Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance

Background: The New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasis-causing flies, causing severe losses to the livestock industry. In its current geographical distribution, this species has been controlled by the application of insecticides, mainly organophosphate (OP) compounds, but a number of lineages have been identified that are resistant to such chemicals. Despite its economic importance, only limited genetic information is available for the NWS. Here, as a part of an effort to characterize the C. hominivorax genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology. Results: Deep sequencing on the 454 platform of three normalized libraries (larval, adult male and adult female) generated a total of 548,940 reads. Eighteen candidate genes coding for three metabolic detoxification enzyme families, cytochrome P450 monooxygenases, glutathione S transferases and carboxyl/cholinesterases were selected and gene expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Of the investigated candidates, only one gene was expressed differently between control and resistant larvae with, at least, a 10-fold down-regulation in the resistant larvae. The presence of mutations in the acetylcholinesterase (target site) and carboxylesterase E3 genes was investigated and all of the resistant flies presented E3 mutations previously associated with insecticide resistance. Conclusions: Here, we provided the largest database of NWS expressed sequence tags that is an important resource, not only for further studies on the molecular basis of the OP resistance in NWS fly, but also for functional and comparative studies among Calliphoridae flies. Among our candidates, only one gene was found differentially expressed in resistant individuals, and its role on insecticide resistance should be further investigated. Furthermore, the absence of mutations in the OP target site and the high frequency of mutant carboxylesterase E3 indicate that metabolic resistance mechanisms have evolved predominantly in this species.

Validation of dot blot hybridization and denaturing high performance liquid chromatography as reliable methods for TP53 codon 72 genotyping in molecular epidemiologic studies

Background: Mutations in TP53 are common events during carcinogenesis. In addition to gene mutations, several reports have focused on TP53 polymorphisms as risk factors for malignant disease. Many studies have highlighted that the status of the TP53 codon 72 polymorphism could influence cancer susceptibility. However, the results have been inconsistent and various methodological features can contribute to departures from Hardy-Weinberg equilibrium, a condition that may influence the disease risk estimates. The most widely accepted method of detecting genotyping error is to confirm genotypes by sequencing and/or via a separate method. Results: We developed two new genotyping methods for TP53 codon 72 polymorphism detection: Denaturing High Performance Liquid Chromatography (DHPLC) and Dot Blot hybridization. These methods were compared with Restriction Fragment Length Polymorphism (RFLP) using two different restriction enzymes. We observed high agreement among all methodologies assayed. Dot-blot hybridization and DHPLC results were more highly concordant with each other than when either of these methods was compared with RFLP. Conclusions: Although variations may occur, our results indicate that DHPLC and Dot Blot hybridization can be used as reliable screening methods for TP53 codon 72 polymorphism detection, especially in molecular epidemiologic studies, where high throughput methodologies are required.

Innovation concept for measurement gross error detection and identification in power system state estimation

In this study, the innovation approach is used to estimate the measurement total error associated with power system state estimation. This is required because the power system equations are very much correlated with each other and as a consequence part of the measurements errors is masked. For that purpose an index, innovation index (II), which provides the quantity of new information a measurement contains is proposed. A critical measurement is the limit case of a measurement with low II, it has a zero II index and its error is totally masked. In other words, that measurement does not bring any innovation for the gross error test. Using the II of a measurement, the masked gross error by the state estimation is recovered; then the total gross error of that measurement is composed. Instead of the classical normalised measurement residual amplitude, the corresponding normalised composed measurement residual amplitude is used in the gross error detection and identification test, but with m degrees of freedom. The gross error processing turns out to be very simple to implement, requiring only few adaptations to the existing state estimation software. The IEEE-14 bus system is used to validate the proposed gross error detection and identification test.

Thermal error evaluation and modelling of a CNC cylindrical grinding machine

With the relentless quest for improved performance driving ever tighter tolerances for manufacturing, machine tools are sometimes unable to meet the desired requirements. One option to improve the tolerances of machine tools is to compensate for their errors. Among all possible sources of machine tool error, thermally induced errors are, in general for newer machines, the most important. The present work demonstrates the evaluation and modelling of the behaviour of the thermal errors of a CNC cylindrical grinding machine during its warm-up period.

