One-dimensional TRFLP-SSCP is an effective DNA fingerprinting strategy for soil Archaea that is able to simultaneously differentiate broad taxonomic clades based on terminal fragment length polymorphisms and closely related sequences based on single stranded conformation polymorphisms

DNA fingerprinting methods provide a means to rapidly compare microbial assemblages from environmental samples without the need to first cultivate species in the laboratory. The profiles generated by these techniques are able to identify statistically significant temporal and spatial patterns, correlations to environmental gradients, and biological variability to estimate the number of replicates for clone libraries or next generation sequencing (NGS) surveys. Here we describe an improved DNA fingerprinting technique that combines terminal restriction fragment length polymorphisms (TRFLP) and single stranded conformation polymorphisms (SSCP) so that both can be used to profile a sample simultaneously rather than requiring two sequential steps as in traditional two-dimensional (2-D) gel electrophoresis. For the purpose of profiling Archaeal 16S rRNA genes from soil, the dynamic range of this combined 1-D TRFLP-SSCP approach was superior to TRFLP and SSCP. 1-D TRFLP-SSCP was able to distinguish broad taxonomic clades with genetic distances greater than 10%, such as Euryarchaeota and the Thaumarchaeal clades g_Ca. Nitrososphaera (formerly 1.1b) and o_NRP-J (formerly 1.1c) better than SSCP. In addition, 1-D TRFLP-SSCP was able to simultaneously distinguish closely related clades within a genus such as s_SCA1145 and s_SCA1170 better than TRFLP. We also tested the utility of 1-D TRFLP-SSCP fingerprinting of environmental assemblages by comparing this method to the generation of a 16S rRNA clone library of soil Archaea from a restored Tallgrass prairie. This study shows 1-D TRFLP-SSCP fingerprinting provides a rapid and phylogenetically informative screen of Archaeal 16S rRNA genes in soil samples.     

Effects of Dietary Non-Digestible Oligosaccharides on Microbial Characteristics of Ileal Chyme and Faeces in Weaner Pigs

Fructooligosaccharides (FOS) and transgalactooligosaccharides (TOS), which are non-digestible oligosaccharides (NDO), were included at 10 and 40g/kg in an NDO-free control diet at the expense of purified cellulose. Each of the 5 diets was fed to 4 weaner pigs and microbial characteristics of their ileal chyme and faeces were assessed. The NDO-pigs had lower ileal pH than the control pigs. Dietary NDO did not affect the ileal volatile fatty acid concentration, though FOS-pigs had a higher concentration of lactic acid and relatively more iso-valeric acid and less acetic acid than TOS-pigs. The NDO-pigs had lower ileal aerobic bacterial counts than the control pigs, whilst the FOS-pigs had a larger ileal anaerobic bacterial counts than the TOS-pigs. The NDO-pigs had an higher faecal pH and their faecal volatile fatty acid pool contained relatively more iso-butyric acid and iso-valeric acid than the control pigs. The TOS-pigs tended to have higher faecal anaerobic bacterial counts and had a smaller concentration of faecal volatile fatty acid than the FOS-pigs. We concluded that whilst effects at the faecal level may have been partly due to a reduced intake of cellulose, dietary NDO can exert precaecal prebiotic effects in weaner pigs.     

Antimicrobial enzymes: An emerging strategy to fight microbes and microbial biofilms

With the increasing prevalence of antibiotic resistance, antimicrobial enzymes aimed at the disruption of bacterial cellular machinery and biofilm formation are under intense investigation. Several enzyme-based products have already been commercialized for application in the healthcare, food and biomedical industries. Successful removal of complex biofilms requires the use of multi-enzyme formulations that contain enzymes capable of degrading microbial DNA, polysaccharides, proteins and quorum-sensing molecules. The inclusion of anti-quorum sensing enzymes prevents biofilm reformation. The development of effective complex enzyme formulations is urgently needed to deal with the problems associated with biofilm formation in manufacturing, environmental protection and healthcare settings. Nevertheless, advances in synthetic biology, enzyme engineering and whole DNA-Sequencing technologies show great potential to facilitate the development of more effective antimicrobial and anti-biofilm enzymes.     

