PCR-denaturing Gradient Gel Electrophoresis Analysis Research Articles

Reductive deiodination of 2,4,6-triiodophenol by Vallitalea sp. strain TIP-1 isolated from the marine sponge

An anaerobic microbial consortium capable of reductively dehalogenating 2,4,6-triiodophenol (2,4,6-TIP) was enriched from the marine sponge Hymeniacidon sinapium. The enrichment reductively deiodinated 100μM of 2,4,6-TIP to 4-iodophenol (4-IP) and 2-iodophenol (2-IP) in the presence of sterile sponge tissue as the sole carbon source and electron donor. PCR-denaturing gradient gel electrophoresis and 16S rRNA gene sequence analysis revealed that bacteria closely related with Vallitalea guaymasensis and Oceanirhabdus sediminicola, both of which are members of the order Clostridiales, were predominant in the enrichment. When glucose was added to the enrichment as alternative carbon source, one of these bacteria grew predominantly, which was subsequently isolated as a pure culture. The strain, designated as TIP-1, showed 99.7% 16S rRNA gene sequence similarity with V. guaymasensis. In the presence of glucose, strain TIP-1 reductively deiodinated 2,4,6-TIP to 2-IP and 4-IP at a molar ratio of 3:1, during which 2,4-diiodophenol (2,4-DIP) and 2,6-diiodophenol (2,6-DIP) were observed as deiodinated intermediates. Glucose was required for 2,4,6-TIP deiodination, but 2,4,6-TIP was not essential for growth of strain TIP-1. The strain also deiodinated 2,4-DIP to 2-IP and 4-IP at a molar ratio of 1:1, and 2,6-DIP to 2-IP, but further deiodination of the monoiodophenols was not observed. These results suggest that strain TIP-1 removed both ortho- and para-substituted iodines equally. Such deiodinating bacteria could be applied to the mineralization or dehalogenation of triiodobenzene derivatives, which are widely used as X-ray contrast media.

Community structure of actively growing bacteria in a coastal fish-farming area.

In fish-farming areas, copious amounts of organic matter are released into the surrounding environment. Although it is well-known that bacterial community structures and activities are tightly coupled with organic conditions in the environment, actively growing bacteria (AGB) species that are responsible are still largely unknown. Here, we determined seasonal variations in the community structures of free-living and particle-attached AGB in surface and bottom seawater, and also in the easily resuspendable sediment boundary layer. Accordingly, we used bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) analysis. Whereas overall bacterial communities in the resuspendable sediment were quite different from those of the free-living and particle-attached bacteria, the AGB community structures were similar among them. This result suggests that sediment resuspension in aquaculture environments functions as an organic source for bacteria in the water column, and that bacterial species contributing to the environmental capacity and carbon cycle are limited. We identified 25 AGB phylotypes, belonging to Alphaproteobacteria (Roseobacter clade, nine phylotypes), Gammaproteobacteria (five phylotypes), Deltaproteobacteria (one phylotype), Bacteroidetes (seven phylotypes), and Actinobacteria (three phylotypes). Among them, some AGB phylotypes appeared throughout the year with high frequency and were also identified in other coastal environments. This result suggests that these species are responsible for the environmental capacity and carbon cycle, and are key species in this fish-farming area, as well as other coastal environments.

Development of an S-layer gene-based PCR-DGGE assay for monitoring dominant Lactobacillus helveticus strains in natural whey starters of Grana Padano cheese

Monitoring L. helveticus strain dynamics in natural whey starters is of great interest at the industrial level due to the key role that this bacterial population plays in Grana Padano cheese production. In this study, we aimed to develop a PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay based on the slpH locus, in parallel with performing culture-dependent analysis of whey samples using optimized media to maximize the number of isolated strains. We designed new primers targeting the slpH locus to amplify a gene region that would be suitable for PCR-DGGE analysis and discriminating strains. Our results confirmed that the developed PCR-DGGE method was rapid and reliable for monitoring the L. helveticus population in whey starter cultures. All sequences of bands detected in the PCR-DGGE profiles from whey samples showed high similarity to S-layer genes of L. helveticus, and perfectly matched with the slpH locus sequences of dominant strains. Overall, our findings indicated that the target region of the slpH locus was sufficiently heterologous to discriminate L. helveticus strains, and that our PCR-DGGE analysis provided a more accurate picture of the population composition of whey starters compared to culture-dependent techniques that often fail to isolate the most abundant strains.

