- Volume 1, Issue 1A, 2019
Volume 1, Issue 1A, 2019
- Oral Abstract
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- Virology Workshop: Pathogenesis
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Aedes aegypti(Aag2)-derived clonal mosquito cell lines reveal impact of pre-existing persistent co-infection with the insect-specific Bunyavirus Phasi Charoen-like virus on arbovirus replication
Aedes aegyptiis a vector mosquito of major public health importance, transmitting arthropod-borne viruses (arboviruses) such as chikungunya, dengue, yellow fever and Zika viruses. Wild mosquito populations are persistently infected at high prevalence with insect-specific viruses that do not replicate in vertebrate hosts. In experimental settings, acute infections with insect-specific viruses have been shown to modulate arbovirus infection and transmission in Ae. aegypti and other vector mosquitoes, however the impact of persistent insect-specific virus infections that more accurately mimic the situation in nature has not been investigated extensively. Cell lines are useful models for studying virus-host interactions, however the available Ae. aegypti cell lines are poorly defined and heterogenous cultures. We generated single cell-derived clonal cell lines from the commonly used Ae. aegypticell line Aag2. Two of the fourteen Aag2-derived clonal cell lines studied in detail harboured markedly and consistently reduced levels of the insect-specific bunyavirus Phasi Charoen-like virus (PCLV) known to persistently infect Aag2 cells. In contrast to studies with acute insect-specific virus infections in cell culture and in vivo, we found that pre-existing persistent PCLV infection had no major impact on the replication of the flaviviruses dengue virus and Zika virus, the alphavirus Sindbis virus or the rhabdovirus vesicular stomatitis virus. Our findings highlight the need for further in vivo studies that more closely recapitulate natural arbovirus transmission settings in which arboviruses encounter mosquitoes harbouring persistent rather than acute insect-specific virus infections. Furthermore, we provide well-characterised Aag2-derived clonal cell lines as a valuable resource to the arbovirus research community.
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- Virus Infections of the Central Nervous System
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A novel enterovirus protein modulates infection in gut epithelial cells
Enteroviruses comprise a large group of mammalian pathogens that includes poliovirus. Pathology in humans ranges from sub-clinical to acute flaccid paralysis, myocarditis and meningitis. Until now, all the enteroviral proteins were thought to derive from proteolytic processing of a polyprotein encoded in a single open reading frame (ORF). We report that many enterovirus genomes also harbor an upstream ORF (uORF) that is subject to strong purifying selection. Using echovirus 7 and poliovirus 1, we confirmed expression of uORF protein (UP) in infected cells. Using ribosome profiling (a technique for global footprinting of translating ribosomes), we also demonstrated translation of the uORF in representative members of the predominant human enterovirus species, namely Enterovirus A, B and C. In differentiated human intestinal organoids, UP-knockout echoviruses are attenuated compared to wild-type virus at late stages of infection where membrane-associated UP facilitates virus release. Thus we have identified a previously unknown enterovirus protein that facilitates virus growth in gut epithelial cells – the site of initial viral invasion into susceptible hosts. These findings overturn the 50-year-old dogma that enteroviruses use a single-polyprotein gene expression strategy, and have important implications for understanding enterovirus pathogenesis.
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Influence of gestational and developmental age on human airway epithelial innate immune responses to Respiratory Syncytial Virus (RSV) in early life
More LessBackgroundRSV is a major respiratory pathogen in infants, with preterm and young infants being at increased risk of severe disease. Airway epithelial cell (AEC) innate immune responses represent an important first line of defence. Development of these responses in early life is poorly understood.
MethodsWell differentiated cultures were generated using nasal AECs harvested from term and preterm infants at birth and from the same infants at one-year old. Cultures were infected with RSV and resulting cytopathology and innate immune responses studied.
ResultsRSV growth kinetics were similar between preterm and term newborn cohorts at birth and in the one-year-old repeat cohort. Secretion of interferon lambda-1 (IFN-l1), CXCL10 (IP-10), CCL5 (RANTES) and CXCL8 (IL-8) were similar between RSV-infected preterm and term newborn AECs. Following RSV infection, significantly higher secretion of IFN-l1 (P=0.0034), CXCL10 (P<0.05) and CCL5 (P<0.05) was noted in one-year-derived AECs compared to newborn AECs. We also identified differential expression of a novel endogenously expressed anti-RSV protein, pleiotrophin (PTN), which interacts with nucleolin, a cofactor for RSV entry. PTN expression/secretion was significantly decreased in preterm versus term AECs (P=0.014) and in newborn compared to one-year WD-PNECs (P=0.0008).
ConclusionThese exciting findings represent the first description of age- and prematurity-related differences in AEC innate immune responses, demonstrating greater RSV-induced expression of pro-inflammatory chemokines with increasing age and developmental differences in expression of the novel anti-RSV protein PTN. These findings may, in part, explain the increased susceptibility of preterm and very young infants to severe RSV disease.
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High susceptibility, viral dynamics and persistence of South American Zika virus in New World monkeys
South American Zika virus (ZIKV) emerged as a novel enzootic pathogen linked with neonatal brain defects and acute neuropathies, its natural history characterised by a sylvatic transmission cycle with arboreal mosquitoes and African primates. Yet infectivity studies in New World primates indigenous to Zika-endemic regions including host range, susceptibility, replication dynamics, tissue tropism and virus persistence are currently lacking. New World species Callithrix jacchus (marmosets) and Saguinus labiatus (red-bellied tamarins) were highly susceptible to sub-cutaneous challenge with South American ZIKVPRVABC59 inducing rapid, high, acute viraemia in each species. Differences in acute phase vRNA in blood were highly statistically significant at day 3 between these New and two Old World species Macaca mulatta (rhesus macaque) and Macaca fascicularis (cynomolgus macaque), Mann-Whitney U testp=0.00058. Comparative quantitative RT-PCR and RNAscope in situ hybridisation analysis of tissue viral copy number and cellular localisation patterns with Old World NHP revealed early, widespread distribution across multiple skin biopsies distant to the inoculation site, lymphoid organs, reproductive sites and the brain. While viraemia was cleared from blood by day 42 in all individuals, ZIKV persistence in most tissues was identified 100 days post-inoculation. Early neuroinvasion and persistence, following acutely resolved infection characterises ZIKV susceptibility in multiple hosts, especially New World species. Establishment of a ZIKV sylvatic cycle in the Americas may provide persistent animal reservoirs for future outbreak resurgence. New World monkeys represent viable models to understand ZIKV pathogenesis, potential therapeutic interventions and vaccine development strategies.
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- Poster Presentation
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- Biobased Circular Economy and Bioremediation
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A preliminary investigations of novel dehalogenase producing bacteria from antarctic psychrotropic Bacillus sp.IH1
More LessA preliminary investigations of novel dehalogenase producing bacteria from Antarctic Psychrotropic Bacillus sp.Ih1 2, 2 dichloropropionic acids (Dalapon) like most halogenated compounds are commonly used as herbicides and employed in agricultural areas and industries. Toxicity of these xenobiotic compounds causes serious environmental problems. Bacillus sp. ih1 was isolated from top cliff soil collected from Antarctica. The bacteria was first grown on Antarctic bacterial medium and later transferred to a minimal medium containing 2,2, dichloropropionic acid as carbon source. It grew slowly in the minimal media in different concentrations of 10 mM, 20 mM, 30 mM and 40 mM of 2,2 DCP. The best growth was observed in 20 mM of 2,2-DCP with 32 h as doubling time. To monitor the degradative activity of the bacteria, halide ion assay was carried out to check the release of chloride ion. The best release of chloride was 0.657 mMol/L in 20 mM of 2,2-DCP. The bacteria was identified using 16S rRNA, genomic DNA extraction and PCR amplification of 16S rRNA was performed using universal primers 27F and1492R. Nucleotide blast (BLASTn) showed 97 % similarity with Bacillus sp. Results from biochemical tests further confirm the bacteria as Bacillus sp. Using phylogeny.fr, sequences from nucleotide blast result were used to build a phylogeny tree based on neighbor to neighbor joining.
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Applying transposon-directed insertion site sequencing to industrially important, solventogenic species Clostridium saccharoperbutylacetonicum
More LessSolventogenic Clostridium spp. have significant potential as a source of renewable biochemicals. Production of solvents such as butanol and acetone from members of this genus is already being commercialised by industry. However, the scale of biological knowledge of these Clostridia lags behind the scale of their application. With the advent of next generation sequencing methods, transposon mutagenesis now provides a large-scale, high-throughput forward approach to understanding the genetics of these species. We are applying transposon directed insertion-site sequencing (TraDIS) to the industrially-relevant solventogenic species Clostridium saccharoperbutylacetonicum. We are utilising this robust TraDIS pipeline to uncover the essential genome of the species under laboratory conditions. Furthermore, we are investigating the species’ tolerance to butanol, a key limiting step in the fermentation process. In collaboration with Green Biologics, we are also examining the essential genome of the species under industrially important fermentation conditions. Understanding the genes that are conditionally essential in these contexts will be key in advancing the biological knowledge of the species as well as providing information that can improve the fermentation process.
