communities between the O and A horizons. Significant couplings between bacterial and fungal DGGE profiles (p <0.05 for O horizon; p <0.01 for A horizon), and between bacterial and fungal CLPPs (p = 0.001 for O horizon; p <0.01 for A horizon), were observed in the O and A horizons, implying that common factors strongly influenced the bacterial and fungal communities in each horizon. Although a significant correlation was observed between bacterial community composition and the potential functioning in the A horizon (p <0.01), such a correlation was not observed for the fungal community in the A horizon, and for the bacterial and fungal communities in the O horizon. This finding suggested that potential functioning, which would reflect only rapidly growing microorganisms, was not strongly associated with the composition of the entire microbial community. Further studies are needed to unravel the factors shaping the composition and functioning of microbial communities in forest soils.

The effect of auxin status driven by bacterivorous nematodes on root growth of Arabidopsis thaliana

Bacterivorous nematodes are one of the dominant microbial organisms and play an important role in shaping the bacterial community and hormone dynamics in rhizosphere soils. It has been found that the presence of bacterivorous nematodes potentially regulate indole-3-acetic acid (IAA) content in soils by interacting with the status of IAA-producing bacteria, and eventually act on the growth of plant roots. However, the mechanism behind this interaction in broad soil ecosystems remains unknown. This study reveals the interaction between bacterivorous nematodes and the soil bacterial community, especially IAA-producing bacteria, based on denaturing gradient gel electrophoresis (DGGE) analysis. The results of DGGE showed that the bacterial diversity in soil was increased by inoculation of nematodes during certain periods of plant culture, and was also affected by the genotype of the Arabidopsis thaliana (Johannes Thal, 1577) plants. The variation in soil bacterial communities among the treatments with different genotypes of A. thaliana and nematodes indicated that the soil bacterial community structure was affected by both genotype and nematode grazing. Furthermore, we measured the root architectures of four genotypes of A. thaliana, including wild type (Col-0), two IAA-insensitive mutants (Axr5 and Axr3–1), and DR5::GUS transgenic type (IAA sensitive) under different conditions of bacterivorous nematode inoculation. We found that the root architecture of the IAA-sensitive A. thaliana developed more tips and slender roots under inoculation with activated bacterivorous nematodes, while the root architectures of auxin-insensitive A. thaliana mutants showed no significant differences between alive and inactivated bacterivorous nematodes, even though the IAA content increased in the soil under inoculation with alive nematodes. This study confirms that the presence of bacterivorous nematodes in soils increases the soil IAA content significantly by regulating the bacterial community structure, which leads to an accumulation of auxin in the root tips of A. thaliana, and eventually enhances the plant root growth.

Molecular Analysis of Soil Bacterial Community Structures for Environmental Risk Assessment with Varieties of Genetically Modified Soybean and Hot Pepper

With the advance in gene technology, genetically modified (GM) crops have increased in recent years. GM crops offer us various benefits. However, there are potential risks of GM crops for the environment. In this study, the impacts of transgenic plants on soil microbial community structures were assessed. Two varieties of soybean (Glycine max L.) and hot pepper (Capsicum annuum L.), which introduced the herbicide-resistant gene, bar, were used in this study. The effects of GM crops on soil microbial community structures were investigated using a cultural method, the denaturing gradient gel electrophoresis (DGGE) procedure, and 16S rRNA gene sequencing on the Illumina platform. Additionally, the persistence of transgenes was monitored using a quantitative real-time PCR procedure. The cultural method, DGGE analysis, and the amplicon-based community profile indicated that soil microbial communities were not significantly different between GM and non-GM lines. The level of the bar gene in GM soybean plots greatly increased when the crops were actively growing, but thereafter gradually decreased to the initial level. Meanwhile, the level of the bar gene in GM hot pepper plots repeatedly increased and decreased according to the flowering stages. These results indicated that soil microbial community structures were not significantly affected.