Deriving metabolic engineering strategies from genome-scale modeling with flux ratio constraints

Optimized production of bio-based fuels and chemicals from microbial cell factories is a central goal of systems metabolic engineering. To achieve this goal, a new computational method of using flux balance analysis with flux ratios (FBrAtio) was further developed in this research and applied to five case studies to evaluate and design metabolic engineering strategies. The approach was implemented using publicly available genome-scale metabolic flux models. Synthetic pathways were added to these models along with flux ratio constraints by FBrAtio to achieve increased (i) cellulose production from Arabidopsis thaliana; (ii) isobutanol production from Saccharomyces cerevisiae; (iii) acetone production from Synechocystis sp. PCC6803; (iv) H₂ production from Escherichia coli MG1655; and (v) isopropanol, butanol, and ethanol (IBE) production from engineered Clostridium acetobutylicum. The FBrAtio approach was applied to each case to simulate a metabolic engineering strategy already implemented experimentally, and flux ratios were continually adjusted to find (i) the end-limit of increased production using the existing strategy, (ii) new potential strategies to increase production, and (iii) the impact of these metabolic engineering strategies on product yield and culture growth. The FBrAtio approach has the potential to design “fine-tuned” metabolic engineering strategies in silico that can be implemented directly with available genomic tools.     

Glucose‐based microbial production of the hormone melatonin in yeast Saccharomyces cerevisiae

Melatonin is a natural mammalian hormone that plays an important role in regulating the circadian cycle in humans. It is a clinically effective drug exhibiting positive effects as a sleep aid and a powerful antioxidant used as a dietary supplement. Commercial melatonin production is predominantly performed by complex chemical synthesis. In this study, we demonstrate microbial production of melatonin and related compounds, such as serotonin and N‐acetylserotonin. We generated Saccharomyces cerevisiae strains that comprise heterologous genes encoding one or more variants of an L‐tryptophan hydroxylase, a 5‐hydroxy‐L‐tryptophan decarboxylase, a serotonin acetyltransferase, an acetylserotonin O‐methyltransferase, and means for providing the cofactor tetrahydrobiopterin via heterologous biosynthesis and recycling pathways. We thereby achieved de novo melatonin biosynthesis from glucose. We furthermore accomplished increased product titers by altering expression levels of selected pathway enzymes and boosting co‐factor supply. The final yeast strain produced melatonin at a titer of 14.50 ± 0.57 mg L^?1 in a 76h fermentation using simulated fed‐batch medium with glucose as sole carbon source. Our study lays the basis for further developing a yeast cell factory for biological production of melatonin.     

Extraction and Analysis of Microbial Phospholipid Fatty Acids in Soils

Phospholipid fatty acids (PLFAs) are key components of microbial cell membranes. The analysis of PLFAs extracted from soils can provide information about the overall structure of terrestrial microbial communities. PLFA profiling has been extensively used in a range of ecosystems as a biological index of overall soil quality, and as a quantitative indicator of soil response to land management and other environmental stressors.The standard method presented here outlines four key steps: 1. lipid extraction from soil samples with a single-phase chloroform mixture, 2. fractionation using solid phase extraction columns to isolate phospholipids from other extracted lipids, 3. methanolysis of phospholipids to produce fatty acid methyl esters (FAMEs), and 4. FAME analysis by capillary gas chromatography using a flame ionization detector (GC-FID). Two standards are used, including 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine (PC(19:0/19:0)) to assess the overall recovery of the extraction method, and methyl decanoate (MeC10:0) as an internal standard (ISTD) for the GC analysis.     

Assessment of artificial substrates for evaluating groundwater microbial quality

Protection of groundwater resources requires the development of reliable ecological indicators. Microorganisms involved in ecological services or being associated with particular hosts or habitats could be used for this purpose. Nevertheless, their tracking remains limited because of sampling issues, and a lack of devices for their long term monitoring. In the present study, three artificial substrates (glass and clay beads, and gravel particles) were tested in terms of efficacy at favoring bacterial growth, and at capturing bacterial diversity of waters (i.e., groundwater, surface water and wastewater). Total proteins, total carbohydrates, dehydrogenase and hydrolytic activities were used to monitor biofilm development on these artificial substrates. Fingerprinting analyses based on rrs (16S rRNA) − rrl (23S rRNA) spacer analyses (ARISA) and next generation sequencing (NGS) of partial rrs DNA segments (V5-V6) were used to compare operating taxonomic units (OTUs), and infer bacterial genera trapped on these substrates. Glass beads were found less efficient than the other two artificial substrates at increasing protein contents and microbial activities (hydrolytic and dehydrogenase activities). ARISA showed a discrimination of bacterial communities developing on artificial substrates that was matching water types. An incubation period of 7 days allowed a reliable assessment of bacterial diversity. From this incubation period, around 75% of water genera with more than four V5-V6 rrs DNA sequences detected in a water type were recovered from biofilms growing on artificial substrates. Based on relative abundances of genera, clay beads and gravel particles were more efficient than glass beads to capture and obtain bacterial communities matching those of the initial waters. Between 45–67% of similarities were found for these artificial substrates while it was between 36 and 43% for glass beads. This study demonstrated clay beads and gravel particles as being efficient tools for capturing bacterial diversity and monitoring bacterial growth. Overall, clay beads appeared the best choice for field monitoring because of the ease of their size standardization in comparison with gravel particles.     

