Generating and evaluating salinity and temperature resilient cyanobacteria for tropical outdoor cultivation in Australia

Global population levels, anticipated to increase to >9 billion by 2050, present serious worldwide challenges, such as energy-, food- and freshwater security. In addition, rising greenhouse gas (GHG) emissions lead to climatic instability, reduce the availability of freshwater and challenge agricultural productivity, which is exacerbated by decreasing arable land availability. Hence, the carbon- and freshwater-constrained economy demands industries to limit freshwater usage and carbon emissions. In this context, photosynthetic microalgae or cyanobacteria offer great promise for remediating carbon-dioxide emissions and high-nutient wastewaters, which can be coupled with renewable resource production to cater for large-volume low-value markets, such as animal feed, bio-fertiliser, and energy-production. The required scale of production for these markets, however, has to date not been realised, as outdoor cultivation presents severe challenges, including access to sufficient non-arable land in close proximity to water, nutrients (inorganic fertilisers: nitrogen and phosphate) and carbon-dioxide sources. In addition, high temperature and variable salinities are major limitations to cost-effective commercial microalgal production, as these factors are challenging to control. Ectoine, a valuable osmolyte, is produced by extremophile microbes in response to variable salinities and high temperature stress. Ectoine synthesis is mediated by an ectABC gene cassette. Given this, my research aimed at engineering a de novo biosynthesis pathway for ectoine production into the freshwater cyanobacterium Synechococcus elongatus PCC 7942 - to examine: a) its effect on temperature and/or salinity tolerance and b) potential downstream effects of ectoine on fertilisation requirements and biochemical profiles of this cyanobacterium, as the latter affects bio-product potential. Synechococcus elongatus PCC7942 was chosen as a model cyanobacterium, as its genome is small and fully sequenced, commercial vectors for transformation are available and it is exempt from restriction of laboratory transformation experiments by the Office of Gene Technology Regulator (OGTR). For ectABC transformation of S. elongatus PCC7942, a codon-optimised ectABC_pSyn_6 plasmid was constructed, based on the ectABC gene nucleotide sequence from the temperature- and salinity-tolerant bacterium Halomonas elongata DSM4043. ectABC-transformed, untransformed pSyn_6 vector (lacking ectABC insert) controls and wild-type (no vector, WT) S. elongatus PCC 7942 were subjected to a three temperature (35, 40, and 45°C), three salinity (0, 18, 36 ppt) factorial design experimental challenge without acclimation. Our data confirmed that ectABC-transformed S. elongatus PCC7942 had improved temperature tolerance up to 45°C and salinity tolerance up to 18 ppt at 35°C, compared to WT and pSyn_6 empty vector controls. Limited growth was observed at 36 ppt salinity in WT, pSyn-6 and ectABC transformants, irrespective of temperature. ectABC-transformant population growth rates were highest at 35°C. High pressure liquid chromatography analysis of these ectABC transformants confirmed ectoine production, albeit minimal. Further studies are necessary at the molecular level to resolve impediments associated with the low level of ectoine expression, should ectoine be chosen as a high-value co-product for the cosmetics industry. In terms of commercial production, it is vital to assess ectABCtransformed S. elongatus PCC 7942 fertilisation requirements. Results showed that nitrogen-requirements of ectABC-transformants were higher than that of WT and pSyn_6 empty vector controls at an elevated salinity of 18 ppt, but lower at 45°C temperature stress. Phosphate uptake was lowest in ectABC-transformants at temperature and salinity stress of 45°C and 18 ppt, respectively. Fertilisation costs require serious consideration for commercial-scale cultivation of large-volume, low-value bio-products markets. Thus, the stress-induced increased nitrogen fertilisation requirements of ectABC-transformants suggest that co-location with nitrogen-rich wastewater streams would be beneficial, thereby also reducing nutrient run-off into the local river systems. Regarding the biochemical profile of hydrocarbon-based biofuel production, ectABC transformants had increased lipid and fatty acid production under both temperature (45°C) and salinity (18 ppt) stress. Thus, this research addresses an area of importance for transitioning to a bio‐economy as a whole and for implementing environmentally and economically sustainable production of renewable biofuels, animal feed, bio-fertilisers, which are perhaps best achieved through co-production of some high-value bio-products, such as ectoine or the high-value pigment - c-phycocyanin. To investigate this potential, a modelling approach using multi-criteria analysis and geographical information system analysis was adopted. ArcGIS was used to evaluate potential sites suitable for co‐locating microalgal and sugarcane production in the Great Barrier Reef (GBR) catchment region in Queensland, Australia – whilst taking into account climatic, land-use and economic factors that consider energy balances for each facility. Critical resource inputs such as land, water, CO₂, energy and climatic factors such as temperature and rainfall were considered when estimating the available resources at sugar mills in the Wet Tropics region, adjacent to the GBR. Our economic analysis revealed that co-locating microalgal biomass production with such an industry is economically feasible in the Wet Tropics, by achieving significant cost-reductions and improved economic performance. As such, this research produces valuable information for investors, policy makers, government and industry to make informed decisions about the location potential for microalgal production sites that focus on salinity and temperature-resilient microalgal cultivation for high-value compounds (e.g. the osmolyte ectoine) or low-value animal feed as their principal commodity, whilst reducing CO₂ emissions and nutrient runoff to the GBR, both of which attract tradeable credits which offer additional economic returns over and above the returns from the production and sales process

