Quantitative image analysis as a diagnostic tool for monitoring structural changes of anaerobic granular sludge during detergent shock loads

Two shock loads of a commercial detergent (I-150 mg chemical oxygen demand (COD)/L, fed for 56 h; II-300 mg COD/L fed for 222 h) were applied in a lab-scale Expanded Granular Sludge Blanket (EGSB) reactor, fed with 1,500 mg COD/L of ethanol. The impact of the surfactant was assessed in terms of granular sludge morphology, specific methanogenic activity (SMA) in the presence of individual substrates, and reactor performance. COD removal efficiency remained unaffected in the shock I, but 80 h after starting exposure to the shock II, the COD removal efficiency decreased drastically from 75 to 17%. In the first 8 h of operation of shock I, the SMA was stimulated and decreased afterwards, being recovered 5 days after the end of exposure time. Concerning to shock II, the SMA was immediately and persistently reduced during the exposure time, although, the inhibition of SMA in presence of H(2)/CO(2) showed a trend to increase after the exposure time. Acetoclastic bacteria were observed as the most sensitive to the toxic effects of surfactant whereas the hydrogenotrophic bacteria were less affected. The inhibitory effects were dependent on surfactant concentration and exposure time. The ratio filaments length per total aggregates area (LfA) was an early-warning indicator of biomass washout, since it increased 3 and 5 days before effluent volatile suspended solids (VSS) rise, respectively, in shocks I and II.     

Raman spectroscopy as a diagnostic tool for monitoring acute nephritis

Both acute nephritis and chronic nephritis account for substantial morbidity and mortality worldwide, partly due to the lack of reliable tools for detecting disease early and monitoring its progression non‐invasively. In this work, Raman spectroscopy coupled with multivariate analysis are employed for the first time to study the accelerated progression of nephritis in anti‐GBM mouse model. Preliminary results show up to 98% discriminant accuracy for the severe and midly diseased and the healthy among two strains of mice with different susceptibility to acute glomerulonephritis. This technique has the potential for non‐invasive or minimally‐invasive early diagnosis, prognosis, and monitoring of renal disease progression.

     

Biochemical Evidence for Formate Transfer in Syntrophic Propionate-Oxidizing Cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei

The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H₂ lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H₂ lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.     

Biochemical evidence for formate transfer in syntrophic propionate-oxidizing cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei

The hydrogenase and formate dehydrogenase levels in Syntrophobacter fumaroxidans and Methanospirillum hungatei were studied in syntrophic propionate-oxidizing cultures and compared to the levels in axenic cultures of both organisms. Cells grown syntrophically were separated from each other by Percoll gradient centrifugation. In S. fumaroxidans both formate dehydrogenase and hydrogenase levels were highest in cells which were grown syntrophically, while the formate-H(2) lyase activities were comparable under the conditions tested. In M. hungatei the formate dehydrogenase and formate-H(2) lyase levels were highest in cells grown syntrophically, while the hydrogenase levels in syntrophically grown cells were comparable to those in cells grown on formate. Reconstituted syntrophic cultures from axenic cultures immediately resumed syntrophic growth, and the calculated growth rates of these cultures were highest for cells which were inoculated from the axenic S. fumaroxidans cultures that exhibited the highest formate dehydrogenase activities. The results suggest that formate is the preferred electron carrier in syntrophic propionate-oxidizing cocultures of S. fumaroxidans and M. hungatei.     

Survival of Salmonella spp. in a simulated acid-phase anaerobic digester treating sewage sludge

The presence of pathogenic microorganisms in municipal waste sludge may create a serious outbreak of water borne diseases if the sludge is used for agricultural purpose. An attempt to decrease the number of pathogenic microorganisms, Salmonella spp. using a simulated acid-phase anaerobic digester was tested in a laboratory-scale batch experiment. Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of a two-phase anaerobic digestion process. A high concentration of organic acid at a pH value of 5.5-6.0 prevents a decrease in Salmonella spp. concentration. Almost complete destruction of Salmonella spp. is observed within two days if the pH value is maintained below 5.5.     