Full-scale anaerobic sequencing batch biofilm reactor for sulfate-rich wastewater treatment

This paper describes the performance and biofilm characteristics of a full-scale anaerobic sequencing batch biofilm reactor (ASBBR; 20 m(3)) containing biomass immobilized on an inert support (mineral coal) for the treatment of industrial wastewater containing a high sulfate concentration. The ASBBR reactor was operated during 110 cycles (48 h each) at sulfate loading rates ranging from 6.9 to 62.4 kgSO(4)(2-)/cycle corresponding to sulfate concentrations of 0.58-5.2 gSO(4)(2-)/L. Domestic sewage and ethanol were utilized as electron donors for sulfate reduction. After 71 cycles the mean sulfate removal efficiency was 99%, demonstrating a high potential for biological sulfate reduction. The biofilm formed in the reactor occurred in two different patterns, one at the beginning of the colonization and the other of a mature biofilm. These different colonization patterns are due to the low adhesion of the microorganisms on the inert support in the start-up period. The biofilm population is mainly made up of syntrophic consortia among sulfate-reducing bacteria and methanogenic archaea such as Methanosaeta spp.

Influence of carbon sources and C/N ratio on EPS production in anaerobic sequencing batch biofilm reactors for wastewater treatment

The objective of this work was to evaluate the influence of different carbon sources and the carbon/nitrogen ratio (C/N) on the production and main composition of insoluble extracellular polymers (EPS) produced in an anaerobic sequencing batch biofilm reactor (ASBBR) with immobilized biomass in polyurethane foam. The yield of EPS was 23.6 mg/g carbon, 13.3 mg/g carbon, 9.0 mg/g carbon and 1.4 mg/g carbon when the reactor was fed with glucose, soybean oil. fat acids, and meat extract, respectively. The yield of EPS decreased from 23.6 to 2.6 mg/g carbon as the C/N ratio was decreased from 13.6 to 3.4 gC/gN, using glucose as carbon source. EPS production was not observed under strict anaerobic conditions. The results suggest that the carbon source, microaerophilic conditions and high C/N ratio favor EPS production in the ASBBR used for wastewater treatment. Cellulose was the main exopolysaccharide observed in all experimental conditions. (C) 2009 Elsevier Ltd. All rights reserved.

Nitrogen removal in a sequencing batch biofilm reactor with liquid circulation: Effect of feed strategy and carbon source in the denitrifying step

Ammonium nitrogen removal from a synthetic wastewater by nitrification and denitrification processes were performed in a sequencing batch biofilm reactor containing immobilized biomass on polyurethane foam with circulation of the liquid-phase. It was analyzed the effect of four external carbon sources (ethanol, acetate, carbon synthetic medium and methanol) acting as electron donors in the denitrifying process. The experiments were conducted with intermittent aeration and operated at 30+/-1 degrees C in 8-h cycles. The synthetic wastewater (100 mgCOD/L and 50 mgNH(4)(+)-N/L) was added batch-wise, while the external carbon sources were added fed-batch-wise during the periods where aeration was suspended. Ammonium nitrogen removal efficiencies obtained were 95.7, 94.3 and 97.5% for ethanol, acetate and carbon synthetic medium, respectively. As to nitrite, nitrate and ammonium nitrogen effluent concentrations, the results obtained were, respectively: 0.1, 5.7 and 1.4 mg/L for ethanol; 0.2, 4.1 and 1.8 mg/L for acetate and 0.2, 6.7 and 0.8 for carbon synthetic medium. On the other hand using methanol, even at low concentrations (50% of the stoichiometric value calculated for complete denitrification), resulted in increasing accumulation of nitrate and ammonium nitrogen in the effluent over time.