Effect of β-1/3,1/6-glucan upon immune responses and bacteria in the gut of healthy common carp (Cyprinus carpio).

β-glucans are frequently included in the diet of healthy common carp Cyprinus carpio as a pre-emptive measure for combatting disease. In order to study the effect this has on the relationship between the gut bacteria and host immune response, carp were maintained on either a β-glucan free diet or feed containing 0.1% MacroGard®, a β-1/3, 1/6-glucan, for up to 7 weeks and analysis of innate immune gene expression and molecular analysis of the gut bacteria was performed. The data reveals feeding of MacroGard® to healthy carp does not induce bactericidal innate immune gene expression in the gut but does appear to alter bacterial species richness that did not have a negative effect on overall health. Analysis of innate immune gene expression within the upper midgut revealed that there were significant changes over time in the expression of Interleukin (il)-1β, inducible nitric oxide synthase (inos), mucin (muc2) and C-reactive protein (crp2). Diet did not affect the number of copies of the bacterial 16s rDNA gene in the gut, used as a as a measure of total bacteria population size. However, PCR-denaturing gradient gel electrophoresis (DGGE) analysis revealed a shift in bacterial species richness with MacroGard feeding. Bactericidal immune gene expression of crp2, muc2 and il-1β was weakly correlated with gut bacteria population size indicating a potentially limited role of these genes in interacting with the gut bacteria in healthy carp in order to maintain gut homeostatic conditions. These findings highlight the importance of considering both host immunity and the microbiome together in order to fully elucidate the effeect of immunomodulants, such as β-glucans, upon gut health.

Effects of the cultivable bacteria attached to Microcystis colonies on the colony size and growth of Microcystis

The interactions between bacteria and algae may play a significant part in the formation and development of algal blooms. The bloom-forming cyanobacterium Microcystis occurs mainly as colonial form in natural waters, and thus it is necessary to study the interaction between bacteria and colonial Microcystis. This paper aimed to investigate effects of the cultivable bacteria attached to Microcystis colonies on the colony size and growth of colonial Microcystis aeruginosa. Eleven bacterial strains were isolated from M. aeruginosa colonies collected from Lake Taihu. Among these bacteria, seven bacterial isolates significantly influenced the colony size of M. aeruginosa, and four bacterial isolates significantly influenced the growth rate of M. aeruginosa. Four isolates, related to the Exiguobacterium, Delftia, Bacillus and Stenotrophomonas, significantly decreased the colony size of M. aeruginosa by 36-51%, and two isolates, assigned to the Chryseobacterium and Pseudomonas chengduensis, significantly increased the M. aeruginosa colony size by 89% and 63%, respectively, while these isolates had no effect on the cyanobacterial growth rate. Two isolates, belonged to the Rheinheimera and Pseudomonas, significantly decreased the growth rate of M. aeruginosa by 47% and 36%, respectively, and one bacterial strain related to Aeromonas increased the cyanobacterial growth rate by 22%, while these isolates had no effect on the cyanobacterial colony size. One isolate belonged to Sphingomonas was found to significantly increase the colony size of M. aeruginosa by 80% and significantly decrease the growth rate of M. aeruginosa by 21%. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis showed that all of the bacterial isolates were able to colonize M. aeruginosa colonies. Our data suggested that the bacteria attached to Microcystis colonies might influence the cyanobacterial colony size and growth, and thus influence the formation and development of Microcystis blooms.

