Quantifying in situ growth rate of a filamentous bacterial species in activated sludge using rRNA:rDNA ratio.

If the in situ growth rate of filamentous bacteria in activated sludge can be quantified, researchers can more accurately assess the effect of operating conditions on the growth of filaments and improve the mathematical modeling of filamentous bulking. We developed a method to quantify the in situ specific growth rate of Sphaerotilus natans (a model filament) in activated sludge using the species-specific 16S rRNA:rDNA ratio. Primers targeting the 16S rRNA of S. natans were designed, and real-time PCR and RT-PCR were used to quantify DNA and RNA levels of S. natans, respectively. A positive linear relationship was found between the rRNA:rDNA ratio (from 440 to 4500) and the specific growth rate of S. natans (from 0.036 to 0.172 h-1) using chemostat experiments. The in situ growth rates of S. natans in activated sludge samples from three water reclamation facilities were quantified, illustrating how the approach can be applied in a complex environment such as activated sludge.

Investigation of iron-containing products from natural and laboratory cultivated Sphaerotilus-Leptothrix bacteria.

Bacterial biomass collected from sheath-forming bacteria of the genera Sphaerotilus and Leptothrix was collected from a high-mountain natural stream water source. The elemental constitution and oxide phases of the products after selective cultivation of the bacteria on two different elective media using neutron activation analysis (NAA), electron microscopy (SEM, TEM), and X-ray diffraction (XRD) were studied. A high enrichment level of iron was revealed by the NAA technique in cultivated isolates as compared to the reference sample from nature. Three types of iron oxide compounds were established after cultivation in Adler's medium: lepidocrocite (γ-FeOOH), magnetite (Fe3O4), and goethite (α-FeOOH). The cultivation in the Isolation medium yielded a single phase, that of goethite, excluding one sample with a distinguishable amount of lepidocrocite. XRD and EM investigations show that the biogenic oxides are nanosized. Our study exemplifies the possibilities of the biotechnology approach for obtaining, under artificial conditions, large quantities of iron-containing by-products that could be of further used in appropriate nano- and biotechnologies.

Moderate oxygen depletion as a factor favouring the filamentous growth of Sphaerotilus natans.

Sphaerotilus natans is a neutrophilic iron-related sheath-forming filamentous microorganism that presents dual morphotype: single cells and ensheathed cells forming filaments. As S. natans has been proposed as a sorbent for inorganic pollutants and it is occasionally involved in bulking episodes, elucidating factors affecting its filamentous growth is of crucial interest. The purpose of this work was to evaluate the effect of dissolved oxygen (DO) as a factor affecting S. natans filamentation from single cells. A method to quantify S. natans in its filamentous and single-cell morphotypes, based on a differential filtration procedure coupled with quantitative real-time PCR, was developed here. Scanning Electron Microscopy was used to validate the filtration step. Under actively aerated conditions (DO maintained at 7.6 ± 0.1 mg l(-1)), S. natans grew mainly as single cells throughout the experiment, while a depletion in DO concentration (to ~3 mg l(-1)) induced its filamentous growth. Indeed, when oxygen was reduced the proportion of single cells diminished from 83.3 ± 5.9 to 14.3 ± 3.4% while the filaments increased from 16.7 ± 5.9 to 85.7 ± 3.4%. Our results suggest that oxygen plays a key role in S. natans filamentation and contribute to better understanding of the filamentous proliferation of this bacterium. In addition, the proposed method will be helpful to evaluate other factors favouring filamentous growth.

Filamentous bacteria existence in aerobic granular reactors.

Filamentous bacteria are associated to biomass settling problems in wastewater treatment plants. In systems based on aerobic granular biomass they have been proposed to contribute to the initial biomass aggregation process. However, their development on mature aerobic granular systems has not been sufficiently studied. In the present research work, filamentous bacteria were studied for the first time after long-term operation (up to 300 days) of aerobic granular systems. Chloroflexi and Sphaerotilus natans have been observed in a reactor fed with synthetic wastewater. These filamentous bacteria could only come from the inoculated sludge. Thiothrix and Chloroflexi bacteria were observed in aerobic granular biomass treating wastewater from a fish canning industry. Meganema perideroedes was detected in a reactor treating wastewater from a plant processing marine products. As a conclusion, the source of filamentous bacteria in these mature aerobic granular systems fed with industrial effluents was the incoming wastewater.