Antiobesity, Antihyperglycemic, and Antidepressive Potentiality of Rice Fermented Food Through Modulation of Intestinal Microbiota.

The present study has been aimed at evaluating the antiobesity, antihyperglycemic, and antidepressive potentials of Asparagus racemosus starter-based rice fermented foods. High-throughput NGS technology has revealed a number of bacterial genera in the prepared fermented rice, such as Lactobacillus (29.44%), Brevundimonas (16.21%), Stenotrophomonas (6.18%), Pseudomonas (3.11%), Bacillus (2.88%), and others (<2%). Eight-week administration of rice fermented food has increased food intake, whole-body weight, organ weight, different fat masses, serum lipid profiles, and histology of liver and adipose tissues in HFD-induced obese mice. In addition, upregulation of fatty acid oxidation and downregulation of adipocytogenesis- and lypogenesis-related genes along with the expression of their regulatory nuclear factors such as PPARα, PPARγ, PPARδ, and SREBP-1c have also been noted. Moreover, fermented food decreases fasting blood glucose level and improves glucose and insulin tolerance as well as the expression of GLUT4 receptor. Antiobesity and antihyperglycemic effects are also supported by the changes in insulin, leptin, and adiponectin hormone levels. The real-time polymerase chain reaction (RT-PCR) and denaturing gradient gel electrophoresis (DGGE) analyses have clearly demonstrated the intense colonization of Bacteroides, Lactobacillus, and Bifidobacterium, as well as the suppressed growth rate of γ- and δ-Proteobacteria and Firmicutes in the gut after fermented food intake. In the intestine, the latter group of microorganisms possibly modulate short-chain fatty acid (SCFA) levels such as acetate, butyrate, and propionate more than twofold. The impairment of memory-learning and anxiety-like obesity-associated cognitive phenotypes is mitigated significantly (p < 0.01) by fermented food as well. Thus, the formulated fermented food could be used as a natural therapeutic to alleviate obesity and its associated psychological and pathophysiological ailments.

Assessment of Bacterial Communities Within the Biofilm of Bladder Calculi in the Neurogenic Bladder Rat Model Following Spinal Cord Injury.

PurposeTo develop a rat model of bladder calculi in the neurogenic bladder following spinal cord injury (SCI) and assess bacterial communities within the biofilm of bladder calculi using denaturing gradient gel electrophoresis (DGGE).MethodsThe silk tied to a small segment of the Teflon IV catheter was implanted through the urethra into the bladder of rats with SCI induced by T9 laminectomy. After 6 months, the rats were sacrificed and their bladder calculi were collected by opening the bladders through the low-midline incision. Genomic DNA was extracted from the biofilm of bladder calculi followed by DGGE to obtain bacterial DNA. The DNA sequences were compared and analyzed using BLAST (Basic Local Alignment Search Tool) to identify bacteria.ResultsAfter placing silk nidus in the bladder for 6 months, all 6 rats developed bladder calculi. According to DGGE analysis, Pseudomonas aeruginosa was the most dominant strain, while Clostridium sp. and Lactobacillus sp. were relatively dominant strains within the biofilm of bladder calculi in the rats with SCI.ConclusionsDGGE analysis showed various microorganisms in the biofilm of calculi arising from a neurogenic bladder rat model. This research design can be the basis for clinical studies and may be applied to calculi in patients with neurogenic bladder following SCI.

Easy Removal of Epiphytic Bacteria on Ulva (Ulvophyceae, Chlorophyta) by Vortex with Silica Sands.