Cucumber (Cucumis sativus L.) Seedling Rhizosphere Trichoderma and Fusarium spp. Communities Altered by Vanillic Acid.

Root exudates mediate soil microbiome composition and diversity, which might further influence plant development and health. Vanillic acid from root exudates is usually referred as autotoxin of cucumber, however, how vanillic acid affect soil microbial community diversities and abundances remains unclear. In this study, vanillic acid (VA; 0.02, 0.05, 0.1, and 0.2 μmol g-1 soil) was applied to soil every other day for a total of five applications. We used Illumina MiSeq sequencing, quantitative PCR (qPCR) and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) to test the effects of VA on the total fungi community composition as well as the Trichoderma and Fusarium spp. community abundances and structures in the cucumber rhizosphere. Illumina MiSeq sequencing showed that VA (0.05 μmol g-1 soil) increased the relative abundance of the fungal phylum Basidiomycota while decreasing the relative abundance of Ascomycota (P < 0.05), and not altered the diversity of the soil fungal community. VA (0.05 μmol g-1 soil) also increased the relative abundances of the fungal genera with plant pathogens, such as Conocybe and Spizellomyces spp.(P < 0.05). A qPCR analysis showed that VA (0.05 to 0.2 μmol g-1 soil) exerted promoting effects on Trichoderma spp. community abundance and stimulated Fusarium spp. abundance at low concentrations (0.02 to 0.05 μmol g-1 soil) but inhibited it at high concentrations (0.1 to 0.2 μmol g-1 soil). The PCR-DGGE analysis showed that all concentrations of VA altered the community structures of Trichoderma spp. and that the application of VA (0.02 and 0.05 μmol g-1 soil) changed the band number and the Shannon-Wiener index of the Fusarium spp. community. This study demonstrated that VA changed the total fungal community in the cucumber seedling rhizosphere and that the Trichoderma and Fusarium spp. communities showed different responses to VA.

Influences of quorum-quenching probiotic bacteria on the gut microbial community and immune function in weaning pigs.

The aim of this study is to investigate the dynamic gut microbial diversity in weaning swine after administering feed supplemented with probiotic bacteria that specifically inhibit the activity of quorum molecules. Initially, the universal quorum molecule autoinducer-2 (AI-2) bioassay results indicated that AI-2 activity was profoundly inhibited in enterohemorrhagic Escherichia coli (EHEC) O157:H7 in the presence of Lactobacillus acidophilus strain 30SC cell extract, although the growth of EHEC was not affected. Based on plate counting results, bacterial community analysis revealed a specific reduction in coliforms compared to the control, whereas the population of lactobacilli increased in weaning swine in invivo trials. Supplementation with L.acidophilus strain 30SC did not affect the counts of other communities, such as total aerobes and yeast/mold. In addition, PCR-denaturing gradient gel electrophoresis analysis showed a significant difference in the 16S rRNA gene products after administering L.acidophilus strain 30SC. Selected bands were sequenced, and most of them were identified as uncultured bacterium clones or a Lactobacillus- and Bifidobacterium-specific community. Therefore, our results indicate that quorum-quenching probiotic bacteria can significantly modulate the gut microbiota of swine and these beneficial effects can contribute to the improvement of performance and health in the gastrointestinal tract of weaning pigs.