[Mechanism of malate dehydrogenase isoform formation in Sphaerotilus natans D-507 under different cultivation conditions].

Electrophoretically homogenous preparations of malate dehydrogenase (MDH) isoforms of the bacteria Sphaerotilus natans D-507 with specific activity 7.46 U/mg and 5.74 U/mg with respect to protein concentration have been obtained. The dimeric isoform of the enzyme was shown to function under organotrophic growth conditions, whereas the tetrameric isoform was induced under mixotrophic cultivation conditions. PCR-analysis revealed a single gene encoding the malate dehydrogenase molecule. The topography of the MDH isoform surface was studied by atomic-force microscopy, and a 3D-structure of the enzyme was obtained. Spectraphotometric analysis data allowed us to suggest that stabilization of the tetrameric form of MDH is due to additional bounds implicated in the quaternary structure formation.

[Structural-functional transformation of the malate dehydrogenase system of the bacterium Sphaerotilus sp. strain D-507 depending on nutritional mode].

High-purity preparations of malate dehydrogenase (EC 1.1.1.37) were obtained by multistage purification from the bacterium Sphaerotilus sp. strain D-507 growing under different conditions. Under organotrophic conditions, the enzyme was dimeric; under mixotrophic conditions, dimeric and trimeric. On the basis of studied properties of the enzyme preparations, data on the activity of enzymes of the glyoxylate and tricarboxylic-acid cycles, and analysis of published data, it can be concluded that malate dehydrogenase isoforms are implicated in the adaptive response of bacteria to changing culturing conditions.

About the performance of Sphaerotilus natans to reduce hexavalent chromium in batch and continuous reactors.

The hexavalent chromium biological reduction constitutes a safe and economical detoxification procedure of wastewaters containing Cr(VI). However, little research has been done to evaluate Cr(VI) tolerance and reduction capacity of microbial cultures under different growth conditions. The aims of this work were (a) to evaluate the capacity of Sphaerotilus natans to reduce Cr(VI) to Cr(III) in a continuous system limited in carbon and energy source or in nitrogen source, (b) to evaluate the toxic effect of Cr(VI) on this microorganism, (c) to carry out a complete analysis of Cr(VI) reduction by S. natans not only in continuous regime but also in batch system, and (d) to model the obtained results mathematically. S. natans exhibited great resistance to Cr(VI) (19-78 mg l(-1)) and optimal growth in continuous and batch systems using a mineral medium supplemented only with citric acid as organic substrate. In carbon- and energy-limited continuous systems, a maximum percentual decrease in Cr(VI) by 13% was reached for low influent Cr(VI) concentration (4.3-5.32 mg Cr(VI)l(-1)); the efficiency of the process did not notoriously increase as the length of cellular residence time was increased from 4.16 to 50h. A nitrogen-limited continuous operation with a cellular residence time of 28.5h resulted in a Cr(VI) decrease of approximately 26-32%. In batch system, a mathematical model allowed to predict the Cr(VI) concentration as a function of time and the ratio between the initial Cr(VI) concentration and that of the biomass. High concentrations of initial Cr(VI) and biomass produced the highest performance of the process of Cr(VI) reduction reached in batch system, aspects which should be considered in detoxification strategies of wastewaters.

Relationship of species-specific filament levels to filamentous bulking in activated sludge.

To examine the relationship between activated-sludge bulking and levels of specific filamentous bacteria, we developed a statistics-based quantification method for estimating the biomass levels of specific filaments using 16S rRNA-targeted fluorescent in situ hybridization (FISH) probes. The results of quantitative FISH for the filament Sphaerotilus natans were similar to the results of quantitative membrane hybridization in a sample from a full-scale wastewater treatment plant. Laboratory-scale reactors were operated under different flow conditions to develop bulking and nonbulking sludge and were bioaugmented with S. natans cells to stimulate bulking. Instead of S. natans, the filament Eikelboom type 1851 became dominant in the reactors. Levels of type 1851 filaments extending out of the flocs correlated strongly with the sludge volume index, and extended filament lengths of approximately 6 x 10(8) micro m ml(-1) resulted in bulking in laboratory-scale and full-scale activated-sludge samples. Quantitative FISH showed that high levels of filaments occurred inside the flocs in nonbulking sludge, supporting the "substrate diffusion limitation" hypothesis for bulking. The approach will allow the monitoring of incremental improvements in bulking control methods and the delineation of the operational conditions that lead to bulking due to specific filaments.