Towards The Development of Biosensors for the Detection of Microbiologically Influenced Corrosion (MIC)

Corrosion is one of the biggest concerns for mechanical integrity of infrastructure and infrastructural components, such as oil refineries, bridges and roads. The economic cost of corrosion is typically estimated to be between 1 to 5 % of the gross national product (GNP) of countries, of which the contribution of microbiologically influenced corrosion (MIC) is estimated to be between 10% and 50%. Current state-of-the-art approaches for detecting MIC primarily rely on ex-situ tests, including bacterial test kits (bug bottles); corrosion coupons, pigging deposits analysis and destructive analysis of MIC affected sites using SEM, TEM, and XRD. These ex-situ measurements do not capture the complexities and time sensitivities underlying MIC. This is owed to the fact that the proliferation of the microbial contamination is a dynamic and rapid process, and any delay can prove expensive as it is estimated that once the biofilm formation takes place the amount of biocides needed is magnitude of orders more as compared to when the bacteria are in planktonic form. Additionally, the field environment is a complex biotic and abiotic environment which is often difficult to replicate even in high fidelity laboratory models. Hence a real-time/pseudo real-time method of detection would greatly help reduce the costs and optimize biocide-based mitigation of MIC. To overcome the above-mentioned shortcomings associated with the state-of-the-art; this work is aimed at the development of a sensor substrate whereby highly specific detection can be carried out in the environment where the corrosion exists, in a real-time/pseudo real-time basis. More specifically, the research is aimed at the development of sensors based on a nanowire matrix functionalized with biomolecules which can perform this specific and real-time detection of MIC in the pipeline environment. Here, the detection of MIC is based on the binding of specific biomolecules causing MIC to organic molecules anchored on top of the nanowires. These sensors also need to be inexpensive (made of low-cost, earth abundant materials), have low power consumption, and robustly deployable. The primary component of the detection platforms are copper oxide nanowire arrays (CuONWs with lengths of 25 to 30 m, 50 to 100 nm in diameter) and silicon nanowires arrays (SiNWs with lengths of 5 to 8 m, 45 to 100 nm in diameter). They are synthesized using facile and scalable techniques and are selected for their robust electrical and mechanical properties. Electrochemical degradation studies of the NWs were performed in 3.5 wt. % NaCl solution and simulated produced water using polarization and electrochemical impedance spectroscopy (EIS). The NWs systems showed robust resistance to degradation despite higher surface area (as compared to bulk counterparts), and both diffusion limitations and charge transfer resistance was observed on the analysis of the impedance response. The ability to immobilize a variety of moieties on the nanowire platforms gives them the ability to detecting a wide variety of MIC biomarkers. The Biotin-Streptavidin (SA) complex was used as a proof of concept to test the viability of the NW arrays as a substrate for sensing. A custom test bed was built for the functionalized NW thin films, and cyclic voltammetry studies revealed a stable current response with time for 10nM and 10,000 nM SA concentrations. The use of different probes such as aptamers to larger immunoglobulin probes provides the flexibility to detect the full spectrum of biomarkers. The development of these next generation sensor platforms along with the methodologies employed to stabilize them and assemble them into functional devices are explored in detail in this dissertation

Gene probing reveals the widespread distribution, diversity and abundance of isoprene-degrading bacteria in the environment

Background: Approximately 500 Tg of isoprene are emitted to the atmosphere annually, an amount similar to that of methane, and despite its significant effects on the climate, very little is known about the biological degradation of isoprene in the environment. Isolation and characterisation of isoprene degraders at the molecular level has allowed the development of probes targeting isoA encoding the α-subunit of the isoprene monooxygenase. This enzyme belongs to the soluble diiron centre monooxygenase family and catalyses the first step in the isoprene degradation pathway. The use of probes targeting key metabolic genes is a successful approach in molecular ecology to study specific groups of bacteria in complex environments. Here, we developed and tested a novel isoA PCR primer set to study the distribution, abundance, and diversity of isoprene degraders in a wide range of environments. Results: The new isoA probes specifically amplified isoA genes from taxonomically diverse isoprene-degrading bacteria including members of the genera Rhodococcus, Variovorax, and Sphingopyxis. There was no cross-reactivity with genes encoding related oxygenases from non-isoprene degraders. Sequencing of isoA amplicons from DNA extracted from environmental samples enriched with isoprene revealed that most environments tested harboured a considerable variety of isoA sequences, with poplar leaf enrichments containing more phylogenetically diverse isoA genes. Quantification by qPCR using these isoA probes revealed that isoprene degraders are widespread in the phyllosphere, terrestrial, freshwater and marine environments. Specifically, soils in the vicinity of high isoprene-emitting trees contained the highest number of isoprene-degrading bacteria. Conclusion: This study provides the molecular ecology tools to broaden our knowledge of the distribution, abundance and diversity of isoprene degraders in the environment, which is a fundamental step necessary to assess the impact that microbes have in mitigating the effects of this important climate-active gas