Effects of seven different companion plants on cucumber productivity, soil chemical characteristics and Pseudomonas community

AbstractCompanion cropping can influence cucumber productivity by altering soil chemical characteristics and microbial communities. However, how these alterations affect the growth of cucumber is still unknown. In this study, seven different plant species were selected as companion plants for testing their effects on cucumber productivity. The effects of different companion plants on changes in soil chemical properties such as electrical conductivity (EC) and contents of essential nutrients as well as the structure and abundance of the soil Pseudomonas community were evaluated. The results showed a higher cucumber yield in the wheat/cucumber companion system than that in the cucumber monocultured and other companion cropping systems. The lowest phosphorus (P) and potassium (K) contents in the soil were found in the cucumber monocultured system, and the highest NO3+-N and NH4+-N contents were observed in the rye/cucumber companion system. PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR analysis showed that the trifolium/cucumber companion system increased the diversity of the soil Pseudomonas community, while the chrysanthemum/cucumber companion system increased its abundance. Interestingly, plant-soil feedback trials revealed that inoculating the soil of the wheat/cucumber companion system increased the growth of cucumber seedlings. In conclusion, the effects of different companion plants on cucumber productivity, soil chemical characteristics and the soil Pseudomonas community were different, and wheat was a more suitable companion plant for increasing cucumber productivity. In addition, the altered microbial community caused by companion cropping with wheat contributed to increased cucumber productivity.

Analysis of stable 1,2-dichlorobenzene-degrading enrichments and two newly isolated degrading strains, Acidovorax sp. sk40 and Ralstonia sp. sk41.

Stable degrading 1,2-dichlorobenzene (1,2-DCB) enrichments were generated from original contaminated soil and groundwater via enrichment procedures using a mineral salt medium containing 1,2-DCB as the sole carbon and energy source. Four transferred enrichments showed stable 1,2-DCB-degrading ability and completely degraded 1,2-DCB within 32h. PCR-denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone library analyses indicated that two bacterial strains, belonging to Acidovorax spp. and Ralstonia spp., respectively, were the predominant organisms in each enrichment. Moreover, these strains maintained a stable coexistence in the four transferred enrichments. These two bacteria were subsequently identified as Acidovorax sp. strain sk40 and Ralstonia sp. strain sk41. Strain sk40 was more tolerant to higher concentrations of 1,2-DCB than strain sk41, while strain sk41 maintained a shorter degradation time under lower concentrations of 1,2-DCB. Notably, however, both strains exhibited similar growth rates and degradation rates in media containing 40mg/l 1,2-DCB, as well as complete degradation of the 1,2-DCB (40mg/l) within 32h. It is expected that these two strains will be used in future applications of bioremediation of 1,2-DCB contamination.

Microbial Diversity during Fermentation of Sweet Paste, a Chinese Traditional Seasoning, Using PCR-Denaturing Gradient Gel Electrophoresis.

The aim of this study was to elucidate the changes in the microbial community and biochemical properties of a traditional sweet paste during fermentation. PCR-denaturing gradient gel electrophoresis (DGGE) analysis showed that Aspergillus oryzae was the predominant species in the koji (the fungal mixture), and the majority of the fungi isolated belonged to two Zygosaccharomyces species in the mash. The bacterial DGGE profiles revealed the presence of Bacillus subtilis during fermentation, and Lactobacillus acidipiscis, Lactobacillus pubuzihii, Lactobacillus sp., Staphylococcus kloosi, and several uncultured bacteria were also detected in the mash after 14 days of main fermentation. Additionally, during main fermentation, amino-type nitrogen and total acid increased gradually to a maximum of 6.77 ± 0.25 g/kg and 19.10 ± 0.58 g/kg (30 days) respectively, and the concentration of reducing sugar increased to 337.41 ± 3.99 g/kg (7 days). The 180-day fermented sweet paste contained 261.46 ± 19.49 g/kg reducing sugar and its pH value remained at around 4.65. This study has used the PCR-DGGE technique to demonstrate the microbial community (including bacteria and fungi) in sweet paste and provides useful information (biochemical properties) about the assessment of the quality of sweet paste throughout fermentation.