Macroalgae-associated bacteria play an important role in their algal hosts’ biological processes. They are localized on surfaces of the host thalli, as well as between and even within algal cells. To examine the differences in community structures and functions between epi- and endo- bacteria, an effective approach for maximizing epiphyte removal from delicate seaweeds while retaining endophyte fidelity must be developed. In this study, a variety of surface sterilization methods for Ulva prolifera were compared, including mechanical, chemical, and enzymatical treatments. According to the results of scanning electron microscope (SEM) and denaturing gradient gel electrophoresis (DGGE) analysis, almost complete removal of epiphytic bacteria on Ulva was obtained simply by co-vortex of seaweeds with silica sands, causing minimal disturbance to endosymbionts when compared to previous published methods. In addition, the adaptability was also confirmed in additional U. prolifera strains and Ulva species with blade-like or narrow tubular thallus shapes. This easy mechanical method would enable the analysis of community composition and host specificity for Ulva-associated epi- and endo-bacteria separately.

Effect of Jhum Cycles on &lt;i&gt;Acidobacteria&lt;/i&gt; and &lt;i&gt;Burkholderia&lt;/i&gt; Community in the Rhizosphere of Mixed Crops of Different Jhum Fields

In the present study, culture-dependent and independent [Denaturing gradient gel electrophoresis (DGGE)] approach were used to analyze the effect of fallow cycle on bacterial populations associated with mixed crops selected from three different jhum fallow cycles (5-, 10- and 20- years) of Changki village, Nagaland. Two important bacterial phyla viz. Acidobacteria and Burkholderia were selected for DGGE analysis. According to the DGGE banding profile, Acidobacteria showed more richness compared Burkholderia. The presence and absence of few of the bands in the profile indicate the community shifts in the selected jhum fields. Shannon diversity index was higher in Acidobacteria than Burkholderia. Multivariate analysis showed that Acidobacteria community structure was similar among the crop plants of the same fallow cycle. Burkholderia community did not show clear separation among fallow cycles although crop of same fallow cycle tend to group together. ANOSIM and PERMANOVA showed significant differences in rhizosphere community pattern among fallow cycles and the crop plants of same fallow cycles indicated that both fallow cycle and crop plants are factors determining the Acidobacteria and Burkholderia community composition in jhum soils which was also confirmed by the culture-dependent analysis. Culturable bacterial counts were dependent on media used and significantly different at different fallow cycles. Bacterial communities (both culturable and DGGE patterns) were correlated with soil nutrients suggesting their importance in stability of jhum soil. The present study revealed that both fallow cycle and host plant may be the key factors in shaping many bacterial communities in jhum soils.

Microbial Communities of Hydrothermal Guaymas Basin Surficial Sediment Profiled at 2 Millimeter-Scale Resolution.

The surficial hydrothermal sediments of Guaymas Basin harbor complex microbial communities where oxidative and reductive nitrogen, sulfur, and carbon-cycling populations and processes overlap and coexist. Here, we resolve microbial community profiles in hydrothermal sediment cores of Guaymas Basin on a scale of 2 millimeters, using Denaturing Gradient Gel Electrophoresis (DGGE) to visualize the rapid downcore changes among dominant bacteria and archaea. DGGE analysis of bacterial 16S rRNA gene amplicons identified free-living and syntrophic deltaproteobacterial sulfate-reducing bacteria, fermentative Cytophagales, members of the Chloroflexi (Thermoflexia), Aminicenantes, and uncultured sediment clades. The DGGE pattern indicates a gradually changing downcore community structure where small changes on a 2-millimeter scale accumulate to significantly changing populations within the top 4 cm sediment layer. Functional gene DGGE analyses identified anaerobic methane-oxidizing archaea (ANME) based on methyl-coenzyme M reductase genes, and members of the Betaproteobacteria and Thaumarchaeota based on bacterial and archaeal ammonia monooxygenase genes, respectively. The co-existence and overlapping habitat range of aerobic, nitrifying, sulfate-reducing and fermentative bacteria and archaea, including thermophiles, in the surficial sediments is consistent with dynamic redox and thermal gradients that sustain highly complex microbial communities in the hydrothermal sediments of Guaymas Basin.