Survival of Salmonella spp. in a simulated acid-phase anaerobic digester treating sewage sludge

The presence of pathogenic microorganisms in municipal waste sludge may create a serious outbreak of water borne diseases if the sludge is used for agricultural purpose. An attempt to decrease the number of pathogenic microorganisms, Salmonella spp. using a simulated acid-phase anaerobic digester was tested in a laboratory-scale batch experiment. Reduction of Salmonella spp. was demonstrated in a mixture of sludge and organic acid, simulating an acid digester of a two-phase anaerobic digestion process. A high concentration of organic acid at a pH value of 5.5-6.0 prevents a decrease in Salmonella spp. concentration. Almost complete destruction of Salmonella spp. is observed within two days if the pH value is maintained below 5.5.     

Stabilometry as a diagnostic tool in clinical medicine.

Stabilomety, a method of measuring stability of stance or postural equilibrium in man, consists of transforming the mechanical oscillations of man''s "physiologic gravicentre" into electric signals, then amplifying, recording and analysing the signals. The frequency, duration and mean and maximum amplitudes of oscillations, and coefficients reflecting the influence of vision, differ in patients with various neurologic diseases and from values in healthy subjects. The method is highly sensitive and accurate, simple and rapid to use, lacks danger and discomfort and permits screening of a large number of people in a short time.     

Decreased activity of a propionate degrading community in a UASB reactor fed with synthetic medium without molybdenum,tungsten and selenium

The composition and dynamics of the propionate degrading community in a propionate-fed upflow anaerobic sludge bed (UASB) reactor with sludge originating from an alcohol distillery wastewater treating UASB reactor was studied. The rather stable propionate degrading microbial community comprised relatives of propionate degrading Syntrophobacter spp., the hydrogen and formate consuming Methanospirillum hungatei and the acetate consuming Methanosaeta concilii. The effect of the long-term absence of molybdenum, tungsten and selenium from the feed to the UASB reactor on microbial community dynamics and activity was examined. Measurements for metal concentrations of the sludge and specific methanogenic activity tests with supplied molybdenum, tungsten and selenium were found to be unsuitable to detect the potential limitation of the microbial activity of the UASB sludge by these trace metals. During a long-term absence of molybdenum, tungsten and selenium from the feed to the UASB reactor, the methanogenic activity decreased while relatives of Smithella propionica and Pelotomaculum spp. competed with Syntrophobacter spp. for propionate consumption.     

Effect of tungsten and molybdenum on growth of a syntrophic coculture of <Emphasis Type="Italic">Syntrophobacter fumaroxidans</Emphasis> and <Emphasis Type="Italic">Methanospirillum hungatei</Emphasis>

The effect of tungsten (W) and molybdenum (Mo) on the growth of Syntrophobacter fumaroxidans and Methanospirillum hungatei was studied in syntrophic cultures and the pure cultures of both the organisms. Cells that were grown syntropically were separated by Percoll density centrifugation. Measurement of hydrogenase and formate dehydrogenase levels in cell extracts of syntrophically grown cells correlated with the methane formation rates in the co-cultures. The effect of W and Mo on the activity of formate dehydrogenase was considerable in both the organisms, whereas hydrogenase activity remained relatively constant. Depletion of tungsten and/or molybdenum, however, did not affect the growth of the pure culture of S. fumaroxidans on propionate plus fumarate significantly, although the specific activities of hydrogenase and especially formate dehydrogenase were influenced by the absence of Mo and W. This indicates that the organism has a low W or Mo requirement under these conditions. Growth of M. hungatei on either formate or H₂/CO₂ required tungsten, and molybdenum could replace tungsten to some extent. Our results suggest a more prominent role for H₂ as electron carrier in the syntrophic conversion of propionate, when the essential trace metals W and Mo for the functioning of formate dehydrogenase are depleted.     

Raman spectroscopy as a potential tool for detection of Brucella spp. in milk

Detection of Brucella, causing brucellosis, is very challenging, since the applied techniques are mostly time-demanding and not standardized. While the common detection system relies on the cultivation of the bacteria, further classical typing up to the biotype level is mostly based on phenotypic or genotypic characteristics. The results of genotyping do not always fit the existing taxonomy, and misidentifications between genetically closely related genera cannot be avoided. This situation gets even worse, when detection from complex matrices, such as milk, is necessary. For these reasons, the availability of a method that allows early and reliable identification of possible Brucella isolates for both clinical and epidemiological reasons would be extremely useful. We evaluated micro-Raman spectroscopy in combination with chemometric analysis to identify Brucella from agar plates and directly from milk: prior to these studies, the samples were inactivated via formaldehyde treatment to ensure a higher working safety. The single-cell Raman spectra of different Brucella, Escherichia, Ochrobactrum, Pseudomonas, and Yersinia spp. were measured to create two independent databases for detection in media and milk. Identification accuracies of 92% for Brucella from medium and 94% for Brucella from milk were obtained while analyzing the single-cell Raman spectra via support vector machine. Even the identification of the other genera yielded sufficient results, with accuracies of >90%. In summary, micro-Raman spectroscopy is a promising alternative for detecting Brucella. The measurements we performed at the single-cell level thus allow fast identification within a few hours without a demanding process for sample preparation.     