Potential application of genetically identical somatic cell nuclear transfer-cloned dogs for gastrointestinal microbiota analysis

This study investigated the gastrointestinal microbiota in three genetically identical cloned dogs (A, B and C) by somatic cell nuclear transfer. We collected feces from three cloned dogs and their feed to investigate gastrointestinal microbiota using both culture-dependent and culture-independent methods. A total of 962 strains from the feces of cloned dogs were isolated using aerobic and anaerobic culture methods. The dominant microorganisms were Enterococcus faecalis and Enterococcus faecium in all fecal samples. In particular, the fecal sample from cloned dog C had similar proportions of three species (E. faecalis, E. faecium and Lactobacillus murinus). In all, 29 DNA fragments were identified by PCR-denaturing gradient gel electrophoresis (DGGE) analysis. The highest DGGE band intensities were for E. faecalis from cloned dogs A and C and for Clostridium sordellii from cloned dog B, with relative intensities of 15.2, 17.7 and 14.4%, respectively. The other strains identified from the cloned dogs were Chryseobacterium soldanellicola, Escherichia coli, L. murinus, Streptococcus alactolyticus, Weissella confusa and uncultured bacterium. Some microbes isolated from the fecal samples, including C. soldanellicola and W. confuse, were derived from the feed. Overall, gastrointestinal microbiota of all genetically identical cloned dogs, maintained under the same environmental and feeding conditions, showed similar profiles in terms of species diversity analyzed by PCR-DGGE, although there were proportional differences in the amounts of bacterial species. To our knowledge, this is the first report to investigate and compare gastrointestinal microbiota of three genetically identical dogs.

Effects of different ratios of glucose to acetate on phosphorus removal and microbial community of enhanced biological phosphorus removal (EBPR) system.

In this study, the effects of different ratios of glucose to acetate on enhanced biological phosphorus removal (EBPR) were investigated with regard to the changes of intercellular polyhydroxyalkanoates (PHAs) and glycogen, as well as microbial community. The experiments were carried out in five sequencing batch reactors (SBRs) fed with glucose and/or acetate as carbon sources at the ratios of 0:100%, 25:75%, 50:50%, 75:25%, and 100:0%. The experimental results showed that a highest phosphorus removal efficiency of 96.3% was obtained with a mixture of glucose and acetate at the ratio of 50:50%, which should be attributed to more glycogen and polyhydroxyvalerate (PHV) transformation in this reactor during the anaerobic condition. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of sludge samples taken from different anaerobic/aerobic (A/O) SBRs revealed that microbial community structures were distinctively different with a low similarity between each other.

Potential ecological effects of Piriformospora indica, a possible biocontrol agent, in UK agricultural systems

Piriformospora indica (syn. Serendipita indica), a root endophytic fungus, was originally isolated from an arid sub-tropical soil. P. indica forms mutualistic symbioses with a broad range of host plants, increases biomass production, resistance and tolerance to fungal pathogens and abiotic stresses. These characteristics make it a very attractive component of more sustainable agriculture. So, it is desirable to understand its wider ecosystem effects. We determined how long P. indica could survive in the soil and how it interacts with other soil microorganisms and some important arable weeds.Survival of P. indica in the soil, under winter and summer conditions in the UK was tested by isolating DNA and RNA of P. indica from pots of soil which had been left open to winter-summer weather conditions without host plants, followed by PCR and reverse transcription-PCR (RT-PCR) with P. indica-specific primers. P. indica effects on other soil and root microorganisms were tested by PCR-denaturing gradient gel electrophoresis analysis of DNA extracted from soil and roots from pots in which P. indica-infected wheat had been grown. The effect of P. indica on growth of black-grass (Alopecurus myosuroides), wild-oat (Avena fatua) and cleavers (Galium aparine) was tested alone and in competition with wheat.In soil P. indica-mRNA and DNA could still be detected after eight months, but not after 15months. Soils from P. indica-inoculated pots had distinct fungal and bacterial species communities which were more diverse than non-inoculated controls. P. indica infected A. myosuroides and A. fatua but was not detected in G. aparine. The average above-ground competitiveness of the weeds with wheat was decreased.If applied to field crops in the UK, P. indica would be persistent for up to 15months and likely to alter competitive relations within vegetation. Increased soil microbial diversity during the first eight weeks after inoculation, although usually desirable, could alter soil composition or functioning.

