Electricity-powered artificial root nodule.

Root nodules are agricultural-important symbiotic plant-microbe composites in which microorganisms receive energy from plants and reduce dinitrogen (N2) into fertilizers. Mimicking root nodules using artificial devices can enable renewable energy-driven fertilizer production. This task is challenging due to the necessity of a microscopic dioxygen (O2) concentration gradient, which reconciles anaerobic N2 fixation with O2-rich atmosphere. Here we report our designed electricity-powered biological|inorganic hybrid system that possesses the function of root nodules. We construct silicon-based microwire array electrodes and replicate the O2 gradient of root nodules in the array. The wire array compatibly accommodates N2-fixing symbiotic bacteria, which receive energy and reducing equivalents from inorganic catalysts on microwires, and fix N2 in the air into biomass and free ammonia. A N2 reduction rate up to 6.5 mg N2 per gram dry biomass per hour is observed in the device, about two orders of magnitude higher than the natural counterparts

Influence of cancer associated microbiome on volatile organic compound production in oesophago-gastric adenocarcinoma

Oesophago-gastric cancer is a significant health problem with poor prognosis in Western countries. This is due to a paucity of alarm symptoms in early stages of the disease resulting in late clinical presentation and associated delays in initiation of treatment. The development of non-invasive breath tests using exhaled Volatile Organic compounds (VOCs) to determine oesophago-gastric cancer risk would help facilitate earlier diagnosis and potentially improve patient survival. Whilst many of the biochemical pathways relating to the origin of these VOCs within humans are as yet unknown, it is postulated that that specific VOCs are produced directly by cancer tissues. Contributions from other endogenous sources including the intestinal microbiome and healthy tissues within the intestinal tract as well as other organ systems. The aim of this thesis was to understand the interaction between the upper gastrointestinal microbiome and VOC production in patients with oesophago-gastric cancer and to explore how this onco-microbial axis can be exploited to augment VOC production. The production of cancer associated VOCs (fatty acids and phenol) were investigated by analysing the ex vivo headspace above un-derivatised tissue samples as well as in vivo mixed breath, isolated bronchial breath and gastric endoluminal air. Increased concentrations of these VOCs were detected in the headspace of cancer tissue samples as well as isolated endoluminal air adjacent to tumours. Findings therefore implicate that the tumour and its local environment are the likely source of upregulated VOCs in oesophago-gastric cancer. The relative contribution of the tumour associated microbiome remains unknown. 16S RNA sequencing analysis for 185 oesophago-gastric tissue samples from cancer and control subjects were performed in order to assess the microbial diversity. Results revealed higher abundance of Firmicutes (e.g. Streptococcus salivarius, Escherichia coli and Streptococcus anginosus) in oesophago-gastric cancer samples compared to controls. The headspace of in vitro and patient derived (ex vivo) cultures of specific targeted bacteria was subsequently found to contain similar VOCs as those previously detected in oesophago-gastric cancer. To increase the sensitivity of breath testing, further work was performed to augment the diagnostic response using simple metabolic substrates (sugars, proteins, lipids). When added to in vitro cultures of cancer-associated bacteria, these nutrients resulted in upregulated VOC production. Oesophago-gastric cancer patients who were given the same substrates orally were found to have a transient rise in the same VOCs that was greater than observed in healthy controls. This thesis provides new insight into the biological origin of VOC production in oesophago-gastric cancer. Experiments linking the cancer-associated microbiome, exogenous substrates to upregulated VOC production in cancer patients offers the potential for a future augmented breath test for this disease. The augmented breath test is expected to increase earlier cancer detection leading to improvement in overall survival.Open Acces

Analytical techniques and instrumentation: A compilation

Technical information is presented covering the areas of: (1) analytical instrumentation useful in the analysis of physical phenomena; (2) analytical techniques used to determine the performance of materials; and (3) systems and component analyses for design and quality control

Establishment of High Cell Density Fed-Batch Microbial Cultures at the Microwell Scale