The treatment of sulfate-rich wastewater using an anaerobic sequencing batch biofilm pilot-scale reactor

This paper presents the results from 92 cycles of an anaerobic sequencing batch biofilm reactor containing biomass immobilized on inert support (mineral coal) applied for the treatment of an industrial wastewater containing high sulfate concentration. The pilot-scale reactor, with a total volume of 1.2 m(3), was operated at sulfate loading rates ranging from 0.15 to 1.90 kgSO(4)(2-)/cycle (48 It - cycle) corresponding to sulfate concentrations of 0.25 to 3.0 gSO(4)(2-) l(-1). Domestic sewage and ethanol were utilized as electron donors for sulfate reduction. Influent sulfate concentrations were increased in order to evaluate the minimum COD/sulfate ratio at which high reactor performance could be maintained. The mean sulfate removal efficiency remained between the range of 88 to 92% at several sulfate concentrations. Temporal profiles along the 48 h cycles were carried out under stable operation at sulfate concentrations of 1.0, 2.0 and 3.0 gSO(4)(2-) l(-1). Sulfate removal reached 99% for cycle times of 15, 25, and 30 h, and the effluents sulfate concentrations were lower than 8 mgSO(4)(2-) l(-1). The results demonstrate the potential applicability of the anaerobic configuration for the biological treatment of sulfate-rich wastewaters. (C) 2009 Elsevier B.V. All rights reserved.

Effect of specific feed volume on the performance of an anaerobic sequencing batch biofilm reactor (AnSBBR) with circulation treating different wastewaters under different organic loads

The objective of this research was to study the behavior of two anaerobic sequencing batch reactors, containing immobilized biomass (AnSBBR), as a function of the ratio of the volume of treated medium in each cycle to the total volume of reaction medium. The reactors, in which mixing was accomplished by recirculation of the liquid phase, were maintained at 30 +/- 1 degrees C and treated different wastewaters in 8-h cycles. The operational conditions imposed had the objective to investigate whether maintenance of a residual volume in the reactor would affect, at the end of each cycle, process efficiency and stability, as well as to verify the intensity of the effect for different types of wastewaters and organic loading rates. The first reactor, with work volume of 2.5 L, treated reconstituted cheese whey at an organic loading rate of 12 g COD.L(-1).d(-1) and presented similar effluent quality for the four conditions under which it was operated: renewal of 100, 70, 50 and 25 % of its work volume at each cycle. Despite the fact that reduction in the renewed volume did not significantly affect effluent quality, in quantitative terms, this reduction resulted in an increase in the amount of organic matter removed by the first reactor. The second reactor, with work volume of 1.8 L, treated synthetic wastewater at organic loading rates of 3 and 5 g COD.L(-1).d(-1) and operated under two conditions for each loading: renewal of 100 and 50 % of its work volume. At the organic loading rate of 3 g COD.L(-1).d(-1), the results showed that both effluent quality and amount of organic matter removed by the second reactor were independent of the treated volume per cycle. At the organic loading rate of 5 g COD.L(-1).d(-1), although the reduction in the renewed volume did not affect the amount of organic matter removed by the reactor, effluent quality improved during reactor operation with total discharge of its volume. In general, results showed process stability under all conditions, evidencing reactor flexibility and the potential to apply this technology in the treatment of different types of wastewater.

Degradation of formaldehyde in anaerobic sequencing batch biofilm reactor (ASBBR)

The present study evaluated the degradation of formaldehyde in a bench-scale anaerobic sequencing batch reactor, which contained biomass immobilized in polyurethane foam matrices. The reactor was operated for 212 days at 35 C with 8 h sequential cycles, under different affluent formaldehyde concentrations ranging from 31.6 to 1104.4 mg/L (formaldehyde loading rates from 0.08 to 2.78 kg/m(3) day). The results indicate excellent reactor stability and over 99% efficiency in formaldehyde removal, with average effluent formaldehyde concentration of 3.6 + 1.7 mg/L. Formaldehyde degradation rates increased from 204.9 to 698.3 mg/L h as the initial concentration of formaldehyde was increased from around 100 to around 1100 mg/L. However, accumulation of organic matter was observed in the effluent (chemical oxygen demand (COD) values above 500 mg/L) due to the presence of non-degraded organic acids, especially acetic and propionic acids, This observation poses an important question regarding the anaerobic route of formaldehyde degradation, which might differ Substantially from that reported in the literature. The anaerobic degradation pathway can be associated with the formation of long-chain oligomers from formaldehyde. Such long- or short-chain polymers are probably the precursors of organic acid formation by means of acidogenic anaerobic microorganisms. (C) 2008 Elsevier B.V. All rights reserved.