Insights into the Anaerobic Biodegradation Pathway of n-Alkanes in Oil Reservoirs by Detection of Signature Metabolites

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Deep Learning Approach for Large-Scale, Real-Time Quantification of Green Fluorescent Protein-Labeled Biological Samples in Microreactors

Absolute quantification of biological samples entails determining expression levels in precise numerical copies, offering enhanced accuracy and superior performance for rare templates. However, existing methodologies suffer from significant limitations: flow cytometers are both costly and intricate, while fluorescence imaging relying on software tools or manual counting is time-consuming and prone to inaccuracies. In this study, we have devised a comprehensive deep-learning-enabled pipeline that enables the automated segmentation and classification of GFP (green fluorescent protein)-labeled microreactors, facilitating real-time absolute quantification. Our findings demonstrate the efficacy of this technique in accurately predicting the sizes and occupancy status of microreactors using standard laboratory fluorescence microscopes, thereby providing precise measurements of template concentrations. Notably, our approach exhibits an analysis speed of quantifying over 2,000 microreactors (across 10 images) within remarkably 2.5 seconds, and a dynamic range spanning from 56.52 to 1569.43 copies per micron-liter. Furthermore, our Deep-dGFP algorithm showcases remarkable generalization capabilities, as it can be directly applied to various GFP-labeling scenarios, including droplet-based, microwell-based, and agarose-based biological applications. To the best of our knowledge, this represents the first successful implementation of an all-in-one image analysis algorithm in droplet digital PCR (polymerase chain reaction), microwell digital PCR, droplet single-cell sequencing, agarose digital PCR, and bacterial quantification, without necessitating any transfer learning steps, modifications, or retraining procedures. We firmly believe that our Deep-dGFP technique will be readily embraced by biomedical laboratories and holds potential for further development in related clinical applications.Comment: 23 pages, 6 figures, 1 tabl

Diversity of isoprene-degrading bacteria in phyllosphere and soil communities from a high isoprene-emitting environment: a Malaysian oil palm plantation

Background: Isoprene is the most abundantly produced biogenic volatile organic compound (BVOC) on Earth, with annual global emissions almost equal to those of methane. Despite its importance in atmospheric chemistry and climate, little is known about the biological degradation of isoprene in the environment. The largest source of isoprene is terrestrial plants, and oil palms, the cultivation of which is expanding rapidly, are among the highest isoprene-producing trees. Results: DNA stable isotope probing (DNA-SIP) to study the microbial isoprene-degrading community associated with oil palm trees revealed novel genera of isoprene-utilising bacteria including Novosphingobium, Pelomonas, Rhodoblastus, Sphingomonas and Zoogloea in both oil palm soils and on leaves. Amplicon sequencing of isoA genes, which encode the α-subunit of the isoprene monooxygenase (IsoMO), a key enzyme in isoprene metabolism, confirmed that oil palm trees harbour a novel diversity of isoA sequences. In addition, metagenome assembled genomes (MAGs) were reconstructed from oil palm soil and leaf metagenomes and putative isoprene degradation genes were identified. Analysis of unenriched metagenomes showed that isoA-containing bacteria are more abundant in soils than in the oil palm phyllosphere. Conclusion: This study greatly expands the known diversity of bacteria that can metabolise isoprene and contributes to a better understanding of the biological degradation of this important but neglected climate-active gas

Pyrosequencing of Bacterial Symbionts within Axinella corrugata Sponges: Diversity and Seasonal Variability

Background: Marine sponge species are of significant interest to many scientific fields including marine ecology, conservation biology, genetics, host-microbe symbiosis and pharmacology. One of the most intriguing aspects of the sponge ‘‘holobiont’’ system is the unique physiology, interaction with microbes from the marine environment and the development of a complex commensal microbial community. However, intraspecific variability and temporal stability of sponge-associated bacterial symbionts remain relatively unknown. Methodology/Principal Findings: We have characterized the bacterial symbiont community biodiversity of seven different individuals of the Caribbean reef sponge Axinella corrugata, from two different Florida reef locations during variable seasons using multiplex 454 pyrosequencing of 16 S rRNA amplicons. Over 265,512 high-quality 16 S rRNA sequences were generated and analyzed. Utilizing versatile bioinformatics methods and analytical software such as the QIIME and CloVR packages, we have identified 9,444 distinct bacterial operational taxonomic units (OTUs). Approximately 65,550 rRNA sequences (24%) could not be matched to bacteria at the class level, and may therefore represent novel taxa. Differentially abundant classes between seasonal Axinella communities included Gammaproteobacteria, Flavobacteria, Alphaproteobacteria, Cyanobacteria, Acidobacter and Nitrospira. Comparisons with a proximal outgroup sponge species (Amphimedon compressa), and the growing sponge symbiont literature, indicate that this study has identified approximately 330 A. corrugata-specific symbiotic OTUs, many of which are related to the sulfur-oxidizing Ectothiorhodospiraceae. This family appeared exclusively within A. corrugata, comprising \u3e34.5% of all sequenced amplicons. Other A. corrugata symbionts such as Deltaproteobacteria, Bdellovibrio, and Thiocystis among many others are described. Conclusions/Significance: Slight shifts in several bacterial taxa were observed between communities sampled during spring and fall seasons. New 16 S rDNA sequences and concomitant identifications greatly expand the microbial community profile for this model reef sponge, and will likely be useful as a baseline for any future comparisons regarding sponge microbial community dynamics

