Characterizing the removal of antibiotics in algal wastewater treatment ponds : a case study on tetracycline in HRAPs: a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Environmental Engineering at Massey University, Turitea Campus, Palmerston, New Zealand

Antibiotics are ubiquitous pollutants in wastewater, owing to their usefulness in both animal and human treatment. Antibiotic pollution is a growing concern because of the risk of encouraging antibiotic resistance in wastewater treatment (WWT) systems and downstream of effluent discharge. The aim of this thesis was to investigate the fate of antibiotics in algal WWT ponds, which have unique ecological and environmental characteristics (e.g. presence of algae; diurnal variation in pH, dissolved oxygen, and temperature) compared with conventional biological WWT. The research in this thesis focused on a case study of the fate of tetracycline (TET, an antibiotic) in high rate algal ponds (HRAP). Indoor lab scale HRAP studies were used to investigate the fate of TET under several operating conditions. Outdoor pilot scale studies (900 L and 180 L HRAPs) under Oceanic and Mediterranean climates were used to validate the lab scale findings. Results showed that high removal (85% to >98%) of TET was possible in the lab and pilot scale HRAPs with HRTs of 4 and 7 days. Sorption was consistently a low contributor (3-10% removal by sorption) during continuous HRAP studies, based on the amount of TET extracted from biomass. Batch experimentation was used to further distinguish mechanisms of TET removal. The majority of TET removal was caused by photodegradation. Indirect photodegradation of TET was dominant over direct photolysis, with 3-7 times higher photodegradation observed in wastewater effluent than for photodegradation in purified water during batch tests incubated in sunlight. Under dark conditions sorption was the dominant removal mechanism, and biodegradation was negligible in batch tests since aqueous TET removed was recovered (± 10%) by extraction of sorbed TET from the biomass. Irreversible abiotic hydrolysis was not observed during TET removal batch tests in purified (MQ) water. A kinetic model was developed and used to predict TET removal in the pilot HRAPs, based on parameters derived from batch experiments. The model predictions for aqueous TET concentrations were successfully validated against initial TET pulse tests in the 180 L pilot scale HRAP. However TET removal decreased in subsequent pulse tests in the pilot HRAP, resulting in over-prediction of TET removal by the kinetic model. This decrease in TET removal was associated with decrease in pH, dissolved oxygen concentrations, and biomass settleability, but causal relationships between TET removal and these variables could not be quantified. Until the predictive kinetic model is developed further, this model may serve as a preliminary estimate of TET fate in algal WWT ponds of different design and operation. Future research should also investigate the potential formation and toxicity (including antibiotic efficiency) of TET degradation products, but this was outside the scope of this thesis. Predictions from the model were sensitive to the daily light intensity, suggesting that TET removal would be reduced in the winter months

Genetic Basis of Tetracycline Resistance in Bifidobacterium animalis subsp lactis

All strains of Bifidobacterium animalis subsp. lactis described to date show medium level resistance to tetracycline. Screening of 26 strains from a variety of sources revealed the presence of tet(W) in all isolates. A transposase gene upstream of tet(W) was found in all strains, and both genes were cotranscribed in strain IPLAIC4. Mutants with increased tetracycline resistance as well as tetracycline-sensitive mutants of IPLAIC4 were isolated and genetically characterized. The native tet(W) gene was able to restore the resistance phenotype to a mutant with an alteration in tet(W) by functional complementation, indicating that tet(W) is necessary and sufficient for the tetracycline resistance seen in B. animalis subsp. lactis

Tertiary-Treated Municipal Wastewater is a Significant Point Source of Antibiotic Resistance Genes Into Duluth-Superior Harbor

In this study, the impact of tertiary-treated municipal wastewater on the quantity of several antibiotic resistance determinants in Duluth-Superior Harbor was investigated by collecting surface water and sediment samples from 13 locations in Duluth-Superior Harbor, the St. Louis River, and Lake Superior. Quantitative PCR (qPCR) was used to target three different genes encoding resistance to tetracycline (tet(A), tet(X), and tet(W)), the gene encoding the integrase of class 1 integrons (intI1), and total bacterial abundance (16S rRNA genes) as well as total and human fecal contamination levels (16S rRNA genes specific to the genus Bacteroides). The quantities of tet(A), tet(X), tet(W), intI1, total Bacteroides, and human-specific Bacteroides were typically 20-fold higher in the tertiary-treated wastewater than in nearby surface water samples. In contrast, the quantities of these genes in the St. Louis River and Lake Superior were typically below detection. Analysis of sequences of tet(W) gene fragments from four different samples collected throughout the study site supported the conclusion that tertiary-treated municipal wastewater is a point source of resistance genes into Duluth-Superior Harbor. This study demonstrates that the discharge of exceptionally treated municipal wastewater can have a statistically significant effect on the quantities of antibiotic resistance genes in otherwise pristine surface waters

In vivo manipulation of interleukin-2 expression by a retroviral tetracycline (tet)-regulated system