The rate limiting steps of biopharmaceutical process development are clone evaluation and process optimisation. To improve the efficiency of this step, miniature bioreactors are increasingly being used as a tool for high throughput experimentation. At industrial scale, microbial cultivations are usually performed in fed-batch mode to allow for high cell density cost-effective processes; however, many commercially available miniature bioreactors do not have an inbuilt feeding capacity. There are several challenges that need to be addressed to establish high cell density fed-batch cultivation at microscale: attaining high oxygen mass transfer rates, achieving good mixing for the duration of the culture and implementation of an industrially relevant feeding strategy requiring low volume additions. The overall aim of this project was to develop a scale-down fermentation platform suitable for the study and optimisation of high cell density cultures. The first objective of this work was to evaluate options for fed-batch cultures in a commercially available 24-well shaken microbioreactor. To achieve this, two feeding strategies were evaluated using an E. coli strain expressing a domain antibody: in situ feeding by the enzymatic release of glucose from polymeric starch, and direct feeding using a bespoke feed delivery system. In situ feeding was investigated as it is a simple option that does not require a physical method of feed delivery; cellular productivity was enhanced in comparison to batch cultures, however the glucose release was insufficient to sustain high cell density cultures representative of laboratory and pilot scale processes. To enable direct and continuous feed delivery to the microbioreactor a bespoke 3D-printed feeding system was developed that can operate at flow rates of 20μL h-1 and above, and enables up to twelve fed-batch cultures to be run in parallel. E. coli fermentations were performed on complex medium containing glycerol with direct feeding of a 23% w/v glycerol solution initiated at around 18 hours. The second objective of this project was to establish an industrially relevant feeding strategy in the microbioreactor, comparable to a laboratory scale fed-batch process. To this end, the direct feeding strategy was refined in terms of cell growth and product expression; the feed rate and concentration were modified, the DO set point was increased, and a pre-feeding hold period was implemented to allow for consumption of the inhibitory by-products generated in the batch phase. It was found that direct feeding enhanced biomass production by ~70% and product expression by ~2.4 fold in comparison to non-fed cultures. The third objective of this work was to demonstrate the applicability of the new feeding system as a tool for process optimisation experiments. The effect of IPTG concentration and post-induction temperature on product expression was performed using the both the microbioreactor feeding system and the 1L laboratory scale process. The data trends were consistent between scales; product expression was enhanced at a higher post-induction temperature, and IPTG concentration did not affect product expression over the concentration range tested. This demonstrates that the microbioreactor, is predicative of the 1L laboratory scale process terms of sensitivity to change in process conditions The fourth objective of this work was to characterise the microbioreactor in terms of oxygen transfer capability and fluid mixing. To achieve this aim, the volumetric oxygen mass transfer coefficient (kLa) and liquid phase mixing time (tm) of the microbioreactor were determined. The impact of shaking frequency, total gas flow rate and fill volume on oxygen transfer and fluid mixing were investigated and the optimum operating conditions were determined. Within the operating ranges of the miniature bioreactor system, it was found that oxygen transfer was dependant on both shaking frequency and gas flow rate, but was independent of fill volume. The oxygen mass transfer coefficient, kLa increased with both increasing shaking frequency (500-800rpm) and gas flow rate (0.1-20 mL min-1) over the range 3-101h-1; this is at the lower end of the range for conventional stirred tank reactors. It was demonstrated that the miniature bioreactor system is well mixed under the range of operating conditions evaluated. The liquid phase mixing time, tm under non-aerated conditions increased with shaking frequency and decreased with fill volume over the range 0.5-15s. The final objective this project was to demonstrate suitability of the microbioreactor as a scale-down model of an industrial fermentation process. 50L pilot scale, 1L laboratory scale, and 4mL microbioreactor fed-batch fermentations were performed under optimum conditions. The 4mL microbioreactor fed-batch process was shown to better predict the 50L pilot-scale process than the 1L laboratory-scale process based on cell growth, product expression and product quality. This could be explained by mixing and oxygen mass transfer phenomena. At 1L scale, oxygen mass transfer and fluid mixing are most efficient, meaning cell growth and productivity were the highest of the three processes. It appears that the limitations in oxygen mass transfer in the microbioreactor and fluid mixing in the 50L scale vessel, results in a comparable cellular environment, and therefore cell growth, productivity and product quality. In summary, this work has demonstrated the ability to conduct high cell density, fed-batch microbial cultures in parallel, using a shaken miniature bioreactor system. A bespoke, 3D-printed feed delivery system was developed allowing for twelve industrially-relevant microbial fed-batch cultures to be run in parallel. The microbioreactor fed-batch cultures were shown to be predictive of, a 50L pilot scale process in terms of cell growth, productivity and product quality

Biodesulphurized subbituminous coal by different fungi and bacteria studied by reductive pyrolysis. Part 1: Initial coal

One of the perspective methods for clean solid fuels production is biodesulphurization. In order to increase the effect of this approach it is necessary to apply the advantages of more informative analytical techniques. Atmospheric pressure temperature programming reduction (AP-TPR) coupled with different detection systems gave us ground to attain more satisfactory explanation of the effects of biodesulphurization on the treated solid products. Subbituminous high sulphur coal from ‘‘Pirin” basin (Bulgaria) was selected as a high sulphur containing sample. Different types of microorganisms were chosen and maximal desulphurization of 26% was registered. Biodesulphurization treatments were performed with three types of fungi: ‘‘Trametes Versicolor” – ATCC No. 200801, ‘‘Phanerochaeta Chrysosporium” – ME446, Pleurotus Sajor-Caju and one Mixed Culture of bacteria – ATCC No. 39327. A high degree of inorganic sulphur removal (79%) with Mixed Culture of bacteria and consecutive reduction by 13% for organic sulphur (Sorg) decrease with ‘‘Phanerochaeta Chrysosporium” and ‘‘Trametes Versicolor” were achieved. To follow the Sorg changes a set of different detection systems i.e. AP-TPR coupled ‘‘on-line” with mass spectrometry (AP-TPR/MS), on-line with potentiometry (AP-TPR/pot) and by the ‘‘off-line” AP-TPR/GC/MS analysis was used. The need of applying different atmospheres in pyrolysis experiments was proved and their effects were discussed. In order to reach more precise total sulphur balance, oxygen bomb combustion followed by ion chromatography was used