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Identification and quantification of antibiotic resistant bacteria & Genes in an aquaculture facility which uses a novel bioactive filtering system
More LessThe Advanced Biotechnology for Intensive-Freshwater Aquaculture Wastewater Reuse (ABAWARE) project, which is part of the European Commission’s Water Joint Programming Initiative 2016 Joint Call, aims to increase the efficiency and resilience of water use in aquaculture and minimise its negative impact on the environment and human health. This research, which forms one part of the total ABAWARE project, aimed to ascertain the impact of using microbiota and certain plant species, in conjuncture with a more traditional Recirculated Aquaculture System (RAS), as a filtering system had on the arb and G abundance in various samples taken from an aquaculture facility. Sediment and water samples were taken from the inflow, the main fish basin, after the bioactive ponds before filtration, and after filtration. The resistance genes present in these samples were detected using the Wafergen smartchip real-time qPCR system. This system allows for the simultaneous quantification of 348 distinct Antibiotic Resistance Genes for each sample. The samples also underwent microbiome analysis via 16S rRNA metagenomic sequencing. Mothur was used to analyse the sequencing data. This data informs us of the changes in the microbial population changes that are enacted by the various stages within the aquaculture facility.
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Meeting the rare earth expanding circle
More LessThe increasing demand for rare earth elements (REE) is fuelled by their importance in a green energy future, with the demand for dysprosium predicted to increase by 5 % annually by 2026. Bioleaching approaches are being investigated for the recovery of REE and other precious metals from waste materials, however even 100 % recovery will not be able to meet increasing demand. Therefore, extraction from primary sources will be required. REE do not form high concentration ores, so their extraction can require processing large volumes of material, however REE are frequently associated with other raw materials and REE are sold as by-products of iron mining from Chinese deposits. Bioleaching offers the potential to produce valuable by-products from existing mining operations or to remediate historical mine waste. The diverse nature of REE-bearing minerals means that a variety of established and emerging bioleaching approaches could be applied: organic acid leaching, oxidative leaching of sulphidic ores, and reductive leaching of oxidised ores. We have applied these processes to three bauxites, demonstrating varied responses to bioleaching with each bauxite. The combination of low concentration ores and varied sources provide a challenge to recovery: however, it is one that microbes could take on.
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Formation of selenium- and tellurium-containing nanoparticles during the growth of filamentous fungi
More LessMicrobial Se or Te reduction offers a potential route to biorecovery of these elements from solution. Reduction is often efficient and large amounts of these metalloids can be removed from solution, resulting in extensive precipitation around biomass. This is more effective than biomethylation, which can result in only small amounts of removal, and would necessitate a further trapping step to recover volatilized methylated derivatives. In this research, the fungi Aureobasidium pullulans, Mortierella humilis, Trichoderma harzianum, and Phoma glomeratawere used to investigate the formation of selenium- and tellurium-containing nanoparticles during growth on selenium- and tellurium-containing media. Most organisms were able to grow on both selenium- and tellurium-containing media at concentrations of 1 mM and this resulted in extensive precipitation of elemental selenium and tellurium on fungal surfaces observed by the bright red and black colour changes. Red or black deposits were confirmed as elemental selenium and tellurium, respectively, by X-ray powder diffraction. Apart from elemental selenium and tellurium, selenium oxide and tellurium oxide were also found after growth of Trichoderma harzianumin the presence of 1 mM selenite and tellurite together with the formation ofelemental selenium and tellurium. The hyphal matrix provided nucleation sites for metalloid deposition with extracellular protein and extracellular polymeric substances serving to localize the resultant Se or Te nanoparticles. These findings are relevant to remedial treatments for selenium and tellurium contamination, and possible novel approaches for selenium and tellurium biorecovery from liquid matrices.
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From trash to treasure – turning plastic waste into biodegradable polymers using bacteria
Mountains of plastic waste consisting of carrier bags and medical packaging are buried in landfill sites dumped in rivers around the world annually. Unfortunately plastics generated by the petrochemical industry are not biodegradable and therefore accumulate in the environment at a rate of over 25 million tones year-1. Therefore there is a huge demand for biodegradable plastics. Polystyrene (PS), polyethylene (PE) and polypropylene (PP) are problematic materials, used for appliance housings, disposable cutlery and general packaging. This study investigates the utilisation of waste PS, PE and PP as a potential additional carbon sources using bacteria to synthesise polyhydroxyalkanoates (PHAs); a value-added material, able to replace some conventional fossil-fuel plastics while being non-toxic, fully biodegradable and biocompatible. Prodegraded waste PS and PP, and thermally treated PEwere used as supplementary carbon sources totryptone soya broth (TSB and BSM) for 48 h fermentations [1, 2, 3]. The bacterial strain Cupriavidus necator H16 was selected as it is non-pathogenic, genetically stable, robust and one of the best natural producers of PHA. The accumulation of PHAs varied from 17 % (wt / wt) of dry biomass in TSB controls to 39–66 % for PS, PE and PP thermally treated samples. The polymers obtained were analysed with nuclear magnetic resonance (NMR) and electrospray ionisation tandem mass spectrometry (ESI-MS/MS) to characterise their chemical structure. In conclusion, certain thermal treatment protocols of the waste plastics were shown to be viable for PHA production; with3-hydroxybutyrate and up to 12 mol% of 3-hydroxyvalerate and 3-hydroxyhexanoate co-monomeric units formed.
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Dairy processing wastewater as a feedstock for microbial bioplastic production
More LessThe end of the European milk quotas in 2015 resulted in a steep increase of Irish milk production from 5 to over 7.2 billion litres annually. Dairy processing ads value, but generates up to 10 litres wastewater (WW) per litre processed. Organic pollutants in the WW need to be removed before discharge. Our approach aims to turn this waste into a resource for polyhydroxyalkanoates (PHA), bioplastic production. Bioplastics are promising materials to reduce our dependency on fossil fuels and waste production. The first production step includes an adaption of the dairy processing WW in an anaerobic, hydrolytic reactor, where acidogenic bacteria metabolise the organic fraction of the WW and form volatile fatty acids (VFAs) the building blocks of PHA. The second step comprises the adaption of biomass towards PHA accumulation. The adaption is driven by an aerobic dynamic feeding strategy to increase the formation of the storage molecule PHA. Addition of the WW as substrate is followed by a starvation period, where bacteria capable of PHA storage have a selection advantage. Over time the mixed microbial system is therefore optimised for PHA accumulation. In a final production step the adapted WW and biomass is combined in a fed-batch reactor to produce the end product PHA. The laboratory scale system could be established and is currently optimised. A later scale-up will help to assess the full economic potential of this waste to value approach.
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- Environmental and Applied Microbiology Forum
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Assessing the presence and genetic basis of ESBL-producing bacteria in pre-boil wort, dairy cattle manure and dog faeces
More LessAntimicrobial resistance (AMR) is becoming the biggest problem facing human and animal health care, with a predicted 10 million deaths per year due to antibiotic resistance by 2050 (Review on Antimicrobial resistance, 2014). The focus of many AMR studies is on problems inside clinical settings, such as hospitals and veterinary practices, yet, as this study shows, often the most diverse sources of resistance genes are found in relatively unexplored areas of the environment; in particular dog faeces and dairy cattle manure.
Chromogenic media analysis showed dog faeces had the greatest number and diversity of ESBL-producing bacteria. Multiplex PCR showed inconclusive evidence of what the resistance genes were in both dairy cattle manure and dog faeces. Phylogenetic analysis of the 16S rRNA showed Flavobacteriaceae-like bacteria in dairy cattle manure but the presence of Enterobacteriaceae-like bacteria, most similar to Escherichia coli, in dog faeces.
The major conclusion of this study is that ESBL-producing bacteria are present in high abundance in both dog faeces and dairy cattle manure and that ESBL-producing bacterial species in dog faeces showed higher diversity than first expected while pre-boil wort had no ESBL-producing bacteria.
The results of this study have crucial significance for future studies and the wider community. The statistical, microbiological and phylogenetic data collected provide strong evidence of the role of the environment in the spread of antibiotic resistance. Secondly, the study utilises a holistic visual model to map the spread of resistance and inform the wider community of the issue of AMR in the environment.
