The biodiversity, control and genetic characterisation of the lactococcal 936 group phages

Phages belonging to the 936 group represent one of the most prevalent and frequently isolated phages in dairy fermentation processes using Lactococcus lactis as the primary starter culture. In recent years extensive research has been carried out to characterise this phage group at a genomic level in an effort to understand how the 936 group phages dominate this particular niche and cause regular problems during large scale milk fermentations. This thesis describes a large scale screening of industrial whey samples, leading to the isolation of forty three genetically different lactococcal phages. Using multiplex PCR, all phages were identified as members of the 936 group. The complete genome of thirty eight of these phages was determined using next generation sequencing technologies which identified several regions of divergence. These included the structural region surrounding the major tail protein, the replication region as well as the genes involved in phage DNA packing. For a number of phages the latter genomic region was found to harbour genes encoding putative orphan methyltransferases. Using small molecule real time (SMRT) sequencing and heterologous gene expression, the target motifs for several of these MTases were determined and subsequently shown to actively protect phage DNA from restriction endonuclease activity. Comparative analysis of the thirty eight phages with fifty two previously sequenced members of this group showed that the core genome consists of 28 genes, while the non-core genome was found to fluctuate irrespective of geographical location or time of isolation. This study highlights the continued need to perform large scale characterisation of the bacteriophage populations infecting industrial fermentation facilities in effort to further our understanding dairy phages and ways to control their proliferation.

Isolation and characterisation of antifungal compounds from lactic acid bacteria and their application in wheat and gluten-free bread

As part of the “free-from” trend, biopreservation for bread products has increasingly become important to prevent spoilage since artificial preservatives are more and more rejected by consumers. A literature review conducted as part of this thesis revealed that the evaluation of more suitable antifungal strains of lactic acid bacteria (LAB) is important. Moreover, increasing the knowledge about the origin of the antifungal effect is fundamental for further enhancement of biopreservation. This thesis addresses the investigation of Lactobacillus amylovorus DSM19280, Lb. brevis R2: and Lb. reuteri R29 for biopreservation using in vitro trials and in situ sourdough fermentations of quinoa, rice and wheat flours as biopreservatives in breads. Their contribution to quality and shelf life extension on bread was compared and related to their metabolic activity and substrate features. Moreover, the quantity of antifungal carboxylic acids produced during sourdough fermentation was analysed. Overall a specific profile of antifungal compounds was found in the sourdough samples which were strain and substrate dependently different. The best preservative effect in quinoa sourdough and wheat sourdough bread was achieved when Lb. amylovorus DSM19280 fermented sourdough was used. However, the concentration of the antifungal compounds found in these biopreservatives were much lower when compared with Lb. reuteri R29 as the highest producer. Nevertheless, the artificial application of the highest concentration of these antifungal compounds in chemically acidified wheat sourdough bread succeeded in a longer shelf life than achieved only by acidifying the dough. This evidences their partial contribution to the antifungal activity and their synergy. Additionally, a HRGC/MS method for the identification and quantification of the antifungal active compounds cyclo(Leu-Pro), cyclo(Pro-Pro), cyclo(Met-Pro) and cyclo(Phe-Pro) was successfully developed by using stable isotope dilutions assays with the deuterated counterparts. It was observed that the concentrations of cyclo(Leu-Pro), cyclo(Pro-Pro), and cyclo(Phe-Pro) increased only moderately in MRS-broth and wort fermentation by the activity of the selected microorganism, whereas the concentration of cyclo(Met-Pro) stayed unchanged.

Entry and competition in generic biologics

Patents for several blockbuster biological products are expected to expire soon. The Food and Drug Administration is examining whether biologies can and should be treated like pharmaceuticals with regard to generics. In contrast with pharmaceuticals, which are manufactured through chemical synthesis, biologies are manufactured through fermentation, a process that is more variable and costly. Regulators might require extensive clinical testing of generic biologies to demonstrate equivalence to the branded product. The focus of the debate on generic biologies has been on legal and health concerns, but there are important economic implications. We combine a theoretical model of generic biologies with regression estimates from generic pharmaceuticals to estimate market entry and prices in the generic biologic market. We find that generic biologies will have high fixed costs from clinical testing and from manufacturing, so there will be less entry than would be expected for generic pharmaceuticals. With fewer generic competitors, generic biologies will be relatively close in price to branded biologies. Policy makers should be prudent in estimating financial benefits of generic biologies for consumers and payers. We also examine possible government strategies to promote generic competition. Copyright © 2007 John Wiley & Sons, Ltd.