Efficient molasses fermentation under high salinity by inocula of marine and terrestrial origin

BACKGROUND: Molasses is a dense and saline by-product of the sugar agroindustry. Its high organic content potentially fuels a myriad of renewable products of industrial interest. However, the biotechnological exploitation of molasses is mainly hampered by the high concentration of salts, an issue that is nowadays tackled through dilution. In the present study, the performance of microbial communities derived from marine sediment was compared to that of communities from a terrestrial environment (anaerobic digester sludge). The aim was to test whether adaptation to salinity represented an advantage for fermenting molasses into renewable chemicals such as volatile fatty acids (VFAs) although high sugar concentrations are uncommon to marine sediment, contrary to anaerobic digesters. RESULTS: Terrestrial and marine microbial communities were enriched in consecutive batches at different initial pH values (pH(i); either 6 or 7) and molasses dilutions (equivalent to organic loading rates (OLRs) of 1 or 5 g(COD) L(−1) d(−1)) to determine the best VFA production conditions. Marine communities were supplied with NaCl to maintain their native salinity. Due to molasses inherent salinity, terrestrial communities experienced conditions comparable to brackish or saline waters (20–47 mS cm(−1)), while marine conditions resembled brine waters (>47 mS cm(−1)). Enrichments at optimal conditions of OLR 5 g(COD) L(-1) d(-1) and pH(i) 7 were transferred into packed-bed biofilm reactors operated continuously. The reactors were first operated at 5 g(COD) L(-1) d(-1), which was later increased to OLR 10 g(COD) L(−1) d(−1). Terrestrial and marine reactors had different gas production and community structures but identical, remarkably high VFA bioconversion yields (above 85%) which were obtained with conductivities up to 90 mS cm(−1). COD-to-VFA conversion rates were comparable to the highest reported in literature while processing other organic leftovers at much lower salinities. CONCLUSIONS: Although salinity represents a major driver for microbial community structure, proper acclimation yielded highly efficient systems treating molasses, irrespective of the inoculum origin. Selection of equivalent pathways in communities derived from different environments suggests that culture conditions select for specific functionalities rather than microbial representatives. Mass balances, microbial community composition, and biochemical analysis indicate that biomass turnover rather than methanogenesis represents the main limitation to further increasing VFA production with molasses. This information is relevant to moving towards molasses fermentation to industrial application. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13068-017-0701-8) contains supplementary material, which is available to authorized users

JPL spacecraft sterilization technology program - A status report

Facility description and procedures for heat and ethylene oxide sterilization of spacecraft instrumentation, components, and material

Analytical Applications of Bioluminescence and Chemiluminescence

Bioluminescence and chemiluminescence studies were used to measure the amount of adenosine triphosphate and therefore the amount of energy available. Firefly luciferase - luciferin enzyme system was emphasized. Photometer designs are also considered

Small secreted proteins enable biofilm development in the cyanobacterium Synechococcus elongatus.

Small proteins characterized by a double-glycine (GG) secretion motif, typical of secreted bacterial antibiotics, are encoded by the genomes of diverse cyanobacteria, but their functions have not been investigated to date. Using a biofilm-forming mutant of Synechococcus elongatus PCC 7942 and a mutational approach, we demonstrate the involvement of four small secreted proteins and their GG-secretion motifs in biofilm development. These proteins are denoted EbfG1-4 (enable biofilm formation with a GG-motif). Furthermore, the conserved cysteine of the peptidase domain of the Synpcc7942_1133 gene product (dubbed PteB for peptidase transporter essential for biofilm) is crucial for biofilm development and is required for efficient secretion of the GG-motif containing proteins. Transcriptional profiling of ebfG1-4 indicated elevated transcript levels in the biofilm-forming mutant compared to wild type (WT). However, these transcripts decreased, acutely but transiently, when the mutant was cultured in extracellular fluids from a WT culture, and biofilm formation was inhibited. We propose that WT cells secrete inhibitor(s) that suppress transcription of ebfG1-4, whereas secretion of the inhibitor(s) is impaired in the biofilm-forming mutant, leading to synthesis and secretion of EbfG1-4 and supporting the formation of biofilms