Effects of temperature at different organic loading levels on the performance of a fluidized-bed anaerobic sequencing batch bioreactor

An investigation was performed on the effect of temperature and organic load on the stability and efficiency of a 1.8-L fluidized-bed anaerobic sequencing batch reactor (ASBR), containing granulated biomass. Assays were carried out employing superficial up How velocity of 7 m/h, total cycle length of 6 h and synthetic wastewater volume of 1.3 L per cycle. The fluidized-bed ASH was operated at 15, 20, 25 and 30 degrees C with influent organic matter concentrations of 500 and 1000 mgCOD/L The system showed stability under all conditions and presented filtered samples removal efficiency ranging from 79 to 86%. A first-order kinetic model could be fitted to the experimental values of the organic matter concentration profiles. The specific kinetic parameter values of this model ranged from 0.0435 to 0.2360 L/(gTVS h) at the implemented operation conditions. in addition, from the slope of an Arrhenius plot, the activation energy values were calculated to be 16,729 and 12,673 cal/mol for operation with 500 and 1000 mgCOD/L, respectively. These results show that treatment of synthetic wastewater. with concentration of 500 mgCOD/L, was more sensitive to temperature variations than treatment of the same residue with concentration of 1000 mgCOD/L. Comparing the activation energy value for operation at 500 mgCOD/L with the value obtained by Agibert et al. (S.A. Agibert, M.B. Moreira, S.M. Ratusznei, J.A.D. Rodrigues, M. Zaiat, E. Foresti. Influence of temperature on performance of an ASBBR with circulation applied to treatment of low-strength wastewater. journal of Applied Biochemistry and Biotechnology, 136 (2007) 193-206) in an ASBBR treating the same wastewater at the same concentration, the value obtained in the fluidized-bed ASBR showed to be superior, indicating that treatment of synthetic wastewater in a reactor containing granulated biomass was more sensitive to temperature variations than the treatment using immobilized biomass. (c) 2008 Elsevier B.V. All rights reserved.

Post-treatment of effluents from the sulfate reduction process by anaerobic sequencing batch biofilm reactors

The main objective of this research was to evaluate the potential use of a bench-scale anaerobic sequencing batch biofilm reactor (ASBBR) containing mineral coal as inert support for removal Of Sulfide and organic matter effluents from an ASBBR (1.2 m(3)) utilized for treatment of sulfate-rich wastewater. The cycle time was 48 h, including the steps of feeding (2 h), reaction with continuous liquid recirculation (44 h) and discharge (2 h). COD removal efficiency was up to 90% and the effluents total sulfide concentrations (H(2)S, HS(-), S(2-)) remained in the range of 1.5 to 7.5 mg.l(-1) during the 50 days of operation (25 cycles). The un-ionized Sulfide and ionized sulfides were converted by biological process to elemental sulfur (S(0)) under oxygen limited conditions. The results obtained in the bench-scale reactor were used to design an ASBBR in pilot scale for use in post-treatment to achieve the emission standards (sulfide and COD) for sulfate reduction. The pilot-scale reactor, with a total volume of 0.43 m(3), the COD and total sulfide removal achieved 88% and 57%, respectively, for a cycle time of 48 h (70 days of operation or 35 cycles).

Feasibility of a Sequencing Reactor Operated in Batch and Fed-batch Mode Applied to Nitrification and Denitrification Processes

The objective of this work was to study the operational feasibility of nitrification and denitrification processes in a mechanically stirred sequencing batch reactor (SBR) operated in batch and fed-batch mode. The reactor was equipped with a draft-tube to improve mass transfer and contained dispersed (aerobic) and granulated (anaerobic) biomass. The following reactor variables were adjusted: aeration time during the nitrification step; dissolved oxygen concentration, feed time defining batch and fed-batch phases, concentration of external carbon source used as electron donor during the denitrification stage and volumetric ammonium nitrogen load in the influent. The reactor (5 L volume) was maintained at 30 +/- 1 degrees C and treated either 1.0 or 1.5 L wastewater in 8-h cycles. Ammonium nitrogen concentrations assessed were: 50 (condition 1) and 100 mgN-NH(4)(+).L(-1) (condition 2), resulting in 29 and 67 mgN-NH(4)(+).L-1-d(-1), respectively. A synthetic medium and ethanol were used as external carbon sources (ECS). Total nitrogen removal efficiencies were 94.4 and 95.9% when the reactor was operated under conditions 1 and 2, respectively. Low nitrite (0.2 and 0.3 mgN-NO(2)(-).L(-1), respectively) and nitrate (0.01 and 0.3 mgN-NO(3)(-).L(-1), respectively) concentrations were detected in the effluent and ammonium nitrogen removal efficiencies were 97.6% and 99.6% under conditions 1 and 2, respectively.