Using sediment flocculation to reduce the impacts of Chesapeake Bay Microcystis aeruginosa harmful algal blooms

Gemstone Team BREATHE (Bay Revitalization Efforts Against the Hypoxic Environment)

Antisense Gene Silencing and Bacteriophages as Novel Disinfection Processes for Engineered Systems

The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.

Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.

In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.


For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 + 13.2 and 38.2 + 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.

Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of E. coli K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h-1, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 108 PFU/mL and 108 CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.

In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.

Finally, for an industrial application, the use of phages to inhibit invasive Lactobacilli in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of Lactobacillus plantarum during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.

In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.

In vitro fluid dynamics of the Ahmed glaucoma valve modified with expanded polytetrafluoroethylene.

PURPOSE: Long-term intraocular pressure reduction by glaucoma drainage devices (GDDs) is often limited by the fibrotic capsule that forms around them. Prior work demonstrates that modifying a GDD with a porous membrane promotes a vascularized and more permeable capsule. This work examines the in vitro fluid dynamics of the Ahmed valve after enclosing the outflow tract with a porous membrane of expanded polytetrafluoroethylene (ePTFE). MATERIALS AND METHODS: The control and modified Ahmed implants (termed porous retrofitted implant with modified enclosure or PRIME-Ahmed) were submerged in saline and gelatin and perfused in a system that monitored flow (Q) and pressure (P). Flow rates of 1-50 μl/min were applied and steady state pressure recorded. Resistance was calculated by dividing pressure by flow. RESULTS: Modifying the Ahmed valve implant outflow with expanded ePTFE increased pressure and resistance. Pressure at a flow of 2 μl/min was increased in the PRIME-Ahmed (11.6 ± 1.5 mm Hg) relative to the control implant (6.5 ± 1.2 mm Hg). Resistance at a flow of 2 μl/min was increased in the PRIME-Ahmed (5.8 ± 0.8 mm Hg/μl/min) when compared to the control implant (3.2 ± 0.6 mm Hg/μl/min). CONCLUSIONS: Modifying the outflow tract of the Ahmed valve with a porous membrane adds resistance that decreases with increasing flow. The Ahmed valve implant behaves as a variable resistor. It is partially open at low pressures and provides reduced resistance at physiologic flow rates.

Interactive Effects of Plant Species and Organic Carbon on Nitrate Removal in Chesapeake Bay Treatment Wetlands

Nitrate from agricultural runoff are a significant cause of algal blooms in estuarine ecosystems such as the Chesapeake Bay. These blooms block sunlight vital to submerged aquatic vegetation, leading to hypoxic areas. Natural and constructed wetlands have been shown to reduce the amount of nitrate flowing into adjacent bodies of water. We tested three wetland plant species native to Maryland, Typha latifolia (cattail), Panicum virgatum (switchgrass), and Schoenoplectus validus (soft-stem bulrush), in wetland microcosms to determine the effect of species combination and organic amendment on nitrate removal. In the first phase of our study, we found that microcosms containing sawdust exhibited significantly greater nitrate removal than microcosms amended with glucose or hay at a low nitrate loading rate. In the second phase of our study, we confirmed that combining these plants removed nitrate, although no one combination was significantly better. Furthermore, the above-ground biomass of microcosms containing switchgrass had a significantly greater percentage of carbon than microcosms without switchgrass, which can be studied for potential biofuel use. Based on our data, future environmental groups can make a more informed decision when choosing biofuel-capable plant species for artificial wetlands native to the Chesapeake Bay Watershed.