Variability in chemical and microbiological profiles of long-ripened Caciocavallo cheeses.

Five protected designation of origin (PDO) Caciocavallo Silano and 6 non-PDO Caciocavallo cheeses, ripened for 6mo and collected in the 5 Italian regions of the PDO area (Apulia, Basilicata, Calabria, Campania, and Molise, Italy), were studied to assess their physico-chemical (pH, acidity, moisture, fat, ash, protein, and free amino acid composition) and microbiological profiles. Analyses evidenced a certain fluctuation of previous parameters among samples regardless of the kind of cheese evaluated (PDO and non-PDO). The PCR-denaturing gradient gel electrophoresis analysis performed on the DNA directly extracted from cheeses gave different results, but a low number of bands was always observed. Only one band, corresponding to the species Streptococcus thermophilus, was detectable in 1 PDO and in 2 non-PDO cheese samples, whose free amino acid content was the lowest. Analyses were repeated on experimental Caciocavallo cheeses. Specifically, 2 productions were made, one mimicking the industrial technology (pasteurized milk and selected starter culture) and one the artisanal technology (raw milk and natural whey starter). Results obtained on experimental cheeses at 6mo of ripening showed that industrial samples had lower amounts of total free amino acids then the artisanal ones (1,188.2 vs. 7,523.67mg/100 g of dry matter). Moreover, the PCR-denaturing gradient gel electrophoresis analysis evidenced the sole presence of S. thermophilus in the case of the industrial technology. These data sustain the hypothesis that, out of 11 cheeses analyzed previously, 1 PDO Caciocavallo Silano and 2 non-PDO Caciocavallo cheeses were obtained with the industrial technology. These results could be of help in the discrimination of PDO products, taking into account that the PDO production regulation does not allow the milk pasteurization, nor the use of selected starters.

Microbiological and molecular characterization of commercially available probiotics containing Bacillus clausii from India and Pakistan

Probiotics are actively used for treatment of diarrhoea, respiratory infections, and prevention of infectious gastrointestinal diseases. The efficacy of probiotics is due to strain-specific features and the number of viable cells; however, several reports of deviations from the label in the actual content of strains in probiotic products are a matter of concern. Most of the available data on quality focuses on probiotic products containing lactobacilli and/or bifidobacteria, while very few data are available on spore-forming probiotics. The present study evaluates the label claims for spore count and species identification in five commercial probiotic products marketed in India and Pakistan that claim to contain Bacillus clausii: Tufpro, Ecogro, Enterogermina, Entromax, and Ospor. Bacterial enumeration from three batches was done by microbiological plating methods by two independent operators. Species identification was done using PCR amplification and sequence analysis of the 16S rRNA gene, and determination of the total amount of species present in the products was done using PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by DNA sequencing of the excised bands. Plate count methods demonstrated poor correlations between quantitative label indications and bacteria recovered from plates for Tufpro, Ecogro, and Ospor. The 16S rRNA analysis performed on bacteria isolated from plate counts showed that only Enterogermina and Ospor contained homogenous B. clausii. PCR-DGGE analysis revealed that only Enterogermina had a homogenous B. clausii population while other products had mixed bacterial populations. In conclusion, the current analysis clearly demonstrates that of the five analysed commercial probiotics, only Enterogermina followed the label claims.

Pediocin SA-1: A selective bacteriocin for controlling Listeria monocytogenes in maize silages.