Evaluation of Physicochemical and Microbial Properties of Extracts from Wine Lees Waste of Matelica’s Verdicchio and Their Applications in Novel Cosmetic Products

Wine lees are sediments deposited on the walls and bottom of barrels resulting from wine fermentation and mainly consist of yeasts. Saccharomyces cerevisiae extracts, rich in beneficial components for the skin, have already been used in cosmesis, while wine lees have not been well exploited by the cosmetics industry yet. The aim of this work was the full characterization of the wine lees from Verdicchio's wine, with the aim to exploit it as a beneficial ingredient in new cosmetic products. After mapping the microbial composition of the sample waste, the parameters for the sonication extraction process were optimized and the physicochemical properties of the extract were analyzed. The efficiency of the aqueous extraction-and in particular the yeast cell lysis necessary for the release of proteins from the cell-was assessed by evaluating cell shape and size, and protein release, under scanning electron microscopy (SEM), dynamic light scattering (DLS) and Bradford's protein assays. Thus, the total phenol content and antioxidant capacity of the supernatant recovered from native and sonicated lees were determined by Folin-Ciocalteu's and spectrophotometric assays, respectively. To quantify the heavy metals and highlight the presence of microelements beneficial for the skin, inductively coupled plasma-mass spectrometry (ICP-MS) was applied. In vitro metabolic activity and cytotoxicity were tested on both HaCat keratinocytes and human gingival fibroblasts, showing that wine lees are safe for skin's cells. The results show that sonicated lees appear to be more interesting than native ones as a consequence of the release of the active ingredients from the cells. Due to the high antioxidant capacity, content of beneficial elements for skin and an appropriate microbiologic profile, wine lees were included in five new solid cosmetic products and tested for challenge test, compatibility with human skin, sensory analysis, trans epidermal water loss (TEWL) and sebometry

Ecophysiology and genomics of key sulfate-reducing bacteria involved in anaerobic hydrocarbon degradation at marine gas and oil seeps

The diversity, function and community structure of anaerobic hydrocarbon-degrading microorganisms in marine environments were elucidated by methods in molecular ecology, microbiology and microbial genomics. A high diversity of n-alkane degraders was revealed in globally distributed marine seep sediments based on genes encoding (1-methylalkyl)succinate synthase (MasD), the functional marker for anaerobic n-alkane degradation. Both abundant cosmopolitan and specialized variants of MasD were detected as well as novel lineages of n-alkane degraders. It could be shown that the community structure is clearly driven by the available hydrocarbon substrate. Further, the response of the microbial community in Caspian Sea sediments to simulated crude oil seepage using a Sediment-Oil-Flow-Through system was investigated. Sulfate reduction and methanogenesis were important processes in the anaerobic degradation of hydrocarbons during crude oil seepage in these sediments. After oil-flow-through, several groups of SRB exhibited an increase in cell numbers and are likely responsible for the observed decrease in aliphatic hydrocarbon concentration

Single-cell droplet microfluidics for metagenomics and cancer multiomics

Cellular heterogeneity is inherent to many biological systems, across both normal and disease states. For example, diverse ensembles of microbes in the natural environment fulfill distinct roles related to nutrient metabolism and gas fixation. In human cancers, genetic and phenotypic heterogeneity is observed among cells originating from a common oncogenic clone. Understanding biological heterogeneity, whether for metabolic engineering applications or the design of cancer therapeutics, begins at the fundamental unit of the organism: a single cell. Droplet microfluidics enables analyses of single cells at a biologically-relevant scale through rapid compartmentalization and manipulation of millions of parallel reactions. In this thesis, I describe the development and application of single-cell genomics platforms leveraging droplet microfluidics to interrogate many individual genomes. These technologies enable single-cell metagenomics and multiomic analysis of single cancer cells, providing new insights into the extent of cellular heterogeneity and its implications across biology