- Volume 1, Issue 1A, 2019

The 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.

Brown 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.

Extended-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.

Poor 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.

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.

Manure 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.

Geoactive 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.

The 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.

In 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.

The 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.

The 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.

Drinking 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.

Lanthanum 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.

The 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.

Actinobacteria 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.

Environmental 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.

Selenium 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.