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Molecular identification of fungi isolated from coastal regions of Red Sea, Jeddah, Saudi Arabia
More LessTo isolate fungal communities from coastal areas of the Red Sea in Saudi Arabia and identify and classify them by molecular techniques. Samples were collected from the seaside of the Red Sea in Jeddah, Saudi Arabia in March 2012 and stored in sterile screw cap bottles for further analysis. Phenotypic and genotypic characterization of fungal isolates was done using standard techniques. Eight fungal genera including Aspergillus, Penicillium, Thielavia, Fusarium, Emericella, Cladosporium, Scytalidium and Alternaria. Most isolated fungi showed significant growth on petroleum media and were thus considered capable of biodegradation of crude oil-based substances. The fungal genera isolated from the Red Sea had 97 –100 % similarity with the related fungi recorded in the GenBank in which they were deposited. The morphological and molecular structure of these marine fungal isolates closely resembles their terrestrial counterparts in the Genbank. The capabilities of these fungal species to utilize petroleum as a source of carbon speaks to future applications in which marine fungi may be utilized in the breakdown of petroleum-based waste in an ecologically efficient manner.
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Bioinformatics technology in clinical and public health microbiology applying computational methods
More LessThe role of clinical genomics in infectious disease diagnostics and public health microbiology is the topic of discussion during a recent decade. Although much of this work is aimed at describing the structure of outbreak communities, the methodology works equally well to identify pathogens in clinical samples. Clinical genomics is the exploitation of genome sequence data for diagnostic, therapeutic, and public health purposes. Central to this field is the high-throughput DNA sequencing of genomes and metagenomes. The key concept in using clinical genomics methodology is that detection of microbes is independent of culture and is not limited to targets used for in-depth PCR assays. Rather, it is a process of generating large-scale sequence data sets that adequately sample a specimen for microbial content and then of applying computational methods to resolve the sequences into individual species, genes, pathways, or other features.
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Effect of fractions of toxic cyanobacterium, Nodularia spumingena KAC 66 and its purified nodularin and nodulopeptin 901 on inhibition of colorimetric protein phosphatase 1 assay and lethality against two species of Daphnia
More LessThe strains of Nodularia are known to produce potent hepatotoxic nodularins (NOD) and other bioactive metabolites i.e. nodulopeptins and spumigins. Contemporary, three new nodulopeptins were also isolated from N. spumigena KAC 66 collected from the Baltic Sea. The wide distribution and toxicological effects of N. spumigena on food chain, has caused significant attention towards the effects of the species. In the present investigation the toxicity of NOD and nodulopeptin 901, produced by N. spumigena, was fractionated by Reversed Phase Flash Chromatography (RPFC) and their toxicity was determined by their lethality to water fleas, Daphnia pulex and D. magna along with inhibition of protein phosphatase 1 assay (PP1; eukaryotic threonine phosphatase/protein serine). All fractions showed lethality to Daphnids and inhibitory activity against PP1, the toxicity was due to additional compounds as NOD and nodulopeptin 901 were only detected in 7 fractions. The pure NOD was lethal to D. pulex and D. magna LC50=8.4 µg. ml-1 and 5.0 µg .ml-1, respectively. The newly characterized nodulopeptin 901 was also tested against D. magna (LC50=>100 µg .ml-1). NOD and nodulopeptin 901 inhibited PP1 with weak IC500.038 μg .ml-1 and 25 µg .ml-1, respectively. The anabaenopeptin A (ANA) and anabaenopeptin B (ANAB) were used as reference peptides as they have similarity in structure with newly characterized nodulopeptin 901. The ANA, ANB and linear nodularin (LNOD) inhibited PP1 with IC5070μg .ml-1,100μg .ml-1 and20μg .ml-1, correspondingly. This is the first study that indicates the toxic and inhibitory activities of nodulopeptin 901 and fractions of N. spumigena KAC 66 against daphnids and PP1.
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Effect of different biocoagulants on the amino acid content of soft cheese (wara) produced from sheep milk
More LessSoft cheese (wara) is an unripened cheese consumed in several parts of West Africa due to its various nutritional qualities. Soft cheese, a coagulated product of raw milk is usually produced from cow milk using Calotropis procera. This study therefore sought to assess the effect of the different coagulants such as Calotropis procera, Carica papaya, lemon juice and steep water from cereals (maize, millet and sorghum) on the amino acid content of soft cheese produced from sheep milk. Raw milk sample was collected from sheep and processed into soft cheese by these coagulants and the amino acid composition of the sample was carried out using standard methods. The result revealed that Calotropis procera coagulated soft cheese has the highest essential amino acid content Leucine (10.21 g/100 g), while steep water from millet coagulated soft cheese has the lowest essential amino acid content methionine (0.72 g/100 g). However, lemon juice coagulated soft cheese has the highest non essential amino acid glutamic acid (16.27 g/100 g) in all the cheese samples. In conclusion, this study revealed that highly nutritious soft cheese can also be gotten from sheep milk other than the commonly used cow milk and other coagulants such as lemon juice can compete favorably well with Calotropis procera in production of highly nutritious soft cheese. It is therefore recommended that soft cheese produced from sheep milk coagulated by lemon juice should be incorporated into daily diet due to its highly nutritional content.
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Molecular identification of Bacillus spp. as starter culture for the production of ugba (Pentraclethra macrophylla), a Nigerian fermented condiment
More LessThe isolation, physiological and molecular identification of Bacillus spp. for the development of starter culture in Ugba production were carried out. Sixty two bacteria were isolated and 55 Phynotypically characterized as Gram positive, rod shaped, motile, spore-forming and catalase positive typical characteristics of Bacillus spp. Seven isolates were suspected to be Micrococcus and Staphylococcus spp. They were Gram negative, cocci and catalase negative. The genotypic identification of the suspected Bacillus spp. was done using amplification and partial sequencing of the 16S rRNA gene. The sequence analysis of 16S rRNA gene fragment analyzed from isolates confirmed the organisms as Bacillus cereus (30), Bacillus subtilis (17) and Bacillus licheniformis (8). Bacillus subtilis was the dominant species in Ugba fermentation as it had the highest recorded number of isolates. This study indicated that molecular characterization of organisms as a good tool for the identification of organisms for the development of starter culture for use in food fermentation.
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Characterization and identification of corrosive bacterium isolated from petroleum product transporting pipelines
More LessCorrosion is the main feature of the oil pipelines destruction. Biocorrosion has been detected in various industries, especially in the oil industry. The rusted pipes samples were obtained from the Gandomkar petroleum pipeline station, Iran. For screening the corrosion producing bacteria, the samples were cultured in the selective culture medium, manganese agar and Iron oxidizing agar incubated at 30 °C for 18 h. The purified individual colonies were subjected to macroscopic, microscopic and molecular examinations, respectively. The cultivation of corrosion based material on manganese agar isolated cream-coloured colonies, convex with the surrounding smooth. The microscopic examinations showed Gram-negative coccobacilli. Based on macroscopic, microscopic and molecular examinations the bacterial isolate was identified as Stenotrophomonas maltophilia strains PBM-IAUF-4 with the accession number of KU145280.1 in Gene Bank. The cultivation of corrosion based material on Iron oxidizing agar isolated cream-coloured colonies cream-coloured colonies that had swarming, convex. The results showed Gram-negative bacilli. Based on macroscopic, microscopic and molecular examinations the bacterial isolate was identified as Kluyvera intermedia strains PBM-IAUF-1 with the accession number of KU145277.1 in Gene Bank. This is the first report of isolation and identification of corrosion-producing bacteria from, Gandomkar, Iran. The first isolated bacterium was identified as Stenotrophomonas maltophilia. The second isolated bacterium was identified Kluyvera intermedia. Both bacteria were isolated for the first time in the world from pipeline corrosion samples. This study confirmed the role of bacteria in the corrosion of oil pipelines.
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Differential amplicons for the evaluation of RNA integrity extracted from complex environmental samples
More LessReliability and reproducibility of transcriptomics-based studies are dependent on RNA integrity. Microfluidics-based techniques targeting rRNA are currently the only approaches to evaluate RNA integrity. However, the relationship between rRNA and mRNA integrity is unknown. Here we present a new integrity index, the Ratio amplicon, Ramp, to monitor mRNA integrity based on the differential amplification of RT-Q-PCR amplicons of the glutamine-synthetase A (glnA) transcript. We showed, in a suite of experimental degradations of RNA extracted from sediment, that while the RIN generally reflected the degradation status of RNA the Ramp mapped mRNA degradation better. Furthermore, we examined the effect of degradation on transcript community structure byamplicon sequencing of the 16S rRNA, amoA and glnA transcripts. We successful sequenced transcripts for all three targets even from highly-degraded RNA samples. While RNA degradation changed the community structure of the mRNA profiles, no changes were observed for the 16S rRNA transcripts profiles. Since both RT-Q-PCR and sequencing results were obtained, even from highly degraded samples, we strongly recommend evaluating RNA integrity prior to downstream processing to ensure meaningful results. For this both the RIN and Ramp are useful, with the Ramp better evaluating mRNA integrity in this study.