Cultivos de Rhizobium phaseoli en sistema sumergido

p.19-27

Selection of yeast strains for bioethanol production from UK seaweeds

Macroalgae (seaweeds) are a promising feedstock for the production of third generation bioethanol, since they have high carbohydrate contents, contain little or no lignin and are available in abundance. However, seaweeds typically contain a more diverse array of monomeric sugars than are commonly present in feedstocks derived from lignocellulosic material which are currently used for bioethanol production. Hence, identification of a suitable fermentative microorganism that can utilise the principal sugars released from the hydrolysis of macroalgae remains a major objective. The present study used a phenotypic microarray technique to screen 24 different yeast strains for their ability to metabolise individual monosaccharides commonly found in seaweeds, as well as hydrolysates following an acid pre-treatment of five native UK seaweed species (Laminaria digitata, Fucus serratus, Chondrus crispus, Palmaria palmata and Ulva lactuca). Five strains of yeast (three Saccharomyces spp, one Pichia sp and one Candida sp) were selected and subsequently evaluated for bioethanol production during fermentation of the hydrolysates. Four out of the five selected strains converted these monomeric sugars into bioethanol, with the highest ethanol yield (13 g L−1) resulting from a fermentation using C. crispus hydrolysate with Saccharomyces cerevisiae YPS128. This study demonstrated the novel application of a phenotypic microarray technique to screen for yeast capable of metabolising sugars present in seaweed hydrolysates; however, metabolic activity did not always imply fermentative production of ethanol.

Effect of chronic angiotensin converting enzyme inhibition on spatial memory and anxiety-like behavours in rats

Angiotensin converting enzyme inhibitors (ACEis) are widely used anti-hypertensive agents that are also reported to have positive effects on mood and cognition. The present study examined the influence of the ACEi, perindopril, on cognitive performance and anxiety measures in rats. Two groups of rats were treated orally for one week with the ACEi, perindopril, at doses of 0.1 and 1.0mg/kg/day. Learning was assessed by the reference memory task in the water maze, comparing treated to control rats. Over five training days both perindopril-treated groups learnt the location of the submerged platform in the water maze task significantly faster than control rats. A 60s probe trial on day 6 showed that the 1.0mg/kg/day group spent significantly longer time in the training quadrant than control rats. This improved performance in the swim maze task was not due to the effect of perindopril on motor activity or the anxiety levels of the rats as perindopril-treated and control animals behaved similarly in activity boxes and on the elevated+maze. These results confirm the anecdotal human studies that ACEis have a positive influence on cognition and provide possibilities for ACEis to be developed into therapies for memory loss.

Using idiothetic cues to swim a path with a fixed trajectory and distance: Necessary involvement of the hippocampus but not the retrosplenial cortex.

Rats rapidly learned to find a submerged platform in a water maze at a constant distance and angle from the start point, which changed on every trial. The rats performed accurately in the light and dark, but prior rotation disrupted the latter condition. The rats were then retested after receiving cytotoxic hippocampal or retrosplenial cortex lesions. Retrosplenial lesions had no apparent effect in either the light or dark. Hippocampal lesions impaired performance in both conditions but spared the ability to locate a platform placed in the center of the pool. A hippocampal deficit emerged when this pool-center task was run in the dark. The spatial effects of hippocampal damage extend beyond allocentric tasks to include aspects of idiothetic guidance.

Metabolism of Maillard reaction products by the human gut microbiota - implications for health

The human colonic microbiota imparts metabolic versatility on the colon, interacts at many levels in healthy intestinal and systemic metabolism, and plays protective roles in chronic disease and acute infection. Colonic bacterial metabolism is largely dependant on dietary residues from the upper gut. Carbohydrates, resistant to digestion, drive colonic bacterial fermentation and the resulting end products are considered beneficial. Many colonic species ferment proteins but the end products are not always beneficial and include toxic compounds, such as amines and phenols. Most components of a typical Western diet are heat processed. The Maillard reaction, involving food protein and sugar, is a complex network of reactions occurring during thermal processing. The resultant modified protein resists digestion in the small intestine but is available for colonic bacterial fermentation. Little is known about the fate of the modified protein but some Maillard reaction products (MRP) are biologically active by, e.g. altering bacterial population levels within the colon or, upon absorption, interacting with human disease mechanisms by induction of inflammatory responses. This review presents current understanding of the interactions between MRP and intestinal bacteria. Recent scientific advances offering the possibility of elucidating the consequences of microbe-MRP interactions within the gut are discussed.