Development of image analysis techniques as a tool to detect and quantify morphological changes in anaerobic sludge: I. Application to a granulation process

Image analysis techniques were developed and applied to quantify the process of anaerobic granulation in an expanded granular sludge blanket reactor (EGSB) fed with a synthetic substrate based on glucose [60-30% COD (chemical oxygen demand)] and volatile fatty acids (40-70% COD) over 376 days. In a first operation period that lasted 177 days, the aggregation of dispersed sludge was quantitatively monitored through the recognition and quantification of aggregates and filaments. A parameter defined as the ratio between the filaments' length and the aggregates projected area (LfA) has proven to be sensitive to detect changes in the aggregation status of the anaerobic sludge. The aggregation time-defined as the moment when a balance between filaments' length and aggregates' size was established-was recognized through the LfA. The percentage of projected area of aggregates within three size ranges (0.01-0.1 mm, 0.1-1 mm, and >1 mm, equivalent diameter) reflected the granular size spectrum during the aggregation process. When sudden increases on the upflow velocity and on the organic loading rate were applied to the previously formed granules, the developed image analysis techniques revealed to be good indicators of granular sludge stability, since they were sensitive to detected filaments release, fragmentation, and erosion that usually leads to washout. The specific methanogenic activities in the presence of acetate, propionate, butyrate, and H(2)/CO(2) increased along the operation, particularly relevant was the sudden increase in the specific hydrogenophilic activity, immediately after the moment recognized as aggregation time.     

Ultrasonography as a diagnostic tool for Neural Pain in Leprosy

Leprosy is still a prevalent disease in Brazil, representing 93% of all occurrences in the Americas. Leprosy neuropathy is one of the most worrying manifestations of the disease. Acute neuropathy usually occurs during reaction episodes and is called neuritis. Twenty-two leprosy patients were included in this study. These patients had neural pain associated with ulnar sensory neuropathy, with or without adjunct motor involvement. The neurological picture began within thirty days of the clinical evaluation. The patients underwent a nerve conduction study and the demyelinating findings confirmed the diagnosis of neuritis. Ultrasonographic study (US) of the ulnar nerve was performed in all patients by a radiologist who was blinded to the clinical or neurophysiological results. Morphological characteristics of the ulnar nerve were analyzed, such as echogenicity, fascicular pattern, transverse cross-sectional area (CSA), aspect of the epineurium, as well as their anatomical relationships. The volume of selected muscles referring to the ulnar nerve, as well as their echogenicity, was also examined. Based on this analysis, patients with increased ulnar nerve CSA associated with loss of fascicular pattern, epineurium hyperechogenicity and presence of power Doppler flow were classified as neuritis. Therefore, patients initially classified by the clinical-electrophysiological criteria were reclassified by the imaging criteria pre-established in this study as with and without neuritis. Loss of fascicular pattern and flow detection on power Doppler showed to be significant morphological features in the detection of neuritis. In 38.5% of patients without clinical or neurophysiological findings of neuritis, US identified power Doppler flow and loss of fascicular pattern. The US is a method of high resolution and portability, and its low cost means that it could be used as an auxiliary tool in the diagnosis of neuritis and its treatment, especially in basic health units.     

PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp.

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp. Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L. sativa and 13 accessions of the wild species L. serriola, L. saligna, L. virosa, L. perennis, and L. indica. The same genotypes were analyzed as in a previous study that had utilized RFLP markers. The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data. The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis. There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data. AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels.     

Genetic monitoring as a promising tool for conservation and management

In response to ever-increasing anthropogenic changes to natural ecosystems, regional, national and international organizations have established guidelines for monitoring biological diversity. Most monitoring programs, however, do not take full advantage of the potential afforded by molecular genetic markers, which can provide information relevant to both ecological and evolutionary time frames, while costing less and being more sensitive and reliable than traditional monitoring approaches. As several molecular and computational approaches are relatively new, many technical and theoretical issues remain to be resolved. Here, we illustrate how DNA and population genetic data can provide valuable information, often unattainable via other approaches, for monitoring species of management, conservation and ecological interest.     