Seventy-year chronology of Salinas in southern France: Coastal surfaces managed for salt production and conservation issues for abandoned sites

After World War II, twenty-nine coastal Salinas (122 km²), located in the vicinity of coastal lagoons and in deltas, were exploited along the Mediterranean coastlines in South France. Today, only five of these are still actively producing salt, currently representing 175 km². Concomitant with the abandonment of many of the smaller Salinas, the larger Salinas in the Rhône delta (Camargue) strongly increased their surfaces at the expense of natural ecosystems, of which a part has also been abandoned after 2009. This paper documents these changes in landscape use by chronological GIS mapping and describes the fate of the 91 km² of abandoned Salina surfaces. The majority of this area (88 km²) is included in the Natura 2000 network, among which most (74 km²) has been acquired by the French coastal protection agency (Conservatoire du Littoral) to be designated as Protected Areas. Only a very minor part (<1%) has been lost for industry and harbour development. Managing abandoned Salinas as Protected Areas is a challenge, because of the different landscape, biodiversity conservation, natural and cultural heritages issues at stake. In two cases, abandoned Salinas have been brought back again into exploitation by private initiative thus allowing for the protection of original hypersaline biodiversity. In other cases, the shaping of the landscape by natural processes has been privileged. This has facilitated the spontaneous recreation of temporal Mediterranean wetlands with unique aquatic vegetation, and offered opportunities for managed coastal re-alignment and the restoration of hydrobiological exchanges between land and sea. In other areas, former salt ponds continue to be filled artificially by pumping favouring opportunities for waterfowl. This has often been combined with the creation of artificial islets to provide nesting ground for bird colonies protected from terrestrial predators.     

Salivary calculi microbiota: new insights into microbial networks and pathogens reservoir

Sialolithiasis represents the most common disorders of salivary glands in middle-aged patients. It has been hypothesized that the retrograde migration of bacteria from the oral cavity to gland ducts may facilitate the formation of stones. Thus, in the present study, a microbiome characterization of salivary calculi was performed to evaluate the abundance and the potential correlations between microorganisms constituting the salivary calculi microbiota. Our data supported the presence of a core microbiota of sialoliths constituted principally by Streptococcus spp., Fusobacterium spp. and Eikenella spp., along with the presence of important pathogens commonly involved in infective sialoadenitis.     

Microbiological and chemical quality of a traditional salted-fermented fish (Hout-Kasef) product of Jazan Region,Saudi Arabia

The Hout-Kasef is traditional salted fermented fish product of natural fermentation of salted mullet fish of coastal area of Jazan region of Saudi Arabia. The present study was carried out to investigate the microbiological and chemical characteristic of Hout-Kasef. A total of twenty-four salted fish samples were purchased from fish market in Jazan and Abu-Arish at different times of the year. The microbial studies of salted-fermented fish revealed a total bacterial count ranging from 2.81 to 4.72 Log₁₀ CFU/g, yeast and mold counts ranging from 0.48 to 3.14 Log₁₀ CFU/g, total staphylococci count 2.71–3.85 Log₁₀ CFU/g, halophile bacteria count 3.26–5.14 Log₁₀ CFU/g, and coliforms count <1 Log₁₀ CFU/g. However, pathogenic bacteria such as Listeria monocytogenes, Vibrio spp., Campylobacter spp. and Yersinia species were not detected. The major bacteria species isolated and identified from the salted fermented fish were Bacillus Subtilus, Bacillus mycoides, Bacillus licheniformis, Bacillus pumilus, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus xylosus, Staphylococcus saprophticus and Staphylococcus cahnii subsp cahnii. The chemical analysis of salted fermented fish showed high content of moisture (47.96%), protein (25.71%), ash (19.6%) and salt (15.19%) but low contents of lipid (7.25%). The salted-fermented fish also showed high level of total volatile basic nitrogen (78.86 mg/100 gm sample) and thiobarbutric acid number (32.32 mg malonaldehyde/kg) with a pH value of pH 6.3. Finally, this study showed the presence of gram positive and gram negative bacteria in the fish product. The predominant microorganisms found were Bacillus and Staphylococcus spp. The fish product had high content of salt and TVB-N levels.