Role of in vivo-induced genes ilvI and hfq in the survival and virulence of Actinobacillus pleuropneumoniae

ABSTRACTROLE OF IN VIVO-INDUCED GENES ILVI AND HFQ IN THE SURVIVAL AND VIRULENCE OF ACTINOBACILLUS PLEUROPNEUMONIAE By Sargurunathan SubashchandraboseActinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a contagious and often fatal disease of pigs. Infection with this bacterium leads to the development of a fulminant pleuropneumonia resulting in severe damage to the lungs. A. pleuropneumoniae genes that are up-regulated during the infection of pig lungs were previously identified in our laboratory and designated as in vivo-induced genes. The role of two such in vivo-induced genes, ilvI and hfq, in the pathobiology of A. pleuropneumoniae is the subject of this dissertation.The gene ilvI encodes an enzyme involved in the biosynthesis of branched-chain amino acids (BCAAs). The leucine-responsive regulatory protein (Lrp) is a transcriptional regulator of the ilvIH operon. BCAA biosynthetic genes are associated with virulence in pathogens infecting the respiratory tract and blood stream. Also, twenty five percent of the A. pleuropneumoniae in vivo-induced genes were up-regulated under BCAA limitation, suggesting that these BCAAs may be found at limiting concentrations in certain sites of the mammalian body such as the respiratory tract. The concentration of amino acids in the porcine pulmonary epithelial lining fluid was determined and BCAAs were found at limiting concentration in the respiratory tract. Further, the virulence of two BCAA auxotrophs, an ilvI mutant and an lrp mutant, was tested in a pig infection model and both were found to be attenuated. Finally, inhibitors of BCAA biosynthesis were found to prevent the growth of A. pleuropneumoniae. Hfq, encoding the Host factor Q-beta (Hfq) in A. pleuropneumoniae, was identified as an in vivo-induced gene and is also up-regulated under BCAA limitation. Since Hfq is a global regulator which is induced under one of the signals found in the porcine respiratory tract, the role of Hfq in A. pleuropneumoniae was analyzed. An A. pleuropneumoniae hfq mutant strain failed to form biofilm. Levels of the pgaC transcript, encoding the biofilm matrix biosynthetic enzyme, were ~14-fold lower in the hfq mutant compared to wild-type strain. The hfq mutant displayed enhanced sensitivity to superoxide stress and tellurite. Hfq was found to regulate two virulence-associated phenotypes, biofilm formation and resistance to oxidative stress, in A. pleuropneumoniae.As Hfq was associated with at least two virulence-associated phenotypes, the effect of Hfq on the virulence of A. pleuropneumoniae was tested in a pig infection model. Infection with the hfq mutant did not result in the development of pleuropneumonia while pigs infected with the wild-type strain exhibited classic signs of pleuropneumonia. Competitive index analysis revealed that the hfq mutant is severely attenuated, compared to the wild-type strain. In summary, studies described in this dissertation have uncovered a new host signal found in the porcine respiratory tract and provide evidence for the potential of inhibitors of BCAA biosynthesis as antibacterial agents. Hfq, regulated by BCAA limitation, was found to regulate biofilm formation, resistance to oxidative stress and virulence in A. pleuropneumoniae. The role of Hfq in the virulence of a bacterial pathogen during infection of lungs has been examined in the studies described in this dissertation. The hfq mutant is highly attenuated and is a potential candidate for the development of a live-attenuated vaccine against porcine pleuropneumonia.Thesis (Ph. D.)--Michigan State University, Department of Comparative Medicine & Integrative Biology, 2011Includes bibliographical reference

FT-IR, FT-Raman spectral and conformational studies on (E)-2-(2-hydroxybenzylidenamino)-3-(1H-indol-3yl) propionic acid