In this study, we assessed the potential as silage additive of a bacteriocin produced by Pediococcus acidilactici Northern Regional Research Laboratory (NRRL) B-5627 (pediocin SA-1). Maize was inoculated either with a bacterial starter alone (I) or in combination with the bacteriocin (IP), and untreated silage served as control. We monitored the products of fermentation (ethanol, and lactic and acetic acids), the microbial population, and the presence of the indicator strain Listeria monocytogenes Colección Española de Cultivos Tipo (CECT) 4032 (1×10(5) cfu/g) after 1, 2, 5, 8, 16, and 30d of ensiling. Our results indicated antilisterial activity of the bacteriocin, anticipating the disappearance of L. monocytogenes in IP compared with I and control silages. The PCR-denaturing gradient gel electrophoresis analysis revealed the addition of the bacteriocin did not affect the bacterial communities of the spontaneous fermentation, and the inoculant-containing bacteria (Lactobacillus plantarum, Lactobacillus buchneri, and Enterococcus faecium) were found in addition to the bacterial communities of untreated maize silages in I and IP silages. Both treatments increased the concentration of antimicrobial compounds (acetic acid, ethanol, and 1,2-propanodiol) and led to lower residual sugar contents compared with the control, which would provide enhanced aerobic stability. The fact that the identified species L. plantarum, L. buchneri, and E. faecium produce some of these inhibitory compounds, together with their persistence throughout the 30d of fermentation, suggest these bacteria could actively participate in the ensiling process. According to these results, pediocin SA-1 could be used as an additive to control the presence of L. monocytogenes in maize silages selectively, while improving their fermentative quality and eventually their aerobic stability.

Decreased microbial diversity and Lactobacillus group in the intestine of geriatric giant pandas (Ailuropoda melanoleuca)

It has been established beyond doubt that giant panda genome lacks lignin-degrading related enzyme, gastrointestinal microbes may play a vital role in digestion of highly fibrous bamboo diet. However, there is not much information available about the intestinal bacteria composition in captive giant pandas with different ages. In this study, we compared the intestinal bacterial community of 12 captive giant pandas from three different age groups (subadults, adults, and geriatrics) through PCR-denaturing gradient gel electrophoresis (DGGE) and real-time PCR analysis. Results indicated that microbial diversity in the intestine of adults was significantly higher than that of the geriatrics (p < 0.05), but not significant compared to the subadults (p > 0.05). The predominant bands in DGGE patterns shared by the twelve pandas were related to Firmicutes and Proteobacteria. Additionally, in comparison to healthy individuals, antibiotic-treated animals showed partial microbial dysbiosis. Real-time PCR analyses confirmed a significantly higher abundance of the Lactobacillus in the fecal microbiota of adults (p < 0.05), while other bacterial groups and species detected did not significantly differ among the three age groups (p > 0.05). This study revealed that captive giant pandas with different ages showed different intestinal bacteria composition.

Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented quinoa sourdoughs.

To analyse lactic acid bacteria (LAB) diversity and technological-functional and safety properties of strains present during spontaneous fermented quinoa sourdoughs. Fermentation was performed by daily backslopping at 30°C for 10days. Autochthonous LAB microbiota was monitored by a biphasic approach combining random amplified polymorphic DNA (RAPD)-PCR and rRNA gene sequencing with PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Identification and intraspecies differentiation allowed to group isolates within nine LAB species belonging to four genera. A succession of LAB species occurred during 10-days backslopping; Lactobacillus plantarum and Lactobacillus brevis were detected as dominant species in the consortium. The characterization of 15 representative LAB strains was performed based on the acidifying capacity, starch and protein hydrolysis, γ-aminobutyric acid and exopolysaccharides production, antimicrobial activity and antibiotic resistance. Strains characterization led to the selection of Lact.plantarum CRL1905 and Leuconostoc mesenteroides CRL1907 as candidates to be assayed as functional starter culture for the gluten-free (GF) quinoa fermented products. Results on native LAB microbiota present during quinoa sourdough fermentation will allow the selection of strains with appropriate technological properties to be used as a novel functional starter culture for GF-fermented products.