Assessment of the migration of the operational taxonomic units (OTUs) from rice rhizosphere to endosphere

An experiment was conducted to understand the migration of the OTUs from rice rhizosphere to endosphere at vegetative and flowering stages of the crop. The soil and plant samples were studied through culture independent molecular PCR-DGGE technique involving denaturing gradient gel electrophoresis (DGGE) of PCR-amplified V3 region of 16S rRNA gene. The DGGE analysis revealed higher species richness in soil, followed by root, stem and leaf. The analyses for range-weighted richness and Shannon diversity index indicated vegetative stage carries more diverse microflora in soil and root samples than stem and leaf samples. The Sorenson’s similarity index indicates the migration of bacterial population from rhizosphere to endosphere. The soil samples shared about 28 to 35 % similarity with different plant compartments viz., root, stem and leaf at both crop stages. The fingerprinting of rhizosphere and endosphere samples would be a useful resource for plant microbe breeding through understanding of the associated microbial community.st

Sustainable management of paddy crop residues: effects on methanotrophs diversity and value for soil health restoration

AbstractIn recent years, improved paddy crop varieties, advanced tools and technologies have resulted in a huge quantity of crop residues (CRs) production. Hence, appropriate sustainable management of the paddy CRs produced annually is needed. In this study, a field experiment was conducted to evaluate the impact of rice husk biochar (RHB) amendments on soil physico‐chemical characteristics, methanotrophs diversity, and functional pmoA genes numbers in paddy soil. ThepmoA gene (27 kDa) encodes the β‐subunit of the particulate methane monooxygenase (pMMO), an enzyme responsible for methane oxidation found in most of the methanotrophs. A marked variation in methanotrophs diversity, functional pmoA genes abundance, and physico‐chemical parameters in paddy soil have been noted across the given amendments. The RHB significantly improves soil physico‐chemical status and enhances methanotrophs community structure and number of pmoA gene numbers in paddy soils. The various soil physico‐chemical parameters were improved due to RHB + microbial inoculant treatments. Maximum rice grain yield (5.91 ± 0.07 t ha−1) and methanotrophic functional pmoA gene numbers (57.25 ± 0.88 × 107 g−1 paddy soil) were found in the RHB + microbial inoculant treated plot compared to untreated (control) plot. The predominant methanotrophic bacteria identified by the denaturing gradient gel electrophoresis (DGGE) in RHB treated paddy soils were Methylovulum and Methylobacter members of the Methylococcaceae family. Consequently, the methanotrophs community study based on DGGE analysis shows that RHB could be a beneficial amendment to increase the methanotrophic community composition in paddy soils. Overall, the results suggest that the RHB amendment could markedly enhance soil conditions and methanotrophs abundance via regulating soil physico‐chemical properties in nutrient‐deprived and degraded paddy agriculture soils.

Evaluation of anaerobic and anoxic reactors installed in serial to treat fish tank effluent

Effluent from a fish tank with Nile tilapia (Oreochromis niloticus) was treated in a system of two parallel fixed-bed anaerobic reactors followed by a suspended stirred, anoxic reactor for solids, organic matter and nitrogen removal. Microbial community structure was compared between the reactors and an enriched anammox culture originated from anaerobic sludge by using the PCR (Polymerase Chain Reaction) and DGGE (Denaturing Gradient Gel Electrophoresis) techniques. DGGE analysis revealed three large microbial clusters, namely, the biomass in the inoculum, the biomass growing in the reactors, and the enriched anammox culture. The similarity between the microbial community recovered from the anaerobic reactors and the anammox community was considered low (32.7%), suggesting differentiation of the bacterial community as a function of the selection of specific anammox groups. The last step in the anoxic reactor had worsened the effluent quality. The potential of organic matter and ammonia removal in a single anaerobic reactor was demonstrated.

