A modified pH drift assay for inorganic carbon accumulation and external carbonic anhydrase activity in microalgae

The threat of global warming due to CO2 emissions has stimulated research into carbon sequestration and emissions reduction technologies. Alkaline scrubbing allows CO2 to be captured as bicarbonate, which can be photochemically fixed by microalgae. The carbon concentrating mechanism (CCM), of which external carbonic anhydrase is a key component, allows the organisms to utilise this bicarbonate. In order to select a suitable strain for this application, a screening tool is required. The current method for determining carbonic anhydrase activity, the Wilbur and Anderson assay, was found to be unsuitable as a screening tool as the associated error was unacceptably large and tests on whole cells were inconclusive. This paper presents the development of a new, whole cell assay to measure inorganic carbon uptake and external carbonic anhydrase activity, based on classical pH drift experiments. Spirulina platensis was successfully used to develop a correlation between the specific carbon uptake (C) and the specific pH change (dpH). The relationship is described by: C (mmol C (g dry algae)-1 h-1) = 0.064 × (dpH). Inhibitor and salt dissociation tests validated the activity and presence of external carbonic anhydrase, and allowed correlation between the Wilbur and Anderson assay and the new whole cell assay. Screening tests were conducted on Spirulina platensis, Scenedesmus sp., Chlorella vulgaris and Dunaliella salina which were found to have carbon uptake rates of 5.76, 5.86, 3.86 and 2.15 mmol C (g dry algae)-1 h-1 respectively. These results corresponded to the species’ known bicarbonate utilisation abilities and validated the use of the assay as a screening tool

Analysis of the microbial community associated with a bioprocess system for bioremediation of thiocyanate- and cyanide-laden mine water effluents

Gold extraction by cyanidation from refractory gold ores results in the formation of thiocyanate- and cyanide-contaminated wastewater effluents that must be treated before recycle or discard. Activated sludge processes, such as ASTER™, can be used for biodegradation of these effluent streams. The destruction of these compounds is catalyzed by a mixed microbial culture, however, very little is known about the community composition and metabolic potential of the thiocyanate- and cyanide-degrading microorganisms within the community. Here we describe our on-going attempts to better understand the key microorganisms, within the ASTER™ bioprocess, that contribute to the destruction of thiocyanate and cyanide, and how this knowledge relates to further process optimisation

Plant-inducible virulence promoter of the Agrobacterium tumefaciens Ti plasmid

Agrobacterium tumefaciens is the causative agent of crown gall, a plant tumour that can arise on most species of dicotyledonous plants. The tumour-inducing capacity of the bacterium requires the presence of a large plasmid, designated the Ti plasmid, which itself contains two regions essential for tumour formation-the T(umour)-region and the Vir(ulence)-region. The T-region is transferred to plant cells by an unknown mechanism, and becomes stably integrated into the plant genome. The Vir-region has been identified by transposon mutagenesis, but the DNA of this region has never been detected in tumour lines. However, trans-complementation of Vir mutants indicates that genes of the Vir-region are functional in the bacterium. Moreover, the Vir- and T-regions can be physically separated in A. tumefaciens without loss of tumour-inducing capacity. Seven loci, designated virA-F and virO, have been identified in the Vir-region of the octopine Ti plasmid, but their functions are unknown. As virC mutants in the octopine-type plasmid pTiB6 are invariably avirulent in tests on various plant species, this gene seems to be essential for virulence and we are studying it in detail. We report here that the promoter of virC shows no detectable activity in A. tumefaciens and Escherichia coli K-12 grown in standard medium, but that its activity is induced by a plant product.