WOS: 000312690800014PubMed ID: 23099165The (E)-2-(2-hydroxybenzylidenamino)-3-(1H-indol-3yl) propionic acid ((E)-2HBA3IPA) was synthesized. The theoretical conformational analysis was performed to identify the stable structure. Optimized molecular bond parameters were calculated by using B3LYP/6-31G(d,p) basis set. The hyperconjugative interaction energy (E-(2)) and electron densities of donor (i) and acceptor (j) bonds were calculated using NBO analysis. First order hyperpolarizability (beta(o)) was calculated. The band gap energy was analyzed by UV-Visible recorded spectra and compared with theoretical band gap TD-DFT/B3LYP/6-31G(d,p) values. The intra-molecular hydrogen bonding interaction was identified between nitrogen and hydroxyl hydrogen (N center dot center dot center dot H-O). (C) 2012 Elsevier B.V. All rights reserved

Draft genome sequences of five recent human uropathogenic Escherichia coli isolates

This study reports the release of draft genome sequences of five isolates of uropathogenic Escherichia coli (UPEC), isolated from patients suffering from uncomplicated cystitis in 2012 in Ann Arbor, Michigan. Phylogenetic analyses revealed that these strains belonged to E. coli phylogroups B2 and D and are closely related to known UPEC strains. Comparative genomic analysis revealed that more conserved proteins were shared between these recent isolates and UPEC strains causing cystitis than those causing pyelonephritis. Additional genomic comparisons identified that three isolates encode a type III secretion system (T3SS) and a putative T3SS effector gene cluster along with an invasin‐like outer membrane protein. The presence of T3SS genes is a rare occurrence among UPEC strains. These genomes further substantiate the heterogeneity of the gene pool of UPEC and provide a foundation for comparative genomic studies using recent clinical isolates.This publication briefly describes the draft genomes of five recent human uropathogenic (UPEC) Escherichia coli isolates. UPEC are of increasing importance to human health. The genomes of these new isolates are clearly and simply described and will be of great utility and interest to this research community.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136326/1/fim12059.pd

Flexible modified plastic strips coated polyaniline/graphene composite for electrochemical biosensors

616-624Polyaniline(PANI)/graphene modified flexible, disposable polyester overhead projector (OHP) strips for non-enzymatic detection of ascorbic acid (AA) and uric acid (UA) is demonstrated in the current work. The deposition of PANI on the 3-aminopropyltrimethoxy silane (APTMS) modified OHP have been optimized through electrochemical impedance spectroscopy. Graphene is coated on another end. The PANI/graphene coating is confirmed through scanning electron microscopy. UA and AA detection have been carried out using the standard electrochemical techniques like cyclic voltammetry and chronoamperometry. The study reveals that the composite of PANI/graphene exhibits good sensing towards the detection of UA and AA. During the detection of UA and AA, PANI/graphene has showed enhanced current density with quick response and a linear concentration range of 10 to 300 μM for UA and 30 to 80 μM for AA. These flexible sensor strips may be of great potential in real-time UA and AA detection

Distinct Signature of Oxylipid Mediators of Inflammation during Infection and Asymptomatic Colonization by E. coli

Urinary tract infection (UTI) is an extremely common infectious disease. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. Asymptomatic bacteriuric E. coli (ABEC) strains successfully colonize the urinary tract resulting in asymptomatic bacteriuria (ABU) and do not induce symptoms associated with UTI. Oxylipids are key signaling molecules involved in inflammation. Based on the distinct clinical outcomes of E. coli colonization, we hypothesized that UPEC triggers the production of predominantly proinflammatory oxylipids and ABEC leads to production of primarily anti-inflammatory or proresolving oxylipids in the urinary tract. We performed quantitative detection of 39 oxylipid mediators with proinflammatory, anti-inflammatory, and proresolving properties, during UTI and ABU caused by genetically distinct E. coli strains in the murine urinary bladder. Our results reveal that infection with UPEC causes an increased accumulation of proinflammatory oxylipids as early as 6 h postinoculation, compared to controls. To the contrary, ABEC colonization leads to decreased accumulation of proinflammatory oxylipids at the early time point compared to UPEC infection but does not affect the level of proresolving oxylipids. This report represents the first comprehensive investigation on the oxylipidome during benign ABEC colonization observed in ABU and acute inflammation triggered by UPEC leading to UTI

Evaluation of the phenol degradation capacity of microalgae-bacteria consortia from the bay of Cartagena, Colombia