METAGENOMICS (16S AMPLICON SEQUENCING) AND DGGE ANALYSIS OF BACTERIAL DIVERSITY OF ACID MINE DRAINAGE

Acid mine drainages (AMDs) are characterized as low pH water containing metal, which is the best-known habitat for the microorganisms described as acidophiles. Most of these members do not survive above pH 4.0; therefore they are acidophilic. Because of these metabolic properties of acidophilic microorganisms, AMD is accepted as a model system in biogeochemical studies on iron and sulfur cycles for the analysis of microbial ecology.&#x0D; In this study, microbial diversity of the acid mine drainage in Çan (Çanakkale) was investigated by using culture-independent techniques including Denaturing Gradient Gel Electrophoresis (DGGE) and high-throughput sequencing.&#x0D; A total of 105,593,810 and 115,689,952 bases read were performed. Following sequencing, raw data were proceeded in “Quantitative Insights Into Microbial Ecology” (QIIME 1.9.1). Acidiphilium, Acidocella and Acidosoma were genus obtained by bioinformatic analysis. The highest rate belonged to Acidiphilium for each water sample. Acidiphilium cryptum, Acidithiobacillus sp. and Acidibacter ferrireducens were determined according to DGGE analysis.

Analysis of Soil Fungal Community Structure on the Surface of Buried Polyethylene Terephthalate

This study investigated soil fungal communities that develop on the surface of polyethylene terephthalate (PET) while buried in situ. PET samples prepared with file or alkali were buried in mountain soils for up to 9 months. The composition of fungal communities in native soil and on the surface of buried PET was compared using denaturing gradient gel electrophoresis (DGGE). DGGE and subsequent cluster analyses based on band patterns demonstrated the variability of fungal communities between native soil and PET surfaces. Burial for 3 months reduced diversity indices of soil on the surface of filed PET, yet soil on the surface of alkali-treated PET retained high diversity index. Cluster analyses show that fungal community structure was initially dependent on the difference in microstructure after surface treatment. This dependence later switched to macrostructure-the shape of PET samples. Principal component analysis (PCA) was performed to assess relationships between the surface structure of PET sheets and fungal diversity. Results show that hydrophilicity of PET contributed to differences in structures between alkali-treated PET and other PET samples.

Bacterial Community Composition in Produced Water of Diyarbakır Oil Fields in Turkey

Oil fields harbour a wide variety of microorganisms with different metabolic capabilities. To examine the microbial ecology of petroleum reservoirs, a molecular-based approach was used to assess the composition of bacterial communities in produced water of Diyarbakır oil fields in Turkey. Denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 16S rRNA gene fragments was performed to characterise the bacterial community structure of produced water samples and to identify predominant community members after sequencing of separated DGGE bands. The majority of bacterial sequences retrieved from DGGE analysis of produced water samples belonged to unclassified bacteria (50%). Among the classified bacteria, Proteobacteria (29.2%), Firmicutes (8.3%), Bacteroidetes (8.3%) and Actinobacteria (4.2%) groups were identified. Pseudomonas was the dominant genus detected in the produced water samples. The results of this research provide, for the first time, insight into the complexity of microbial communities in the Diyarbakır oil reservoirs and their dominant constituents.

Changes of Enzymatic Activities, Substrate Utilization Pattern, and Microbial Community Diversity in Heavy Metal-Contaminated Soils

Globally, heavy metal (HM) pollution of soil is a serious problem that can lead to long-term toxic effects on soil. In this milieu, the present study investigated the eco-toxicological effects of three trace elements, e.g., cadmium (Cd), copper (Cu), and lead (Pb), on enzyme activities and microbial function and structural diversity in phaeozem and red soil samples. Hormesis effects of Cd, Cu, and Pb on catalase and invertase activities were observed in phaeozem soil, while for red soil, there was an inhibitory effect on the activities of catalase and invertase under Cu- and Pb-contaminated soils. The utilization of carbon sources was inhibited in Cd- and Pb-treated phaeozem soil, but higher utilization of polymers and amines exhibited in Cu-contaminated soil. Although the substrates under the contamination of Cd, Cu, and Pb had high average well color development values across incubation time, the utilization of various substrates did not exhibit a regular trend under different treatments with HMs. The denaturing gradient gel electrophoresis (DGGE) analysis showed that the HMs led to marginal changes in the number and species of soil microbes, while the similarity indices decreased in HM-treated samples, varying from 66.2 to 77.3% in phaeozem soil and from 62.8 to 66.7% in red soil. However, the sequence analysis showed that there existed metal-resistant microbial communities such as Bacillales, Bacillus, and Massilia and so on under the stress of HMs.