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Blessing in disguise: a protocol to quantify biofouling effects on drinking water electrochemical sensors is a gateway to investigate biofilm using electroanalytival methods
More LessProvision of clean drinking water is regarded as being the most significant positive intervention in human health and it plays a significant role in supporting global health. Population growth, economic development and climate change all drive urbanisation and increase demand for clean water and the infrastructure for its delivery. As demand for clean water increases, so will the pressure to ensure its safety. Indwelling sensor networks offer real-time, long-term and intelligent monitoring system, and would enable optimisation of networks for quality. Electrochemical sensors are inexpensive and simple to construct and operate. However, prolonged exposure of the sensors to water causes biofouling which compromise their performance even in weeks or days, making early detection of performance failure critical. By using a combination of electroanalytical methods (cyclic voltammetry, chronoamperometry, electrochemical impedance spectroscopy and hydrodynamic voltammetry) we can quantify the mass transport and kinetics effects of the first six days old biofilm layer to electrode reactions. We used agarose hydrogels and cellulose acetate layer as model biofilms to provide rapid and simplified characterisation of early fouling effects which then compared with lab grown biofilm of Pseudomonas fluorescens. We present a protocol forin situdiagnosis of electrochemical sensors fouling during early stages of biofouling. We also demonstrate that the behaviour of lab grown biofilm can be approached by using a simple model made of hydrogels (i.e. agarose) or a dried layer of an organic solution (i.e. cellulose acetate) which provides a potential application in other fields beyond environmental sensors development such as infectious disease and novel antibiotic researches.
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Root-associated archaea: investigating the niche occupied by ammonia oxidising archaea within the wheat root microbiome
Root associated microbiomes (RAMs) are complex communities which provide benefits to host plants via disease suppression, abiotic stress relief and increased nutrient bioavailability. Most RAM studies have focussed on bacteria and fungi, archaea have largely been overlooked as many studies fail to utilize archaea-specific 16S primers. However, there are reports of archaea being detected and isolated from the rhizosphere and endosphere of crop species, and one report of a plant-growth promoting (PGP) ammonia oxidising archaeon (AOA). Here, we aimed to assess the role of AOA within the wheat (Triticum aestivum) RAM. We applied archaea-specific primers and 16S amplicon sequencing to profile the archaeal community associated with wheat roots grown in agricultural soil. To assess PGP capacity we treated wheat seeds with a concentrated inoculum of model AOA Candidatus Nitrosocosmicus franklandus C13. In contrast to prior reports this had no impact on plant biomass, indicating N. franklandus may be a passive member of the wheat RAM. Stable isotope probing (SIP) experiments have confirmed that bacterial species metabolise Arabidopsis thaliana root exudates. Fractions are being examined to assess whether archaeal species can do the same, and a similar SIP experiment will be performed in wheat. An enrichment culture experiment using root exudates will also be applied to identify and isolate archaea capable of metabolising wheat root exudates. Here we show that AOA are present within the wheat RAM; to understand the niche occupied by these microbes we must further probe how they interact with host metabolites, and whether they contribute to host fitness.
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The selective sequestration of bacterial populations using polymeric glycomaterials
More LessThe ability to selectively sequester bacteria from a mixed population is a desirable aim across a range of fields. Already some technologies exist to attempt to meet these aims spanning microfluidics and automated flow cytometry to antibody conjugated magnetic nanoparticle precipitation. However, these technologies are generally limited to the confinement of small populations in defined locations. Furthermore they may not be able to differentiate morphologically similar but phenotypically divergent cells. We aim to produce a novel technology to isolate bacteria from mixed populations by utilising polymeric carbohydrate ligands which bind to inducible bacterial adhesion proteins. To achieve this aim an inducible mutant of the fim operon in Escherichia coli has be constructed, thus allowing for switchable production of the mannose binding organelle, Type 1 fimbriae. Furthermore, we have previously observed that mannosylated polymers selectively bind to Type 1 fimbriated E. coli and to our induced mutant. Novel mannose functionalised polymers are being synthesised via a reversible addition-fragmentation chain transfer (RAFT) scheme. These polymers contain a catechol terminus which may be conjugated to magnetic Fe3O4 nanoparticles thus facilitating selective bacterial sequestration by magnetic separation. The successful development of this polymer-based bacterial sequestration platform could potentially enable the equivalent of immunoprecipitation in large-scale fermentation processes or the precise manipulation of living cells in laboratory scale procedures.
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Metagenomic analysis reveals significant seasonal variations in the epiphytic bacterial communities associated with different parts of the brown seaweed Laminaria digitata
More LessBrown seaweeds such as Laminaria species are a rich source of polysaccharides such as laminarin and fucoidan which have a variety of functional food and animal feed applications, as well as alginates with demonstrated biological and pharmacological activities. Macroalgal surfaces are rich in carbon-based constituents which provide a suitable environment for growth and colonization by diverse bacterial communities. Several environmental and non-environmental factors can influence the composition and abundance of epibacterial communities associated with seaweeds. In addition to the biological, physical and chemical properties of the macroalgal surface, seasonal variations have been found to play a significant role in the structure of the associated microbial communities. Variations in macroalgal epibacterial communities have also been observed within different parts of the host algal species. However, to date, in-depth studies on bacterial communities associated with macroalgal species, their ecological role and interactions with the algae are still scarce. To gain an insight into the diversity and composition of the microbial communities associated with the brown alga Laminaria digitata, the communities derived from different parts of the alga including the blade, meristem, stipe and holdfast; were investigated using metagenomic Illumina sequencing of 16S rRNA gene amplicons. Seasonal variations in the microbial populations were found in samples taken from the Irish coast in different seasons between 2017 and 2018. This metagenomic-based investigation provides a detailed view of the seasonal variations in the bacterial populations associated with Laminaria digitata and helps provide further insights into potential interaction between this macroalga and its epiphytic bacterial communities.
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Occurrence of extended-spectrum β-lactamase (ESBL)/AmpC producing bacteria in wastewater treatment plant effluent
More LessExtended-spectrum β-lactamase (ESBL)/AmpC producing bacteria are one of the critical priority resistant bacteria that contributes to treatment failure and increased death rates. In this work we aimed to study the role of wastewater treatment plants (WWTPs) as reservoirs of ESBL/AmpC producing faecal coliforms. The effluent samples were collected from two WWTPs and faecal coliforms were isolated from all samples using the membrane filtration method. Bacterial isolates were subjected to antimicrobial susceptibility testing toward cefotaxime and ceftazidime. The isolates that showed a resistance phenotype to these antibiotics were considered as putative ESBL/AmpC producing bacteria. These bacteria were subjected to the AmpC test using a protocol with phenylboronic acid. The AmpC negative strains in the AmpC test served as samples for multiplex PCR containing primers specific for blaTEM, blaSHV and blaCTX-M. In total, 498 faecal coliforms were isolated from WWTP effluent samples. For the antibiotic susceptibility testing 99 isolates were considered as ESBL/AmpC producing bacteria. Among them, 26 isolates were found to be positive in the AmpC test. The PCR results revealed that 49 isolates carried blaTEM, 6 bla SHV12, 1 blaCTX-M1 and 5 blaCTX-M15. The ESBL/AmpC producing faecal coliforms in WWTP effluent are discharged to the receiving water environment. These data need to be considered when analysing the risk of WWTP effluent to the environment and to human health, as many of the bacteria identified are not analysed in assessment of risk of pollution from WWTPs globally.
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The optimization of microbial induced calcium carbonate precipitation in soil improvement using engineered bacteria
More LessPoor soil conditions limit the building of new infrastructure, which is needed for an ageing and expanding population. Current soil strengthening techniques such as chemical grouting have detrimental effects on the environment from greenhouse gas production, soil pH modification and groundwater contamination, therefore there is demand for a sustainable approach to this process. Microbial-induced calcium carbonate precipitation (MICCP) is a technique that utilises the ability of bacteria to precipitate calcium carbonate (CaCO3), which can be used for a variety of applications including binding adjacent soil particles and filling the pore spaces of soils to increase mechanical properties. Commonly used bacteria include Sporosarcina pasteurii and Bacillus subtilis. A range of factors influences MICCP which presents challenges with process optimisation. These factors need to be optimised in the laboratory before they can be applied for engineering purposes. The overall aims of my research are to optimise urease production in S. pasteurii and B. subtilis and to investigate the distribution and binding of these bacteria with various sand particles, by means of syringe and glass column set ups. These bacteria will be compared with engineered bacteria which can overproduce urease to investigate the impact on precipitation efficiency. Factors to control bacteria biofilm formation to influence the morphology of CaCO3 will be investigated to determine the impact of various crystal shapes on soil properties. Ultimately, raw data generated from the project will be used for predicting biocementing at a lab scale for building computational models.
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How are bacteria involved in the detrimental health outcomes caused by exposure to air pollutants?
