Assessment of the viability of Cryptosporidium parvum oocysts with the induction ratio of hsp70 mRNA production in manure.

Determining the presence of viable Cryptosporidium parvum oocysts in complex environmental matrices in hygiene control can prevent the contamination of water resources and food with this pathogen. This study assessed the induction ratio of hsp70 mRNA production by heat shock in different oocysts as a marker of viability. Using different procedures for (m)RNA extraction directly from manure and reverse transcription real-time qPCR, this study found slightly increased hsp70 mRNA contents in viable oocysts that were heat shock induced at 45°C for 20 min compared to not induced oocysts (1.6 fold induction in average). Prolonging the heat shock treatment to 2h did not further increase the copy numbers. Heat shock by consecutive stimuli, such as freezing and then heating, did not yield significantly higher copy numbers than the 45°C treatment. There was a certain background level of hsp70 mRNA in viable oocysts that were not exposed to heat shock, indicating a constitutive production of the transcripts in the oocysts. The production of hsp70 mRNA induced by heat shock in oocysts aged for 9 months that exhibited reduced viability was lower than in fresher oocysts (induction ratio<1.2). No production of hsp70 mRNA by heat shock was detected in 12 months old oocysts that were not viable in the excystation test. Oocysts inactivated at 75°C for 30 min were not able to respond to heat shock, and low amount of copies were occasionally measured only in total RNA extracts, but not in mRNA extracts that were purified directly with an oligo (dT)25 based system. The induction ratio of hsp70 mRNA varied according to the viability of the organisms in a sample. Copy numbers of ß-tubulin mRNA in viable oocysts were lower than hsp70 mRNA, therefore the latter is more suitable to detect low numbers of oocysts by RT-qPCR.

Evaluation of two methods for quantification of hsp70 mRNA from the waterborne pathogen Cryptosporidium parvum by reverse transcription real-time PCR in environmental samples.

We optimized and evaluated two mRNA extraction methods to quantify induced hsp70 mRNA from viable and injured Cryptosporidium parvum oocysts by reverse transcription quantitative real-time PCR (RT-qPCR) in raw and treated manure. Methods based on guanidinium isothiocyanate/phenol/chloroform (GITC-PC) purification and direct mRNA extraction with magnetic oligo(dT)25-coated beads were evaluated for applicability and sensitivity. Both methods proved to be suitable for processing manure samples. With washed manure samples and oocyst disruption by bead beating for 165 s in time intervals with cumulative pooling of the lysate fractions, optimum RT-qPCR results were achieved. On average, 2.6 times more hsp70 mRNA was detected with the oligo(dT)25 method in comparison to the GITC-PC based method using fresh oocysts, whereas less mRNA was detected in aged oocysts. For fresh oocysts, analytical and method detection limits for the oligo(dT)25 based method were 1.7 cDNA copies/qPCR reaction and 5150 oocysts/mL manure, and for the GITC-PC based method 17 cDNA copies/qPCR reaction and 4950 oocysts/mL, respectively. In 12 months old oocysts with reduced viability, mRNA was occasionally detected only by the GITC-PC based method. Failure of or reduced detection with the oligo(dT)25 based method was apparently a result of weakened oocyst walls leading to quicker release of mRNA and therefore mRNA shredding by bead beating in the relatively long stretch between the capture sequence and the RT-qPCR target sites.

Bioaugmentation of microbial communities in laboratory and pilot scale sequencing batch biofilm reactors using the TOL plasmid.

The aim of this study was to investigate the effectiveness of bioaugmentation and transfer of plasmid pWWO (TOL plasmid) to mixed microbial populations in pilot and laboratory scale sequencing batch biofilm reactors (SBBRs) treating synthetic wastewater containing benzyl alcohol (BA) as a model xenobiotic. The plasmid donor was a Pseudomonas putida strain chromosomally tagged with the gene for the red fluorescent protein carrying a green fluorescent protein labeled TOL plasmid, which confers degradation capacity for several compounds including toluene and BA. In the pilot scale SBBR donor cells were disappeared 84 h after inoculation while transconjugants were not detected at all. In contrast, both donor and transconjugant cells were detected in the laboratory scale reactor where the ratio of transconjugants to donors fluctuated between 1.9 x 10(-1) and 8.9 x 10(-1) during an experimental period of 32 days. BA degradation rate was enhanced after donor inoculation from 0.98 mg BA/min prior to inoculation to 1.9 mg BA/min on the seventeenth day of operation. Survival of a bioaugmented strain, conjugative plasmid transfer and enhanced BA degradation was demonstrated in the laboratory scale SBBR but not in the pilot scale SBBR.