Emulsion PCR (ePCR) as a Tool to Improve the Power of DGGE Analysis for Microbial Population Studies.

To the authors’ knowledge, this is the first report of the use of emulsion-Polymerase chain reaction (e-PCR) coupled with denaturing gradient gel electrophoresis (DGGE) analysis. In the present work the effectiveness of ePCR in improving the power of the DGGE technique for microbial population studies was tested. Our results indicated that ePCR results in uniform amplification of several DNA molecules, overcoming the major limitations of conventional PCR, such as preferential amplification and DNA concentration dependence. Moreover, ePCR-DGGE resulted in higher sensitivity when compared to conventional PCR-DGGE methods used for studying microbial populations in a complex matrix. In fact, compared to conventional PCR, the DGGE profiles of ePCR products permitted the detection of a higher number of the species that were present in the tested sample.

Use of radiofrequency electromagnetic radiation to remove biofilms from canvasses

A method based on the use of electromagnetic radiation (EMR) in the radiofrequency band was experimented to eliminate heterotrophic organisms from biofilms encrusting linen canvasses used for paintings. Artificial biofilms were used for exposure to EMR. Biofilms were grown on fragments of canvasses sampled from the backside of a painting. The fragments, after sterilization, were inoculated with several heterotrophic microorganisms previously isolated from other canvasses used for paintings and identified by molecular analyses as the bacteria Bacillus mycoides and Bacillus subtilis, and the microfungi Aspergillus versicolor, Cladosporium cladosporioides, Cladosporium oxysporum and Fusarium oxysporum. The newly formed biofilms resulted to contain the same microorganisms used for the inocula. They were exposed to EMR obtained by an amplitude-modulating radiofrequency sine wave with a train of rectangular pulses with 200 ns repetition time and 10% duty cycle. The exposure protocol consisted of three applications of two hours each, every other day. Denaturing gradient gel electrophoresis analyses carried out on the EMR-treated biofilms showed that all the microorganisms disappeared after the third application. The amplitude of the adopted EMR was not high enough to cause any significant increase in the temperature of the substrates. Therefore, the methodology showed to be non-invasive for the artefacts and its exposure levels not hazardous for operators and the environment compared to other techniques adopted in the field of conservation of cultural heritage. Among the latter techniques, the most common one is based on the use of biocides that, although effective, present problems in all the above-described areas.

Responses of Microbial Community to Di-(2-ethylhcxyl) Phthalate Contamination in Brown Soil.

Di-(2-ethylhcxyl) phthalate (DEHP) is applied as plasticizer, which results in the pollution of environment. In this study, the effects of DEHP on soil microbial functions, structure and genetic diversity were investigated. The concentration of DEHP in the soil were 0, 0.1, 1, 10 and 50 mg/kg, and the experimental period were 28 days. DEHP reduced the quantity, abundance, species dominance and homogeneity of soil microbes during the first 14 days. In addition, microbial utilization efficiency of carbon (carbohydrates, aliphatics, amino acids, metabolites) was impacted after 28 days, though the effects gradually weakened. Based on denaturing gradient gel electrophoresis and clone library analysis, in the presence of DEHP, the dominant microbes in the DEHP-contaminated soil were Sphingomonas and Bacillus, which belonged to the Acidobacteria and Proteobacteriav, respectively. With 0.1 or 1 mg/kg of DEHP, the relative abundances of Acidobacteria were higher, and with 10 or 50 mg/kg of DEHP, the relative abundances of Proteobacteria were higher.