With an annual death toll of 7 million, the effect of air pollution on human morbidity and mortality is a major global problem. Air pollution associated with infectious respiratory disease is responsible for a mortality rate of 1.5 million annually. However, the impact of air pollution on bacterial behaviour is largely unknown. Our work has shown for the first time that black carbon (BC), a major component of air pollution, increases dissemination of colonising Streptococcus pneumoniae and Staphylococcus aureusin in-vivo infection models, and also alters biofilm structure, composition, and function. However, the biological mechanisms responsible for dissemination in the host and biofilm alterations by BC are unknown. BC is known to elicit an oxidative stress in eukaryotic tissues, therefore we hypothesise that the S. aureus oxidative stress response may play an important role in the bacterial response to BC. Our data shows that exposure to BC is toxic to S. aureusand that oxidative stress response of mutant strains show increased sensitivity to BC than the wildtype strain. Transcriptional analysis demonstrated that the expression of key oxidative stress genes in the oxidative stress response pathway are induced in the presence of BC. These findings demonstrate that BC has a metabolic effect on S. aureus and that the oxidative stress response is required for bacterial survival to BC. Furthermore, the induction of the S. aureus oxidative stress response may be important for increased dissemination in the host through adaptation of bacterial cells to the host immune response.
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The impact of manure application on the microbiome of grassland
More LessManure spreading onto land is an important agricultural process. Manure is recycled as organic fertiliser; however it can introduce manure-derived antibiotic resistant bacteria into the environment. Grassland consists of approximately 70 % of global agricultural land and is a vital source of food for livestock. Despite the important role grassland plays in food security, the impact of manure application on its resistome and microbiome is relatively unknown. Antibiotic resistance is a multifactorial issue, involving an intertwining relationship between animals, humans and the environment. Therefore, it is critical to fully understand all potential routes of antimicrobial resistance (AMR) transmission. As the microbiome of grassland is an under-researched area, it is a possible source of AMR transmission to animals which may enter the food chain. A pot trial mesocosm experiment was carried out to investigate the impact of manure application on the microbiome of the phyllosphere of perennial ryegrass (Lolium perenne). Pig slurry was applied to six pots of L. perenne and grass and soil samples were taken two weeks following manure application. Following sonication, viable bacteria were isolated from the soil, manure and grass by plating on selective agars supplemented with antibiotics. Isolates were screened for antibiotic resistant bacteria by antibiotic susceptibility testing. DNA was extracted from the soil, grass and manure and underwent microbial community compositional analysis by 16S rRNA sequencing on the Illumina Miseq platform. The results from this mesocosm experiment will contribute to a further field trial to investigate the impact various manure types have on the microbiome and resistome of grassland.
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Fungal transformation of cobalt-bearing minerals and metal bioprocessing applications
More LessGeoactive fungi such as Aspergillus niger play a significant role in bioweathering processes and element cycling. These organisms are able to secrete a range of organic acids, such as oxalic acid, into their microenvironment. This enables them to mediate mineral dissolution, leading to metal solubilization and precipitation in the form of secondary biominerals. In this investigation, such biotransformation processes were explored as a means of cobalt bioprocessing, an E-tech element identified as being of key strategic importance, in addition to other mineralogically related metals. A range of Co-bearing mineral phases were investigated, including a Co-bearing lithiophorite [(Al,Li)MnO2(OH)2] and erythrite (Co3(AsO4)2·8H2O), in addition to seafloor ferromanganese nodules. Bioleaching and bioprecipitation studies were carried out to investigate the ability of A. nigerto leach cobalt and related metals from Co-bearing minerals, and to precipitate them in biomineral form as a means of cobalt biorecovery. The objective of the work is to investigate the natural biotransformation of cobalt-bearing minerals, to investigate the factors that influence cobalt bioprocessing and to optimise the maximal yield of cobalt biominerals.
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Characterization of Enterococcus faecium SP15, a novel isolate with probiotic and therapeutic potential
More LessAimsTo characterise the genome and enterocin content of Enterococcus faecium SP15, a new isolate from natural spring water.
Methods and ResultsEnterococcus faecium strain SP15 was isolated from natural spring water and its identity confirmed using 16S rDNA sequence. The bacterial strain produced antimicrobial compounds (enterocins) against pathogenic bacteria including Listeria monocytogenesis as determined by the agar spot method. In addition, an anti-cancer activity was observed on human cancer cell line HT-29 by cytotoxicity assay (MTT) and apoptosis study. A draft genome sequence revealed the presence of several enterocin genes capable of activity against a panel of pathogenic bacteria including L. monocytogenesis. The active production of enterocins was also supported by the presence of peptides in part purified fermentates following trypsin digest and mass spectrometry. The genome showed no classical virulence factors or hemolysins and was free of antibiotic resistance genes. To confirm enterocin related killing, select enterocin genes were cloned and expressed as His-tagged proteins in E. coli. Purified enterocin retained activity in L. monocytogenes overlay assay.
ConclusionE. faecium SP15 is a promising strain for probiotic use and/or food preservation. Purified enterocins retain their activity suggesting them a template for structure-function studies and future improvements as antimicrobial and cancer cell therapeutics. Significance and Impact of the Study: New antibacterial and anticancer agents are required to combat antibiotic resistance and a limited drug repertoire. Enterocins from Enterococcus faecium SP15 could fulfil these goals in addition to use of the strain as a whole organism probiotic.
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Penetration of the air–liquid interface is key to the Wrinkly Spreader success
More LessThe adaptive radiation of Pseudomonas fluorescens SBW25 populations in static liquid microcosms results in the appearance of the biofilm–forming Wrinkly Spreader. This adaptive mutant is able to colonise the high O2-rich region at the top of the liquid column established by the metabolic activity of earlier wild-type colonists, and by doing so, enjoys a significant fitness advantage over non-biofilm–forming competitors including the ancestral Pf. SBW25. Although the underlying molecular biology and evolutionary ecology of the Wrinkly Spreader is well understood, we have recently questioned the need for expensive biofilm–formation to colonise the air-liquid (A-L) interface, as O2-directed flagella-mediated swimming (aerotaxis) should be sufficient to maintain cells in this region. Our investigations show that swimming can overcome displacement by Brownian diffusion and microcurrents within the liquid column. However, it is not sufficient to explain the high levels of enrichment at the A-Linterface shown by Wrinkly Spreader cells. A comparison of the liquid surface tension of wild-type and Wrinkly Spreader cultures, supernatants, and washed cells, suggests that the Wrinkly Spreader produces a surface-active compound weakly associated with the cell which helps penetration of the A-L interface and allows cells to remain in the high-O2 region without further expenditure of energy. Our results suggest that this penetration is key to the following biofilm–formation which supports higher populations at the A-L interface and that this explains the adaptive advantage of the Wrinkly Spreader.
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Fungal biodeterioration of copper metal
More LessIn this work, geoactive fungi including Aspergillus niger, Beauveria caledonica and Paecilomyces javanicus, were used to investigate their biocorrosion and deteriorative effects on copper metal to gain an understanding of the roles that fungi may play in biodeterioration of such a material in the built environment. It was clearly demonstrated that the test fungi possessed a high tolerance to copper metal. New biominerals resulted from fungal interactions with copper metal mainly arising from organic acid excretion. Copper oxalate was formed by oxalate excretion from the fungi and different patterns of bioweathering and biomineralization were generated on the copper surfaces. In addition, copper could be dissolved by certain fungi, result in significant biodeterortive effects such as etching and pitting. These results provide compelling evidence for deteriortion of copper metal by fungi and that organic acids, particularly oxalate, play an important role in this process. Such properties of metal biocorrosion and deterioration indicate the potential significance of fungi in biodeterioration of metal substrates and the importance of considering methods of protection and preservation in the built environment.
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Use of cellular and high throughput genetic approaches to unravel the antibacterial mechanism of honey
More LessThe medical importance of honey has been extensively demonstrated. Although high osmolarity, acidity and hydrogen peroxide (H2O2) proved to be the most prevalent factors in honey’s activity, the underlying antimicrobial mechanism remains obscure. Our aim is to provide insight into the physiological changes and genetic responses in honey-treated bacteria, thus improving our understanding of this natural product as a potential novel antimicrobial. A model honey composed of sugars, gluconic acid, and H2O2as they are accumulated in honey after enzymatic reaction happens, was used the investigation of honey’s activity. The bactericidal action of the model was tested on E. coli K-12 strain MG1655. Flow cytometry (FC) and Atomic Force Microscopy (AFM) identified physiological changes such as membrane potential, blebbing, and cell lysis. Reactive Oxygen Species (ROS) accumulation was observed in individual cells by FC. Transposon Directed Insertion Sequencing (TraDIS) identified mutants’ fitness over a time course of E. coli treatment by model honey. The loss of selenocysteine (selAB) and formate dehydrogenase (fdhDE) mutants, proved the redox- balancing activity as essential for the repression of ROS in stressed cells. High susceptibility of energy metabolism (atpABD) and peptidoglycan synthesis (prc) mutants, indicated the strain unable to maintain the reductive cell environment necessary for cellular activities, post honey exposure. Our findings identified some of the honey’s targets when acting as an antimicrobial. The synergies observed support the use of honey as an antimicrobial; however, the identification of mutations that led to enhanced resistance to honey is an important finding that needs further study.