Identification of Archaeal population in the granular sludge of an UASB reactor treating sewage at low temperatures.

Effect of low temperature on up-flow anaerobic sludge bed (UASB) reactor performance treating raw sewage was investigated in terms of the variations in methanogenic diversity using the 16S rRNA based Fluorescence In-Situ Hybridization (FISH) technique. The diversity of microorganisms present in the anaerobic granular sludge and the structure of the granules operated at 13 degrees C have been investigated using FISH combined with CSLM (Confocal Scanning Laser Microscopy). According to FISH results, archaeal cells representing methanogens were found intensively dominant in the bottom sampling port of the UASB reactor and acetoclastic Methanosaeta was the abundant methanogen. Other methanogens such as Methanosarcina and Methanobacterium like species were also observed. The abundance of originally mesophilic Methanosaeta-related Archaea under low temperature at all sampling days revealed the microbial adaptation to psychrophilic conditions. This might be attributed to the enzymatic alterations in Methanosaeta cells originating from seed sludge, which were exposed to sub-mesophilic temperatures at start-up and then to psychrophilic conditions during gradual decreases of temperature. According to CSLM observation, even though the sludge retained in the reactor kept its granular form as a whole, the majority of the granules had a tendency to be partly broken and they lost their rigidity when raw sewage was fed following synthetic sewage. Besides, Methanosaeta related species prevailing in seed sludge have noticeably lost their long filamentous forms and deteriorated during raw sewage feeding. Members of the order Methanobacteriales constituted the major hydrogenothrophic methanogens present in the psychrophilic UASB reactor, whereas the other hydrogenothrophic methanogens--members of the order Methanococcales and Methanogenium relatives--were absent.

Microbial populations associated with fixed- and floating-bed reactors during a two-stage anaerobic process

Microbial populations associated with methanogenic fixed- or floating-bed bioreactors used for anaerobic digestion of lignocellulosic waste were investigated. Fluorescent in situ hybridization (FISH) was used to characterize microorganisms in samples obtained from different heights in the reactors, which were operated in a semi-continuous manner (feeding and mixing once every 2 days). The FISH results showed that Methanosaeta concilii cells were most numerous at the bottom of both reactors. M. concilii cells were more abundant in the fixed-bed reactor (FXBR), which performed better than the floating-bed reactor (FLBR). Species of the Methanosarcina genera (mainly M. barkeri and M. mazei) were also observed in the FLBR but rarely in the FXBR. Methane production in each of the reactors ranged from 0.29 to 0.33 m3 CH(4)/kg COD(rem) (chemical oxygen demand removed). The removal of volatile fatty acids (VFA; 70-75 h) in the FXBR was more efficient than in the FLBR (AU)

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Dual labeling of Pseudomonas putida with fluorescent proteins for in situ monitoring of conjugal transfer of the TOL plasmid.

We describe here a dual-labeling technique involving the green fluorescent protein (GFP) and the red fluorescent protein (DsRed) for in situ monitoring of horizontal gene transfer via conjugation. A GFPmut3b-tagged derivative of narrow-host-range TOL plasmid (pWWO) was delivered to Pseudomonas putida KT2442, which was chromosomally labeled with dsRed by transposon insertion via biparental mating. Green and red fluorescent proteins were coexpressed in donor P. putida cells. Cells expressing both fluorescent proteins were smaller in size than cells expressing GFP alone. Donors and transconjugants in mixed culture or sludge samples were discriminated on the basis of their fluorescence by using confocal laser scanning microscopy. Conjugal plasmid transfer frequencies on agar surfaces and in sludge microcosms were determined microscopically without cultivation. This method worked well for in situ monitoring of horizontal gene transfer in addition to tracking the fate of microorganisms released into complex environments. To the best of our knowledge, this is the first study that discusses the coexpression of GFP and DsRed for conjugal gene transfer studies.

