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

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

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

Survival in amoeba: a major selection pressure on the presence of bacterial copper and zinc resistance determinants?: identification of a "copper pathogenicity island"

The presence of metal resistance determinants in bacteria usually is attributed to geological or anthropogenic metal contamination in different environments or associated with the use of antimicrobial metals in human healthcare or in agriculture. While this is certainly true, we hypothesize that protozoan predation and macrophage killing are also responsible for selection of copper/zinc resistance genes in bacteria. In this review, we outline evidence supporting this hypothesis, as well as highlight the correlation between metal resistance and pathogenicity in bacteria. In addition, we introduce and characterize the "copper pathogenicity island" identified in Escherichia coli and Salmonella strains isolated from copper- and zinc-fed Danish pigs

Virulence factors of Actinobacillus pleuropneumoniae involved in colonization, persistence and induction of lesions in its porcine host

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia. The virulence factors of this microorganism involved in colonization and the induction of lung lesions have been thoroughly studied and some have been well characterized. A. pleuropneumoniae binds preferentially to cells of the lower respiratory tract in a process involving different adhesins and probably biofilm formation. Apx toxins and lipopolysaccharides exert pathogenic effects on several host cells, resulting in typical lung lesions. Lysis of host cells is essential for the bacterium to obtain nutrients from the environment and A. pleuropneumoniae has developed several uptake mechanisms for these nutrients. In addition to persistence in lung lesions, colonization of the upper respiratory tract – and of the tonsils in particular – may also be important for long-term persistent asymptomatic infection. Information on virulence factors involved in tonsillar and nasal cavity colonization and persistence is scarce, but it can be speculated that similar features as demonstrated for the lung may play a role