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Sustainable natural production of vitamins for human consumption in long space missions using synthetic ecology approaches
More LessThe MELiSSA (Micro Ecological Life Support System Alternative) project aims to create a closed loop system, capable of providing all the necessary food, water and oxygen for astronauts on long Space missions. The MELiSSA loop is comprised of four compartments, with compartment IVA containing photoautotrophic bacteria, such as Arthrospira platensis, a good source of oxygen, edible biomass and micronutrients. One important example of the latter is cobalamin (B12) deficiency of which can lead to pernicious anaemia and neurological systems, and thus would be detrimental to astronauts on long Space missions. The molecule is not made by plants or fungi, so the ultimate source of B12 in the environment is prokaryotes, and its synthesis requires over 20 enzymatic reactions. Many eukaryotic algae also require cobalamin for growth, and some species have been shown to accumulate the vitamin when grown in coculture with cobalamin synthesising bacteria. The aim of this project is to extend existing knowledge of algal-bacterial mutualisms involving cobalamin. Eukaryotic species such as Haematococcus pluvialis and Chlorella vulgaris, both certified as safe for human consumption, along with A. platensis will be investigated with different bacterial partners to maximise cobalamin accumulation. Not only will this research help provide adequate nutrition on long Space missions, it will also support nutritional supply on Earth with the rise of veganism, as well as, aiding in understanding the dynamics of algal-bacterial mutualisms, which is of interest in terms of nutrient cycling in the environment.
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Effect of temperature increase in bacterial and fungal communities of chlorinated drinking water distribution systems
More LessDrinking Water Distribution Systems (DWDS) are diverse ecosystems where the majority of microorganisms live forming biofilms, which can alter the water quality if they are mobilised to the bulk water. Biofilm communities can be affected by the increase of temperature due to climate change, thus compromise the distribution of safe water. To understand the effect of temperature on biofilms in DWDS, biofilm was developed for 30 days at 16 °C and 24 °C using a full-scale experimental DWDS facility. Samples were collected at the end of the experiment from removable coupons inserted into the pipes. DNA was extracted and the 16S rRNA and ITS rRNA genes were sequenced and analysed, for the bacterial and fungal diversity respectively. Differences in bacterial and fungal diversity at both temperatures were observed at family level. At 16 °C bacterial community was dominated by Comamonadaceae (21.48 %), Pseudomonadaceae (16.41 %) and Sphingobacteriaceae (12.99 %). However, at 24 °C the most abundant family was Pseudomonadaceae (50.60 %) followed by Sphingomonadaceae (9.59 %) and Sinobacteraceae (7.82 %). Fungal diversity showed that at 16 °C the most abundant family was Nectriaceae (68.9 %), followed by Helotiales (24.5 %) and Filobasidiales (1.5 %). However, at 24 °C the community was dominated by Nectriaceae (98.15 %) and the following families showed a low relative abundance, Rhizopodaceae (0.95 %) and Cryptomycota (0.24 %). Temperature is a key factor for microbial growth in DWDS and affect the composition of the microbial communities. Temperature increase leads changes and a loss in complexity in bacterial and fungal communities of biofilms, which can affect the water quality.
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Role of oxalate in fungal precipitation and biorecovery of lanthanum
More LessLanthanum is an important member of the rare earth elements (REE) that are a global strategic resource and have many technological applications ranging from microelectronics manufacturing to the production of clean and renewable energy. Aspergillus niger is widely used in industrial fermentations due to its production of multiple secondary metabolites including citric and oxalic acids. Since it is a ubiquitous soil inhabitant and produces geoactive agents, it can play a role in the biotransformation of metal-containing minerals. Previous studies found that A. niger is capable of mineral solubilization and secondary mineral formation, many metals being precipitated as oxalates. However, there is limited knowledge about the biotransformation of La mediated by fungi. The aim of this project was to explore the mechanisms and factors determining the interactions between A. niger and La. In this study, fungal growth on La-supplemented solid media was carried out and it was discovered that crystalline deposits were formed around fungal colonies in the presence of LaCl3. These biogenic crystals were recovered and subjected to examination for their elemental composition, morphological features and mineral phases using energy dispersive X-ray analysis (EDXA), scanning electron microscopy (SEM) and X-ray diffraction (XRD) respectively. These confirmed the biotransformation of lanthanum and identified the products as lanthanum oxalate [La2(C2O4)3·10H2O], which was further transformed into La2O3 by thermogravimetric (TG) treatment. Geochemical modelling also supported these results. Our findings provide a new aspect for the bioprecipitation and biorecovery of REE from solution using fungal culture systems.
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MICROALARM: “a new system for rapid quantification of the total bacteria in water samples”
BackgroundAnalytical methods applied in diagnostic microbiology laboratories are usually performed manually and have several drawbacks, the long time-to-results (48–72 h) being the major one. Rapid detection methods may overcome the disadvantages of traditional microbiological methods and achieve fast detection, which will help preventing the spread of waterborne pathogens and outbreaks of waterborne diseases. The objective of the MICROALARM project is to validate a platform for monitoring the quantification of the total bacteria present in water samples.
MethodsThe MICROALARM system integrates a microfluidic cartridge where the microorganisms from the sample are labelled to facilitate the detection and measurement by a fluorescence sensor. The prototype system has been validated according to ISO 16140-2 : 2016 in the laboratory through the comparison with conventional (culture isolation) and alternative methods (flow cytometry) to determine whether the method is suitable for quantifying microorganisms in water samples.
ResultsThe system will be capable of processing, labelling and quantifying automatically the total bacteria of the water sample using a membrane-permeable fluorescence marker. The system has been validated at the laboratory scale using a wide range of water matrices, then installed and validated in different facilities with satisfactory results.
ConclusionThe MICROALARM system is designed to be portable, thus suitable for on-site applications. It will be a low-cost solution for rapid microbiological analysis. The system will avoid water sampling and transport to the laboratory while enabling on-site quantification of the total bacteria in a reduced timeframe for early decision-making.
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Genomic trends on the biogenic CaCO3 production in the genus Bacillus
More LessThe biogenic production of CaCO3 via Microbially Induced Calcite Precipitation (MICP) has been reported in many microbial strains. This process has a wide variety of current and potential applications, namely in civil engineering, agriculture and bio-remediation. The number of species used in such applications remains limited and seems to be biased towards ureolytic strains. Urea degradation is the best documented process leading to MICP, however, there are several alternative processes, possibly more relevant, which are mostly overlooked. In general, and despite being widely reported, the MICP process is still poorly understood, and has been chronically understudied from a genomic perspective. Here we report on the genus-wide analysis of MICP capability, centred on genomic-based analysis, and focusing on the genus Bacillus. This genus harbours several species capable of MICP and is the most widely used regarding its biotechnological application. The very high number of species within this genus, and availability of whole genome sequence data for several makes it an ideal target for this analysis. Our preliminary results uncover a diverse range of MICP-associated genes, identifies similar genomic profiles within phylogenetic subgroups, and questions the importance of urease activity for CaCO3 production in the genus Bacillus. This study is the first of its kind and provides key insights into the genomic basis of MICP, while testing the feasibility of a genomic-based prediction method for fast identification of new strains with such capabilities, which would be applicable to other genera and be particularly useful for downstream applications.
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Genomic mining of thermophilic actinobacteria from compost for novel antimicrobials
More LessActinobacteria have provided a rich source of novel antimicrobial compounds throughout the antibiotic era. Thermophilic Actinobacteria growing at higher temperatures (>50 °C), have not been extensively studied, despite producing important antibiotics such as thermomycin and anthramycin. We are testing the hypothesis that thermophilic Actinobacteria produce new and unusual antimicrobials at higher temperatures, potentially leading to the discovery of novel heat stable compounds; especially those active against life-threatening fungal infections in humans such as invasive aspergillosis caused by Aspergillus fumigatus, which has developed resistance to current treatments. Compost is a rich source of thermophilic Actinobacteria responsible for generating the heat required for decomposition and yet this niche has been overlooked in terms of natural product discovery. A. fumigatusis a fungus that also lives in compost and also contributes to the composting process and we reasoned that Actinobacteria living in this environment might display activity against pathogenic strains of the fungus. Samples from a series of ‘windrows’ at a commercial green waste processing facility yielded 13 thermophilic Actinobacteria, and strains of Aspergillus fumigatus. The phylogeny and species identity of the bacterial strains were determined by 16S rRNA sequencing. Candidate strains were screened for the ability to inhibit ESKAPE pathogens as well as A. fumigatus, using agar overlays and MIC assays. Selected strains were analysed by whole genome DNA sequencing and likely antimicrobials predicted. Compound identification using mass spectrometry and metabolic profiling has been undertaken on strains that display antibiotic activity, providing a path for the development of new antimicrobials for clinical use.