A novel shuttle vector for Streptomyces spp. and Escherichia coli as a tool in site-directed mutagenesis.

This paper describes the construction and utilization of a novel shuttle vector for Streptomyces spp. and Escherichia coli as a useful vector in site-directed mutagenesis. The shuttle vector pIAFS20 (6.7 kb) has the following features: a replicon for Streptomyces spp., isolated from plasmid pIJ702; the thiostrepton-resistance gene as a selective marker in Streptomyces; the ColE1 origin, allowing replication in E. coli; and the ampicillin-resistance gene as a selective marker in E. coli. Vector pIAFS20 also contains the phage f1 intergenic region, which permits production of single-stranded DNA in E. coli after superinfection with helper phage M13K07. Moreover, the lac promoter is located in front of the multiple cloning sites cassette, allowing eventual expression of the cloned genes in E. coli. After mutagenesis and screening of the mutants in E. coli, the plasmids can be readily used to transform Streptomyces spp. As a demonstration, a 3.2-kb DNA fragment containing the gene encoding the xylanase A from Streptomyces lividans 1326 was inserted into pIAFS20, and the promoter region of this gene served as a target for site-directed mutagenesis. The two deletions reported here confirm the efficiency of this new vector as a tool in mutagenesis.     

Thermal-alkaline solubilization of waste activated sludge as a pre-treatment stage for anaerobic digestion

This work studied the hydrolysis kinetics and the solubilization of waste activated sludge under a medium range temperature (50-90 degrees C) and pH in the alkaline region (8-11), as a pretreatment stage for anaerobic digestion. The hydrolysis rate for the solubilization of volatile suspended solids (VSS) followed a first-order rate. A linear polynomial hydrolysis model was derived from the experimental results leading to a satisfactory correlation between the hydrolysis rate coefficient, pH, and temperature. At pH 11 and a temperature of 90 degrees C the concentration of the VSS was 6.82%, the VSS reduction reached 45% within ten hours and at the same time the soluble COD was 70.000 mg/l and the total efficiency for methane production 0.28 l of CH4 per g of VSS loading.     

Minimalist protein model as a diagnostic tool for misfolding and aggregation

We propose a realistic coarse-grained protein model and a technique to "anchor" the model to available experimental data. We apply this procedure to characterize the effect of multiple mutations on the folding mechanism of protein S6. We show that the mutation of a few "gatekeeper" residues triggers significant changes on the folding landscape of S6. These results suggest that gatekeeper residues control the flexibility of critical regions of S6, that in turn regulates the delicate balance between folding and aggregation. Although obtained with a minimalist protein model, these results are fully consistent with experimental evidence and offer a clue to understand the interplay between folding and aggregation in protein S6.     

Effect of propionate toxicity on methanogen-enriched sludge, Methanobrevibacter smithii, and Methanospirillum hungatii at different pH values.

The effect of propionate toxicity at different pH values (6.5, 7.0, and 8.0) on methanogen-enriched sludge. Methanobrevibacter smithii, and Methanospirillum hungatii was studied. Organisms were grown in Balch medium 3 in Hungate tubes, and toxicity was characterized by a decrease in production of methane and in bacterial numbers. Propionate inhibited bacterial growth and cumulative methane production at concentrations as low as 20 mM. In the absence of propionate, the methanogen-enriched sludge and M. smithii showed better cumulative methane production at pH 6.5 and 7.0 than at pH 8.0. However, in the presence of propionate, these organisms showed better cumulative methane production at pH 8.0. M. hungatii differed in its behavior; the best values of cumulative methane production for this organism occurred at pH 7.0. Bacterial numbers reflected the microbial response to the presence of propionate. The highest counts of methanogenic bacteria were observed at pH 6.5 and 8.0. The numbers of methanogens were affected by the presence of propionate even at concentrations as low as 20 or 30 mM; at propionate concentrations above 80 mM, the methanogen count was affected by at least 2 orders of magnitude. Upon comparison of the responses of the pure cultures and the methanogen-enriched sludge to increasing propionate concentrations, it was found that the sensitivity of the pure cultures was similar to that of the methanogens in the sludge.