Disproportionate Intrauterine Growth Intervention Trial At Term: DIGITAT

Contains fulltext : 65628.pdf ( ) (Open Access)BACKGROUND: Around 80% of intrauterine growth restricted (IUGR) infants are born at term. They have an increase in perinatal mortality and morbidity including behavioral problems, minor developmental delay and spastic cerebral palsy. Management is controversial, in particular the decision whether to induce labour or await spontaneous delivery with strict fetal and maternal surveillance. We propose a randomised trial to compare effectiveness, costs and maternal quality of life for induction of labour versus expectant management in women with a suspected IUGR fetus at term. METHODS/DESIGN: The proposed trial is a multi-centre randomised study in pregnant women who are suspected on clinical grounds of having an IUGR child at a gestational age between 36+0 and 41+0 weeks. After informed consent women will be randomly allocated to either induction of labour or expectant management with maternal and fetal monitoring. Randomisation will be web-based. The primary outcome measure will be a composite neonatal morbidity and mortality. Secondary outcomes will be severe maternal morbidity, maternal quality of life and costs. Moreover, we aim to assess neurodevelopmental and neurobehavioral outcome at two years as assessed by a postal enquiry (Child Behavioral Check List-CBCL and Ages and Stages Questionnaire-ASQ). Analysis will be by intention to treat. Quality of life analysis and a preference study will also be performed in the same study population. Health technology assessment with an economic analysis is part of this so called Digitat trial (Disproportionate Intrauterine Growth Intervention Trial At Term). The study aims to include 325 patients per arm. DISCUSSION: This trial will provide evidence for which strategy is superior in terms of neonatal and maternal morbidity and mortality, costs and maternal quality of life aspects. This will be the first randomised trial for IUGR at term. TRIAL REGISTRATION: Dutch Trial Register and ISRCTN-Register: ISRCTN10363217

Site-Directed Mutagenesis in Escherichia coli of a Stable R772::Ti Cointegrate Plasmid from Agrobacterium tumefaciens

The host range of an octopine Ti plasmid is limited to Rhizobiaceae. This has been extended also to Escherichia coli in the form of a stable cointegrate with the wide-host-range plasmid R772. Its structure was studied by constructing a physical map of R772 and of the R772::pTiB6 cointegrate. An insertion sequence present in R772, called IS70, turned out to be involved in cointegrate formation. We found one intact copy of IS70 and a small segment of IS70, respectively, at the junctions of R772 and Ti DNA. The absence of a complete second copy of IS70 is a likely explanation for the stability of the cointegrate plasmid. A procedure for site-directed mutagenesis of this cointegrate plasmid in E. coli is described. The effect of mutations in the Ti plasmid part can be studied subsequently by transferring the cointegrate into Agrobacterium tumefaciens. The advantage of this procedure for Ti plasmids over other methods used at present is discussed.

Effect of sulphate concentration on the community structure and activity of sulphate reducing bacteria

This study investigated the effect of sulphate concentration and residence time on the performance of anaerobic sulphate reduction by a mixed sulphate reducing bacteria (SRB) culture using lactate as the sole carbon source and electron donor. The process perforrnance is related to the population structure of the microbial consortia and dominant metabolic reactions. Laboratory scale chemostat cultures at different residence times (1-4 d) and sulphate concentrations (1.0-10.0 glL) were employed. Lactate oxidation was prevalent at feed sulphate concentrations of 1.0 to 5.0 glL. A colresponding increase in the volumetric sulphate reduction rate with increasing volumetric loading rate was also observed at this range. However, at the higher feed sulphate concentration range (10.0-15.0 glL), sulphate inhibition, lactate fermentation and an increased microbial diversity were evident. At each feed concentration of sulphate in the range 5.0 to 15.0 glL, varying dilution rates resulted in significant shifts in dominant metabolic reactions. Sulphate concentration and residence time have significant effects on both the structure of the microbial population and kinetics of biological sulphate reduction

Functional Microbial Communities in Hybrid Linear Flow Channel Reactors for Desulfurization of Tannery Effluent

Recent research has demonstrated that hybrid linear flow channel reactors (HLFCRs) can desulfurize tannery effluent via sulfate reduction and concurrent oxidation of sulfide to elemental sulfur. The reactors can be used to pre-treat tannery effluent to improve the efficiency of downstream anaerobic digestion and recover sulfur. This study was conducted to gain insight into the bacterial communities in HLFCRs operated in series and identify structure-function relationships. This was accomplished by interpreting the results obtained from amplicon sequencing of the 16S rRNA gene and quantification of the dissimilatory sulfite reducing (dsrB) gene. In an effort to provide a suitable inoculum, microbial consortia were harvested from saline estuaries and enriched. However, it was found that bioaugmentation was not necessary because native communities from tannery wastewater were selected over exogenous communities from the enriched consortia. Overall, Dethiosulfovibrio sp. and Petrimonas sp. were strongly selected (maximum relative abundances of 29% and 26%, respectively), while Desulfobacterium autotrophicum (57%), and Desulfobacter halotolerans (27%) dominated the sulfate reducing bacteria. The presence of elemental sulfur reducing genera such as Dethiosulfovibrio and Petrimonas is not desirable in HLFCRs, and strategies to counter their selection need to be considered to ensure efficiency of these systems for pre-treatment of tannery effluent