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The environmental microbiome toolkit for urban designers
More LessEnvironmental microbiome research shows that microbial communities help to shape complex ecological processes that influence human and environmental health. Researchers are currently investigating the potential health-inducing interactions between humans and the environmental microbiome, particularly in urban areas. However, not only are there inherent technical issues to overcome in implementing the findings of this research, but there are also complex social, political and economic factors that affect the design, construction and management phases. Drawing on a recent paper discussing opportunities for Microbiome-Inspired Green Infrastructure (MIGI) (Robinson et al. 2018), we set out criteria for an ‘Environmental Microbiome Toolkit for Urban Designers’ that could be used by planners, architects, landscape architects and civil engineers. We provide a worked example of this toolkit to design a public space in Sheffield, demonstrating practical design techniques to consider the environmental microbiome and its role in human and ecosystem health. The Landscape Institute (LI) is the professional body that regulates and represents landscape architects, providing guidance across all spheres of the profession. One of the key functions of the LI is to develop and maintain the LI Plan of Work, regulating the work that landscape architects undertake at each stage of a project, from landscape assessment through to conceptual and detailed design, contract administration and landscape management. We apply these industry-standards to show how the environmental microbiome should be considered in landscape assessment, design and management, bridging the gap between research and practice and providing a common reference point for future policy development and industry regulation.
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Selenium and tellurium oxyanion reduction by yeasts
More LessSelenium and tellurium are two metalloids essential for future green energy technologies due to their associated photovoltaic and photoconductive properties. In addition, selenium and tellurium oxyanions can be toxic in the environment and can potentially affect human health. This work aims to examine some geochemical influences on Se/Te reduction carried out by selected yeast strains to identify what limitations there are to the process, and their importance. Several yeast strains, capable of selenite or tellurite reduction, were isolated from environmental soil samples on solid media containing selenite or tellurite, reduction being detected by the colour change of colonies to red (Se) or black (Te). Such reduction resulted in the formation of nanoparticles of elemental Se0 or Te0. Growth was assessed in the presence of selenite or tellurite and minimum inhibitory concentrations determined. Rates of selenite and tellurite depletion were determined in different growth conditions and the production of elemental Se0 or Te0 was analysed using energy dispersion X-ray analysis (EDXA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). This work furthers understanding of selenium and tellurium transformation by yeasts also suggests potential routes for Se/Te biorecovery by the formation of Se/Te nanoparticles.
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Biodiversity and biochemical characterization of bacteria from terrestrial subsurface environment
More LessForty-one aerotolerant subsurface bacterial colonies were isolated from terrestrial subsurface environment at various depth from 5 feet to 50 feet at 5 feet intervals. Out of 41 colonies, 32 morphologically distinct bacterial colonies were selected for gram staining reaction, various sugar utilization pattern and scanning electron microscopic studies. Most of the bacterial colonies were gram-positive rods; some are gram-negative rods, gram-positive cocci and coccobacilli, and gram-negative coccobacilli respectively. Seven bacterial colonies NTN33, NTN34, NTN35, NTN36, NTN01, NTN02 and NTN03 isolated from 40 to 50 feet depth were neither gram positive nor gram negative, and no distinct morphological shape but gigantic structure have identified in that bacteria. The bacterial size were identified on the bacteria NTN36, NTN35, NTN01, NTN30, NTN34, NTN02 as 4.5 µm, 4.1 µm, 3.5 µm, 3.4 µm, 3.2 µm and 2.2 µm. Gigantic rod surrounded by capsular sheath and gigantic rod with footprint like appearance were identified in the bacteria NTN02 and NTN36; those are isolated from 45 and 50 feet depth. Intracellular granules were also found in bacteria NTN34, NTN35 and NTN01. In total, 24 different sugars had used to analyse the sugar utilization pattern of that 32 bacteria. Most of the bacteria have utilized only 03 different types of sugars either maltose, galactose and glycerol or lactose, trehalose and mannose. However, the bacteria NTN16, NTN12, NTN06, NTN24, NTN27, NTN28, NTN34 and NTN02 had utilized 14 different sugars. Nevertheless, very few bacteria have utilized the l-arabinose, α-methyl-d-gluconate and inulin. None of the bacteria had utilized the sugar sodium gluconate, salicin, glucosamine and dulcitol.
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Microbial decomposition of porcine tissue in organic compost at different temperatures
More LessLaboratory experiments were conducted to determine the effect of temperature (20 °C and 30 °C) on the rate of tissue decomposition in organic compost for a period of 28 days. Porcine tissue was used as a substitute for human body parts in burial microenvironment. Measurements of the decay include porcine tissue mass loss, soil pH, the metabolic activity of soil microbes, viable count and Gram stain of associated microflora. Incubation temperature had a significant impact on the post-mortem decay rates and stages at 30 °C in comparison to the samples incubated at 20 °C. Bacterial enumeration demonstrated microbial burden to be higher at higher temperatures, suggesting accelerated decomposition. Gram staining identified mostly Gram-negative bacteria, decomposers that might have originated from the organic meat as opposed to soil bacteria. There was a significant difference in soil pH levels during harvest times and at the end of the experiments in both microenvironments. It is an indication that measured pH of the depository may reveal whether the body parts were recently buried. A fluorescein diacetate (FDA) method that measured metabolic activity in compost demonstrated the highest release of fluorescein during harvests at 30 °C. A non-parametric Friedman test of differences among repeated measures rendered significant results (α=0.00) showing differences in microbial activity according to the temperature level. The results suggest that depositional microenvironment would be significantly modified by the decaying organic matter with the rise of temperature. This way, the temperature would make an impact on the decay of buried remains.
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Gut microbiome of poison arrow frogs and their potential role in toxin sequestration
Poison frogs secrete alkaloid toxins in their skin as defence mechanisms against predators. Numerous studies have shown that the origin of alkaloid toxins in the skin is through ‘sequestration from diet’, i.e. uptake and storage of toxins or their chemical precursors, mostly from consumed arthropods. There exists the intriguing possibility that the gut microbiome of these frogs may play a role in this process. We address this question by looking at the organism together with its associated microbial communities, an effective symbiotic relationship between host and microbiome that could have allowed phenotypic adaptation of the host to a toxic diet. We sequenced the Bacterial and Archaeal 16S rRNA regions of the gut microbiome of 7 Poison frog species and 9 outgroup frog species caught in the rainforest of Eastern Peru. Frog species were selected based on sharing similar microhabitats and comparable individual sizes. A comparative analysis of the microbiome composition across all our samples allowed us to identify a core group of abundant symbiotic microbes unique to poison frogs in spite of intrinsic variation within species. We speculate this group could be associated to their ability to sequester toxins and we carried further metagenomic sequencing to allow us to determine possible functions that may be involved in toxin processing in these frogs.
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High-resolution 16S biogas upgrading communities: contrasting in situ and ex situ setups
More LessAs biogas from anaerobic digestion becomes an increasingly attractive biofuel, the need to improve the quality of biogas has come to the fore. Biological upgrading focuses on adding enough hydrogen to an anaerobic biogas reactor to allow methanation of the remaining carbon dioxide by methanogenic Archaea (in situ upgrading). Alternatively, biogas and hydrogen can be mixed in the absence of feedstock, in a reactor operated exclusively to facilitate methanogenesis (ex situ upgrading). This novel technology can encounter inhibition at high loading rates of hydrogen: however, in contrast to anaerobic digestion, the dynamics of this thermophilic functional microbial community are sparsely characterised. High-resolution 16S rDNA community profiles from four anaerobic biogas upgrading reactors were constructed to determine how feedstock, hydrogen, and CO2 influence biomethanation. Presence/absence of a feedstock led to large differences between in situ and ex situ communities, determining the dominant methanogen genera, and encouraging distinct populations of hydrolysing and fermenting Firmicutes. Although high hydrogen flow rates (∼37 L/day) caused a collapse in methanogenic Methanothermobacter populations in situ, ex situ hydrogen rates greatly exceeded these levels (∼400 L/day) without collapse of Methanobacterium, despite some observed instability and proliferation of likely homoacetogens. Subsequent reduction of hydrogen rates ex situ (259 L/day) appeared to create a niche for hydrogen production, indicated by increased abundance of various syntrophic fermenters known to supply biogenic hydrogen. In either upgrading setup, instability due to increased hydrogen levels manifested as a disruption of fermenting and hydrolysing populations prior to disruption of methanogens.