El desarrollo de nuevas tecnologías de remediación ambiental ha permitido la aplicación de alternativas económicas como la bioremediación, que poseen un alto potencial para tratar ecosistemas contaminados con hidrocarburos. El consorcio microalga-bacteria se ha sido identificado como una eficiente alternativa para la detoxificación de contaminantes orgánicos e inorgánicos y la remoción de diversos compuestos tóxicos. En este trabajo, hemos investigado el potencial de degradación de fenol de varios consorcios microalga-bacteria, donde cada uno involucra un alga (i.e. Chlamydomonas reinhardtii) y una cepa bacteriana osmotolerante resistente al fenol, aislada de la bahía de Cartagena, Colombia. Un total de tres cepas bacterianas fueron evaluadas (i.e. Stenotrophomonas maltophilia, Microbacterium paraoxydans and Paenibacillus lactis) individualmente y en consorcio con C. reinhardtii. Nuestros resultados muestran un incremento significativo en la tasa de crecimiento y una reducción en la fase de latencia para los microorganismos creciendo en consorcio, en comparación con los microorganismos creciendo individualmente. Interesantemente, el inóculo con proporción 2:1 (bacteria-microalga) mostró una alta robustez, basados en el hecho que ambos microorganismos mejoraron su crecimiento. EL potencial de degradación de fenol fue evaluado para cultivos puros y consorcios demostrando una mayor actividad a bajas concentraciones de fenol, en donde el consorcio Microbacterium paraoxydans- Chlamydomonas reinhardtii demostró ser el más efectivo con un porcentaje de 49.89% de remoción de fenol. The development of new technologies for environmental reparation has allowed the application of inexpensive alternatives such as bioremediation, which has a high potential to treat ecosystems polluted with hydrocarbons. Microalgae-bacteria consortia have been identified as an efficient alternative for the detoxification of organic and inorganic contaminants and the removal of toxic compounds. This work investigates the phenol degradation potential of several alga-microbial consortia, which involved the algae Chlamydomonas reinhardtii and an osmotolerant phenol-resistant bacterial strain isolated from the bay of Cartagena, Colombia. A total of three bacterial strains were tested (i.e. Stenotrophomonas maltophilia, Microbacterium paraoxydans and Paenibacillus lactis) individually and in consortium with C. reinhardtii. Our data indicate a significant increase in the growth rate and a reduction in the lag phase of microorganisms in the consortium as compared to microorganisms growing in isolation. Interestingly, the inoculum ratio 2:1 (bacteria-microalgae) was shown to be the most robust taking into account that both microorganisms improved their growth. Afterward, the phenol degradation capacity of pure cultures and consortia in the presence of different phenol concentrations was evaluated. Our results reveal that such consortia perform better at low phenol concentrations; more specifically, the consortium Microbacterium paraoxydans-Chlamydomonas reinhardtii was the most effective: it reached a 49.89% phenol removal

ISOLATION OF ANGIOTENSIN-CONVERTING ENZYME INHIBITOR PRODUCING BACTERIA FROM COW MILK

Objective: To evaluate the potential of protease producing organism for the production of Angiotensin I–converting enzyme (ACE) inhibitor by fermentation of various protein substrates.Methods: Bacterial strains were isolated from cow milk collected in Coimbatore, Tamil Nadu, India by using serial dilution technique, plated on nutrient agar medium. The identity of the strain was ascertained by 16s rRNA gene sequencing method and was submitted to the NCBI GenBank nucleotide database. Various substrates were screened for ACE inhibitor production by the fermentation with the isolated strain.Results: The isolated coded as BUCTL09, which showed a significant zone of clearance was selected and identified as Micrococcus luteus (KF303592.1). Among the seven substrates, only beef extract fermented broth showed an inhibition of 79% and was reported as the best substrate.Conclusion: In the search for non-toxic, and economic ACE inhibitors as an alternative to the synthetic drugs, many natural ACE inhibitors have been isolated from a microbial source. In the present study, isolate BUCTL09 was selected for the production of ACE inhibitor from the beef extract. Findings from this study lead us to investigate this potent ACE inhibitor further for its biological properties and to explore the impending efficacy of the ACE inhibitor which may conceivably be developed into a prospective drug

Flexible modified plastic strips coated polyaniline/ graphene composite for electrochemical biosensors

Polyaniline (PANI)/Graphene modified flexible, disposable polyester overhead projector (OHP) strips for non-enzymatic detection of ascorbic acid (AA) and uric acid (UA) is demonstrated in the current work. The deposition of PANI on the 3-aminopropyltrimethoxy silane (APTMS) modified OHP were optimized through electrochemical impedance spectroscopy. Graphene is coated on another end. The PANI/Graphene coating is confirmed through scanning electron microscopy. UA and AA detection were carried out using the standard electrochemical techniques like cyclic voltammetry (CV) and chronoamperometry. The study reveals that the composite of PANI/Graphene exhibits good sensing towards the detection of UA and AA. During the detection of UA and AA, PANI/Graphene showed enhanced current density with quick response and a linear concentration range of 10 to 300 mM for UA and 30 to 80 mM for AA. These flexible sensor strips may be of great potential in real-time UA and AA detection