Effect of extended idle conditions on structure and activity of granular activated sludge.

In industry and in tourist areas, periods exist during which no or only very little sewage is produced, and the wastewater treatment facilities have to be set into an idle phase over several days and even weeks. When wastewater is generated again and delivered to the treatment plant, the microorganisms in the activated sludge plant may have lost activity, and the activated sludge flocs may have disintegrated. From previous observation, it is assumed that granular activated sludge is more resistant against long-term storage than activated sludge flocs. Experiments using a laboratory-scale sequencing batch reactor (SBR) were conducted to study the impacts of a 7-weeks anaerobic idle time on structural integrity and metabolic activity of granular activated sludge, and the time required to regain the former operational status of the plant. Oxygen consumption rate (OCR) was used as an indicator to evaluate the metabolic activity of the sludge. The results revealed that the size, color and sedimentation characteristics of the granular sludge did hardly change during the storage period. Sludge activity, however, dropped to values as low as 0.17 mg min(-1)L(-1). After restarting the reactor, the OCR increased within 1 day to a level of 0.57 mg min(-1)L(-1), kept rising at a linear rate in the following days, and reached after 1 week, a value of 5.74 mg min(-1)L(-1) typical for the former activity status. These results imply that granular activated sludge can be stored for a considerably long period of time, and brought into service again relatively quickly. After an idle period of 7 weeks, it took less than a week to regain full capacity of the SBR.

Modern scientific methods and their potential in wastewater science and technology.

Application of novel analytical and investigative methods such as fluorescence in situ hybridization, confocal laser scanning microscopy (CLSM), microelectrodes and advanced numerical simulation has led to new insights into micro- and macroscopic processes in bioreactors. However, the question is still open whether or not these new findings and the subsequent gain of knowledge are of significant practical relevance and if so, where and how. To find suitable answers it is necessary for engineers to know what can be expected by applying these modern analytical tools. Similarly, scientists could benefit significantly from an intensive dialogue with engineers in order to find out about practical problems and conditions existing in wastewater treatment systems. In this paper, an attempt is made to help bridge the gap between science and engineering in biological wastewater treatment. We provide an overview of recently developed methods in microbiology and in mathematical modeling and numerical simulation. A questionnaire is presented which may help generate a platform from which further technical and scientific developments can be accomplished. Both the paper and the questionnaire are aimed at encouraging scientists and engineers to enter into an intensive, mutually beneficial dialogue.

Population changes in a biofilm reactor for phosphorus removal as evidenced by the use of FISH.

Induction of denitrification was investigated for a lab-scale phosphate removing biofilm reactor where oxygen was replaced with nitrate as the electron acceptor. Acetate was used as the carbon source. The original biofilm (acclimatised with oxygen) was taken from a well-established large-scale reactor. During the first run, a decrease in the denitrifying bio-P activity was observed after 1 month following a change in the anaerobic phase length. This was initially interpreted as a shift in the microbial population caused by the changed operation. In the second run, biomass samples were regularly collected and analysed by fluorescent in situ hybridisation (FISH) and confocal laser scanning microscopy (CLSM). Concurrently, samples were taken from the original reactor with oxygen as electron acceptor in order to investigate natural microbial fluctuations. A similar decrease in the activity as in the first run was seen after one month, although the phase lengths had not been varied. Hence, the decrease after 1 month in the first and second run should be seen as a start-up phenomenon. FISH could detect a noticeable shift in the microbial population mainly within the first 2 weeks of operation. Almost all bacteria belonging to the alpha subclass disappeared and characteristic clusters of the beta and gamma subclasses were lost. Small clusters of gram-positive bacteria with a high DNA G + C content (GPBHGC) were gradually replaced by filamentous GPBHGC. Most of the bacteria in the denitrifying, phosphate removing biofilm belonged to the beta subclass of Proteobacteria. The applied set of gene probes had been selected based on existing literature on biological phosphate removing organisms and included a recently published probe for a Rhodocyclus-like clone. However, none of the specific probes hybridised to the dominant bacterial groups in the reactors investigated. No noticeable changes were detected in the aerobic bench-scale reactor during this period, indicating that the observed changes in the lab-scale reactor were caused by the changed environment.