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Assessing the impact of chemically engineered surface modifications with respect to attachment, survival and the development of microbes at the cellular level
More LessRecently scientific and industrial interest has grown in relation to antimicrobial surfaces. This interest is mainly due to the persistent and prevalent microbial contamination of industrial and medical surfaces. A critical issue is the dissemination of bacterial colonies across biotic and abiotic surfaces, from surface contact-contact interaction. This ability to colonise materials presents a major problem for cross-contamination and pathogenic bacterial proliferation, resulting in wide-spread distribution and mutation, presenting increased risk to human health. There are two approaches to prevent bacterial spread – disinfection and antimicrobial surfaces. The use of disinfectants presents pollution to the surrounding environment, and the increased development of resistant microbial strains. The beneficial design of antimicrobial polymers enables contact-killing, without the release of biocides into the environment. This project aimed at synthesising sulfur polymeric materials through inverse vulcanisation, with the aim of producing functional antimicrobial materials. As of yet, there is no publication evaluating the antimicrobial effect of diisopropenyl benzene (DIB) and dicyclopentadiene (DCPD) co-polymers as bulk solids, even though elemental sulfur is known to exhibit antimicrobial effects. There is growing demand for materials with antimicrobial capabilities, especially in medical environments, where the epidemiology of hospital acquired infections is of great research interest. The aim of this study was to evaluate the antimicrobial properties of both S-DIB and S. DCPD using Escherichia coli (DSM 1576) and Staphylococcus aureus (DSM 346) against an internationally recognised standard (ISO 22196). To gain further in-depth analysis, confocal microscopy was employed to access the surface impact on bacterial cells.
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Antibiotic resistance transfer in Enterococcus faecalis via pheromone-induced conjugation
More LessIt is acknowledged that the environment facilitates antibiotic resistance development and spread but not much research has been undertaken to understand the mechanisms involved. This study focuses on antibiotic resistance transfer by enterococci, normal inhabitants of the mammalian gut and important healthcare associated pathogens, under a variety of environmental conditions. These are novel environmental strains, isolated from a farm in Monaghan, Ireland. The primary aim of this project is to quantify factors controlling the horizontal gene transfer (HGT) of antibiotic resistance genes via pheromone-induced conjugation. The main objectives include; identifying enterococcal isolates capable of transferring antibiotic resistance genes under laboratory conditions; using whole genome sequencing (WGS) to characterise genetic events involved in conjugal transfer; developing markers of transfer efficiency; and developing environmentally relevant models to measure enterococcal HGT. The transfer of vancomycin resistance genes from a donor to a recipient cell have been demonstrated and transfer efficiencies range from 1.09×10−1 to 9.74×10−5. This data shows that some donors are better at donating vancomycin resistance genes than other donors and some recipients are better than other recipients at receiving them. Preliminary data also shows the transfer of trimethoprim, tetracycline and erythromycin and we can see that within a localised environment Enterococcus faecalis can spread resistance genes very differently. In conclusion this project hopes to further our knowledge of the mechanism of pheromone-induced conjugation in environmental Enterococci and to show that environmental enterococcal strains can become multi-drug resistant just as easily as clinical isolates.
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Investigating methane mitigation in beef cattle fed with natural additives
More LessAntibiotics are extensively fed to beef cattle as they act as rumen modulators, improving animal efficiency and decreasing methane emissions. However, current recommendations by health agencies to limit/ban antibiotic use in animal production call for alternatives such as natural additives. In this study, we aimed to evaluate the efficacy of natural additives in methane mitigation. Bulls (½Angus and ½Nellore) 16±2.2 months old, with average body weight of 385±20.7 kg were fed a basal diet (70 % concentrate, 30 % corn silage) offered ad libitum for 62 days in a feedlot and randomised on five treatments (8 bulls/treatment): control treatment, and addition of 1.5, 3.0, 4.5, or 6.0 g/day/animal of a blend of natural additives containing 37.5 % each of clove essential oil, the commercial blend containing vanillin, eugenol and thymol, 12.5 % and 12.5 % of castor and cashew oils). Methane production from rumen fluid was estimated based on the theoretical fermentation balance for observed molar distribution of VFAs in the rumen. DNA extracted from rumen fluid were sequenced and analysed for methane genes within the MG-RAST database. The natural additives linearly reduced methane production (76 %, P<0.02). Evaluation of Archaeaabundance showed a reduction (79 %, P<0.05) in the major methane producing genera: Halorhabdus, Ferroplasma, Methanoplanus, Picrophilus. A reduction of Fibrobacter and Lactobacillus (71 %), the greatest producers of acetate releasing hydrogen for methane formation was also observed. Our findings suggest that natural additives such as essential oils may be useful in the mitigation of greenhouse gases such as methane in animal production.
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Application of remote real-time monitoring of biological airborne particles to characterise different hospital environments
Conventional microbial sampling of air in hospitals is usually carried out using settle plates or impaction air samples. This provides little information about intermittent contamination events and is unhelpful for source attribution. Direct continuous bioaerosol sampling is an established technology used to characterise ambient external air. Portable instruments such as the Wideband Integrated Bioaerosol Sensor (WIBS) combine laser particle size and shape detection with signals of biological origin (fluorescence from amino acids and NAD(P)H) characteristic of viable bioaerosols. Monitoring is continuous for weeks at a time and data collected remotely over the internet. We present evidence of the utility of WIBS analysis in characterising air in hospitals in three different environments: operating theatres (plenum ventilated and ultraclean), a respiratory ward, and a specialist cystic fibrosis outpatients. The airborne particle profile was quantitatively and qualitatively different in each environment. Plumes of biologically-relevant airborne particles were detected and source investigation of failing conventional counts in an operating theatre aided by the continuous record. Nebulised drugs contributed a detectable effect on airborne particles which lasted for several hours on the ward despite air changes. A significant effect of plasma air treatment on airborne particles in the ward was detected by WIBS and not conventional cultures. Continuous monitoring may in future allow objective standard setting for airborne particles in different hospital environments and facilitate rapid detection of airborne infection risks.
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- Extremophiles: Living Life at the Edge
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The communities that colonise the cold
More LessThe Polar Regions have excellent potential for bioprospecting because the microorganisms that live there have adapted to extreme environmental conditions; and these adaptations can be harnessed in a variety of biotechnological applications. In this study, we describe the community structure of a range of different cryospheric habitats to identify environments appropriate for specific bioprospecting activities. Samples were collected from Midtre Lovénbreen glacier and surrounds in Spitzbergen, Svalbard in late June to early July 2017. Many cryospheric environment types were sampled including air, snow, slush, meltwater, cryoconite, proglacial water, soil and seawater. DNA was extracted and prepared for 16S amplicon sequencing. Amplicon sequence variants (ASVs) were assigned using DADA2 and contaminants were removed using the Decontam package in R. We searched the scientific literature for biotechnological applications of abundant community members in each environment. Each environment type displayed a unique community structure, with some physically linked environment types, like glacial snow, slush and meltwater showing continuity through space, and synchronised changes over time. The snow, slush and meltwater habitats were low in biomass and dominated by Gammaproteobacteria, with Cyanobacteria increasing in abundance as melt progressed. Cryoconite was dominated by Cyanobacteria, while sea water was dominated by Bacteriodetes, Gammaproteobacteria and Cyanobacteria. Soil was by far the most biodiverse habitat, with many phyla represented, and a large number of Actinobacteria suitable for antimicrobial discovery. Many of the current EPS, cold-active enzymes, fatty acids, antioxidants and antifreeze proteins are sourced from Proteobacteria and Cyanobacteria, the most abundant phyla in this study.
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Engineered extremes: microbial interactions with steel and bentonite in a geological disposal facility
More LessThe conditions of the deep subsurface, combined by perturbations caused by geological disposal of radioactive waste create multiple extreme conditions (limited space availability, high pH, high temperature etc.) for microbial communities. Microbial activity has the potential to cause corrosion of steel and alteration of bentonite clays used in geological disposal facilities. To understand the limits on microbial growth, and the potential for microbial activity to affect the swelling behaviour of the bentonite and metal corrosion, a suite of laboratory experiments is being conducted. In situ repository conditions have been replicated in the MIND (Horizon2020) project. Preliminary results show evidence of corrosion in all experiments, an increase in the basal spacings of smectites in the zone immediately surrounding the steel and inoculated samples had evidence of calcite crystal formation, accompanied by differences in the iron phases. These experiments simulate the in situ conditions well, but the complex nature of this experimental design (high pressure and flow) reduces the practicality of varying the environmental conditions. To complement these investigations, a low-tech solution has been implemented with unpressurised, hydrated bentonite batch experiments. The simpler nature of this set-up allows for investigation of more parameters. Microcosms with artificial groundwater used in the MIND set-up are being compared to the MIND groundwater composition, modelled to represent permafrost conditions. The effect of incubation temperature is also being investigated. Combined, these experiments will help to understand the influence of microbes under the extremes of geological disposal facilities and how their behaviour may change by external parameters.
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