Critical stages of the brewing process for changes in antioxidant activity and levels of phenolic compounds in ale

Samples were taken at each stage of brewing (malt, milling, mashing, wort separation, hop addition, boiling, whirlpool, dilution, fermentation, warm rest, chill-lagering, beer filtration, carbonation and bottling, pasteurization, and storage). The level of antioxidant activity of unfractionated, low-molecular-mass (LMM) and high-molecular-mass (HMM) fractions was measured by the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfortic acid) radical cation (ABTS(.+)) and ferric-reducing antioxidant power (FRAP) procedures. Polyphenol levels were assessed by HPLC. The LMM fraction ( 0.001) in catechin and ferulic acid levels. Increases in antioxidant activity levels were observed after mashing, boiling, fermentation, chill-lagering, and pasteurization, in line with previous studies on lager. Additionally, increases in the level of antioxidant activity occurred after wort separation and carbonation and bottling and were accompanied by increases in levels of most monitored polyphenols. Data from the ABTS(.-) and FRAP assays indicated that the compounds contributing to the levels of antioxidant activity responded differently in the two procedures. Levels of ferulic, vanillic, and chlorogenic acids and catechin accounted for 45-61% of the variation in antioxidant activity levels.

Volatile compounds in Bacillus-fermented soybeans

Thua nao, a traditional, proteolytic, fermented soybean condiment of northern Thailand, was prepared from cooked whole soybeans by natural flora fermentation. The microbial flora during the fermentation was dominated by Bacillus species. The formation of volatile compounds during the fermentation was studied. In addition, the volatile compounds of two samples of commercial dried thua nao and two samples of commercial Japanese natto were analysed. Fermentation led to a large increase in the concentration of total volatile compounds, from 35 mug kg(-1) wet weight in cooked soybeans to 3500 mug kg(-1) wet weight in 72h fermented material. The major volatile compounds in fermented beans were 3-hydroxybutanone (acetoin), 2-methlybutanoic acid, pyrazines, dimethyl disulphide and 2-pentylfuran. Sun drying of 72 h fermented material resulted in the loss of 65% of total volatiles, including important aroma compounds. The commercial dried thua nao samples had low concentrations of total volatile compounds (380 mug kg(-1) wet weight). It is suggested that improved drying/preservation methods are needed to retain aroma compounds in the traditional products. The natto samples were devoid of aldehydes, aliphatic acids and esters, and sulphur compounds, whereas the thua nao samples contained a diversity of these compounds. Previous investigators have reported these compounds in natto and it is not possible to suggest the existence of systematic differences between the volatile compounds in traditional thua nao prepared with an undefined, mixed microbial flora and those in natto fermented with Bacillus subtilis. (C) 2001 Society of Chemical Industry.

Under-water scene investigation using ground penetrating radar (GPR) in the search for a sunken jet ski, Northern Ireland

A search for a submerged jet ski and the lost limb of its driver involved in a collision with a speedboat was made in a shallow lake in Northern Ireland. The location of both was crucial to establishing events at the time of the accident. Local intelligence suggested both objects were likely to be partially-buried by lacustrine silt. To avoid sediment churning, this required non-invasive, completely non-destructive assessment and mapping of the scene. A MALA RAMAC ground-penetrating radar system (GPR) mounted on floats for surveying from walkways and jetties or placed in a small rubber dinghy for offshore profiling was used. A grid was established and each line surveyed with 100, 200 and 400MHz antennae. In waters over 6m deep GPR data showed the form of the lake floor but excessive ringing occurred in the data. In waters less than 6m deep ringing diminished on both 100 and 200MHz data, the latter displaying the best trade-off between depth penetration and horizontal object resolution. 400MHz data failed to be of use in waters over 2m deep and at these depths showed only limited improvement of image quality compared to 200MHz data. Surface objects such as a wooden walkway caused interference on 200 and 400MHz data when antennae were oriented both normal and parallel to survey direction; this may be a function of the low attenuation of radar waves in freshwater, allowing excellent lateral and vertical radar wave penetration. On 200MHz data the damaged jet-ski was clearly imaged in a location that contradicted the speedboat driver's account of the accident.