Effects of bed materials on the performance of an anaerobic sequencing batch biofilm reactor treating domestic sewage

The objective of this study was to determine the best performance of an anaerobic sequencing batch biofilm reactor (AnSBBR) based on the use of four different bed materials as support for biomass immobilization. The bed materials utilized were Polyurethane foam (PU), vegetal carbon (VC), synthetic pumice (SP), and recycled low-density polyethylene (PE). The AnSBBR. with I total volume Of 7.2 L, was operated in 8-h batch cycles over 10 months, and fed with domestic sewage with an average influent chemical oxygen demand (COD) of 358 +/- 110 mg/L. The average effluent COD values were 121 +/- 31, 208 +/- 54, 233 +/- 52, and 227 +/- 51 mg/L. for PU, VC, SP, and PE, respectively. A modified first-order kinetic model was adjusted to temporal profiles of COD during a batch cycle, and the apparent kinetic constants were 0.52 +/- 0.05, 0.37 +/- 0.05, 0.80 +/- 0.04, and 0.30 +/- 0.021h(-1) for PU, VC, SP, and PE, respectively. Specific substrate utilization rates of 1.08, 0.11, and 0.86 mg COD/mg VS day were obtained for PU, VC, and PE, respectively. Although SP yielded the highest kinetic coefficient, PU was considered the best support, since SP presented loss of chemical constituents during the reactor`s operational phase. In addition, findings oil the microbial community were associated with the reactor`s performance data. Although PE did not show a satisfactory performance, an interesting microbial diversity was found oil its surface. Based oil the morphology and denaturing gradient gel electrophoresis (DGGE) results, PE showed the best capacity for promoting the attachment of methanogenic organisms, and is therefore a material that merits further analysis. PU was considered the Most suitable material showing the best performance in terms of efficiency of solids and COD removal. (C) 2007 Elsevier Ltd. All rights reserved.

Anaerobic sequencing batch biofilm reactor applied to automobile industry wastewater treatment: Volumetric loading rate and feed strategy effects

This paper presents a technological viability study of wastewater treatment in an automobile industry by an anaerobic sequencing batch biofilm reactor containing immobilized biomass (AnSBBR) with a draft tube. The reactor was operated in 8-h cycles, with agitation of 400 rpm, at 30 degrees C and treating 2.0 L wastewater per cycle. Initially the efficiency and stability of the reactor were studied when supplied with nutrients and alkalinity. Removal efficiency of 88% was obtained at volumetric loading rate (VLR) of 3.09 mg COD/L day. When VLR was increased to 6.19 mg COD/L day the system presented stable operation with reduction in efficiency of 71%. In a second stage the AnSBBR was operated treating wastewater in natura, i.e., without nutrients supplementation, only with alkalinity, thereby changing feed strategy. The first strategy consisted in feeding 2.0 L batch wise (10 min), the second in feeding 1.0 L of influent batch wise (10 min) and an additional 1.0 L fed-batch wise (4 h), both dewatering 2.0 L of the effluent in 10 min. The third one maintained 1.0 L of treated effluent in the reactor, without discharging, and 1.0 L of influent was fed fed-batch wise (4 h) with dewatering 1.0 L of the effluent in 10 min. For all implemented strategies (VLR of 1.40, 2.57 and 2.61 mg COD/L day) the system presented stability and removal efficiency of approximately 80%. These results show that the AnSBBR presents operational flexibility, as the influent can be fed according to industry availability. In industrial processes this is a considerable advantage, as the influent may be prone to variations. Moreover, for all the investigated conditions the kinetic parameters were obtained from fitting a first-order model to the profiles of organic matter, total volatile acids and methane concentrations. Analysis of the kinetic parameters showed that the best strategy is feeding 1.0 L of influent batchwise (10 min) and 1.0 L fed-batch wise (4 h) in 8-h cycle. (c) 2007 Elsevier B.V. All rights reserved.