Decontamination of heavy metal laden sewage sludge with simultaneous solids reduction using thermophilic sulfur and ferrous oxidizing species

A possibility of using simultaneous sewage sludge digestion and metal leaching (SSDML) process at the thermophilic temperature to remove heavy metals and suspended solids from sewage sludge is explored in this study. Though thermophilic sludge digestion efficiently produces a stable sludge, its inability to remove heavy metals requires it to be used in tandem with another process like bioleaching for metal reduction. Previously, different temperature optima were known for the heterotrophs (thermophilic) responsible for the sludge digestion and the autotrophs involved in bioleaching (mesophilic), because of which the metal concentration was brought down separately in a different reactor. In our study, SSDML process was carried out at 50 °C (thermophilic) by using ferrous sulfate (batch-1) and sulfur (batch-2) as the energy source in two reactors. The concentration of volatile suspended solids reduced by >40% in both batches, while that of heavy metals zinc, copper, chromium, cadmium and nickel decreased by >50% in both batch-1 and batch-2. Lead got leached out only in batch-1. Using 16S rRNA gene-based PCR-denaturing gradient gel electrophoresis analysis, Alicyclobacillus tolerans was found to be the microorganism responsible for lowering the pH in both the reactors at thermophilic temperature. The indicator organism count was also below the maximum permissible limit making sludge suitable for agricultural use. Our results indicate that SSDML at thermophilic temperature can be effectively used for reduction of heavy metals and suspended solids from sewage sludge.

Effect of purple loosestrife (Lythrum salicaria) diet supplementation in rabbit nutrition on performance, digestibility, health and meat quality

In this study, 160 Hycole weaned rabbits (35 days old) were randomly divided into four groups of 40. The rabbits were studied throughout a 54-day experimentation period in order to determine the impact of dietary supplementation from herbs composed of 0.2%, 0.4% dry ground Lythrum salicaria leaves (LS) and 0.3% Cunirel® (CR; a commercial herb mixture containing LS as the main ingredient) on performance, digestibility, health and meat quality. The basal diet was given to the control group. No significant differences were found in performance, 10 rabbits from each group were selected for evaluation regarding apparent digestibility. The rabbits fed the control diet and the diet with the low level of LS had a higher level of CP digestibility than did the animals that were supplemented with the high LS levels and CR (85.7% and 84.9% v. 84.0% and 84.0%, respectively; P<0.05). The ether extract digestibility was lower in the treatment group with 0.4%LS addition and CR as compared with the control group (52.2% and 54.5% v. 62.6%, respectively; P<0.05). The slaughter process was performed on 89-day-old rabbits to study the carcass characteristics, meat quality, blood parameters, caecal contents and gut histology. The total leukocyte counts in the control animals were lower than they were in the rabbits fed 0.2%, 0.4%LS and CR (4.06 v. 8.25, 8.63 and 8.21×109/l, respectively; P<0.05). For caecal fermentation, the caecal contents of the rabbits fed 0.4% of LS, showed higher concentrations of total volatile fatty acid (VFA; 24.1 v. 18.9 mg/kg dry matter (DM); P<0.05) and acetic acid (18.3 v. 14.4 mg/kg DM; P<0.05), but lower ammonia levels (594 v. 892 mg/kg DM; P<0.05) as compared with the control group. PCR-denaturing gradient gel electrophoresis analyses were performed to evaluate the microbial community in hard faeces, collected at days 35, 42, 49, 56, 70 and 89, whereas the caecal contents were taken after slaughtering. The results demonstrated that between the treatment groups, the similarity of the microbial communities was higher as compared with the control group. Moreover, only age was shown to influence microbiota diversity. In conclusion, the results of this study indicated that supplementation of LS in rabbit diets leads to an increase in the total white blood cells, total VFA and acetic acid concentration, and a decrease in the ammonia levels, as well as the digestibility when CR and high level of LS were supplemented, without causing any adverse effects on other parameters.