We have used the tetracycline (tet)-regulated system as described previously to evaluate the applicability of controlled gene expression in cancer gene therapy. As a model gene, we used the human interleukin-2 (IL-2) gene, which has been placed under the transcriptional control of the tetO/promoter. Human melanoma cells were transduced by two modified retroviral tet vectors containing the transactivator regulatory unit and the IL-2 gene driven by the tetO/promoter, respectively. In the absence of tet, IL-2 expression in the target cells was stable over several months. IL-2 production was in the range of 40 U/106 cells/24 hours. A fine tuning of IL-2 expression could be achieved by culturing the transduced cells with increasing doses of tet, whereby a concentration of 500 ng/mL tet in the culture medium abrogated IL-2 expression. Most importantly for clinical application, IL-2 expression by the transduced melanoma cells could also be regulated in vivo. When nu/nu mice were inoculated with the transduced tumor cells, they failed to develop tumors. Instead, the inhibition of IL-2 expression in the transduced tumor cells by oral administration of tet led to subcutaneous tumor growth; this growth rate was comparable with the growth rate of subcutaneously inoculated untransduced parental cells. The finding demonstrates the applicability of the tet-regulated system in cancer gene therapy

Functional analysis of the Arabidopsis TETRASPANIN gene family in plant growth and development

TETRASPANIN (TET) genes encode conserved integral membrane proteins that are known in animals to function in cellular communication during gamete fusion, immunity reaction and pathogen recognition. In plants, functional information is limited to one of the 17 members of the Arabidopsis TET gene family and to expression data in reproductive stages. Here, the promoter activity of all 17 Arabidopsis TET genes was investigated by pAtTET::NLS-GFP/GUS reporter lines throughout the life cycle, which predicted functional divergence in the paralogous genes per clade. However, partial overlap was observed for many TET genes across the clades, correlating with few phenotypes in single mutants and therefore requiring double mutant combinations for functional investigation. Mutational analysis showed a role for TET13 in primary root growth and lateral root development, and redundant roles for TET5 and TET6 in leaf and root growth through negative regulation of cell proliferation. Strikingly, a number of TET genes were expressed in embryonic and seedling progenitor cells and remained expressed until the differentiation state in the mature plant, suggesting a dynamic function over developmental stages. cis-regulatory elements together with transcription factor binding data provided molecular insight into the site, conditions and perturbations that affect TET gene expression, and positioned the TET genes in different molecular pathways; the data represent a hypothesis-generating resource for further functional analyses

The mathematical approach for proximity analysis for 3D GIS

Next generation of GIS software would highly depend on 3D analysis in solving geographic problems. 3D analysis is a very important component for GIS as it defines as decision making tools for geographic features. One would like to query about geographic object from numerical calculation or propose optimum solutions for GIS applications. Such desired components in future software or system are to deal with the 3D analytical solutions. This paper presents a portion of the problems, which are 3D solid buffering for 3D GIS. This analytical solution is very important for 3D spatial analysis. The discussion related to the implementation of buffering model for solid object is the main concern in the research, where the primitive objects of point, line, and face will be taken in consideration for the development of 3D solid bufferin

5mC-hydroxylase activity is influenced by the PARylation of TET1 enzyme

5-hydroxymethylcytosine is a new epigenetic modification deriving from the oxidation of 5-methylcytosine by the TET hydroxylase enzymes. DNA hydroxymethylation drives DNA demethylation events and is involved in the control of gene expression. Deregulation of TET enzymes causes developmental defects and is associated with pathological conditions such as cancer. Little information thus far is available on the regulation of TET activity by post-translational modifications. Here we show that TET1 protein is able to interact with PARP-1/ARTD1 enzyme and is target of both noncovalent and covalent PARylation. In particular, we have demonstrated that the noncovalent binding of ADP-ribose polymers with TET1 catalytic domain decreases TET1 hydroxylase activity while the covalent PARylation stimulates TET1 enzyme. In addition, TET1 activates PARP-1/ARTD1 independently of DNA breaks. Collectively, our results highlight a complex interplay between PARylation and TET1 which may be helpful in coordinating the multiple biological roles played by 5-hydroxymethylcytosine and TET proteins

A dilogarithmic 3-dimensional Ising tetrahedron

In 3 dimensions, the Ising model is in the same universality class as $\phi^4$-theory, whose massive 3-loop tetrahedral diagram, $C^{Tet}$, was of an unknown analytical nature. In contrast, all single-scale 4-dimensional tetrahedra were reduced, in hep-th/9803091, to special values of exponentially convergent polylogarithms. Combining dispersion relations with the integer-relation finder PSLQ, we find that $C^{Tet}/2^{5/2} = Cl_2(4\alpha) - Cl_2(2\alpha)$, with $Cl_2(\theta):=\sum_{n>0}\sin(n\theta)/n^2$ and $\alpha:=\arcsin\frac13$. This empirical relation has been checked at 1,000-digit precision and readily yields 50,000 digits of $C^{Tet}$, after transformation to an exponentially convergent sum, akin to those studied in math.CA/9803067. It appears that this 3-dimensional result entails a polylogarithmic ladder beginning with the classical formula for $\pi/\sqrt2$, in the manner that 4-dimensional results build on that for $\pi/\sqrt3$.Comment: 8 pages, LaTeX; Eq(25) simplified; Eqs(27,33) and refs[3,18] adde