Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Microthrix parvicella, a filamentous bacterium isolated from activated sludge: cultivation in a chemically defined medium.

A large number of media have been tested for cultivating Microthris parvicella, a filamentous microorganism often present in the activated sludge of oxidation ditches. The bacterium was found to utilize oleic acid (preferably as Tween 80) as the sole source of carbon and energy. Sulfur is required in the reduced form. The tested media vary from a complex to a chemically defined medium. Growth yields of 1.3 to 1.5 g/liter were obtained on media containing Tween 80 (4 g/liter), reduced nitrogen and reduced sulfur compounds, calcium and magnesium salts, phosphate buffer, trace elements, thiamin, and cyanocobalamin. The optimum temperature for growing the organism is approximately 25 degrees C, and the pH of the nutrient medium should be above 7.     

Ultrastructure of Microthrix parvicella from activated sludge

H.M. STRATTON, R. WEBB, E.M. SEVIOUR, L.L. BLACKALL AND R.J. SEVIOUR. 1996. Filaments of Microthrix parvicella grow very poorly in pure culture and have a characteristic uneven appearance containing large numbers of distinctive spherical swollen cells. This feature was only rarely seen with filaments of this organism in activated sludge biomass samples. Ultrastructurally, these spherical swollen cells do not appear to be bacterial endospores or cysts, but do show features that are consistent with them being some form of resting structure. Their production could be a response of M. parvicella to environmental stress, an explanation proposed because of their relative predominance in pure cultures of M. parvicella compared to their infrequent occurrence in filaments of this bacterium observed in activated sludge biomass.     

Control of Microthrix parvicella growth in activated sludge

Abstract Effects of aerobic and anaerobic conditions on the growth of Microthrix parvicella in the activated sludge were studied to prevent bulking caused by this filamentous bacterium. The study was conducted on a pilot plant with selector and the data were compared with those observed in a full scale plant subjected to severe bulking due to a massive growth of M. parvicella . Both plants were fed with the same settled waste water. A substantial suppression of the growth of M. parvicella was observed in only the experiments where returned activated sludge was mixed with waste water under aerobic conditions. Both the number of filaments and the sludge volume index (SVI) were lower in the pilot plant than in the full scale plant. Under anerobic conditions, the selector was not able to improve the settleability and avoid the growth of M. parvicella .     

"Microthrix parvicella", a filamentous bacterium causing bulking and foaming in activated sludge systems: a review of current knowledge

This review summarizes the microbiology and physiology of "Microthrix parvicella" and the methods of its growth control in activated sludge wastewater treatment plants. This filamentous bacterium is of high interest because of its worldwide involvement in severe bulking and foaming at wastewater treatment plants. We present a critical analysis of physiological and kinetic data on "M. parvicella" and discuss its growth and storage abilities in various environments with the aim of understanding the strategies of this organism to successfully compete with other bacteria in activated sludge. Additionally, this review elaborates on research needs for defining reliable control strategies of bulking and foaming based on key features of "M. parvicella".     

A hypothesis on Microthrix parvicella proliferation in biological nutrient removal activated sludge systems with selector tanks

The objective of this study was to evaluate the ability of Microthrix parvicella for long-chain fatty acids uptake under anaerobic, anoxic, and aerobic conditions as well as its ability to utilize volatile fatty acids and long-chain fatty acids under anoxic and aerobic conditions. According to the results, a hypothesis on the competition between floc-forming microorganisms and M.?parvicella for long-chain fatty acids uptake under aerobic, anoxic, and anaerobic conditions was formulated. According to this hypothesis, M.?parvicella exhibits similar long-chain fatty acids uptake capacity with floc-forming microorganisms even at relatively high floc loading values that are very often imposed at selector tanks. Following this hypothesis, the failure of selector tanks to provide for an effective M.?parvicella control is evidenced. Based on the experimental results, the ability of M.?parvicella to utilize long-chain fatty acids with rates comparable to those of floc formers, even in anoxic conditions, in conjunction with its lower acetate utilization rates, provides a good explanation regarding its preference to slowly biodegradable organic carbon compounds.     

Multiple approaches to assess filamentous bacterial growth in activated sludge under different carbon source conditions

Aims:  To compare molecular and microscopic approaches in determining which filamentous bacteria grow in activated sludge reactors when different carbon sources and different activated sludge mixed liquor inocula are used.
Methods and Results:  Microscopic and molecular (Denaturing Gradient Gel Electrophoresis and Fluorescent In Situ Hybridization) techniques were used to determine which filamentous bacteria became dominant in lab scale reactors treating wastewater composed of different carbon sources. Molecular analysis indicated the presence of Sphaerotilus natans and Thiothrix -related organisms . Microscopy indicated the presence of Nostocoida limicola in some reactors. Sludge volume index increased as filament abundance increased. The detection level of DGGE analysis increased when the abundance levels of the filaments were high.
Conclusions:  Simultaneous application of traditional and molecular methods was effective, and highlighted the advantages and limitations of each method. Readily biodegradable substances favoured the growth of specific filaments in a mixed liquor environment. The origin of inoculum influenced which specific filamentous bacteria grew.
Significance and Impact of the Study:  The study shows the potential problems when using particular techniques, and highlights the need for multiple approaches when studying filaments. The study also provides more information on which filaments will grow under different carbon source conditions for a given inoculum.     

Characterization of a new gram-negative filamentous bacterium isolated from bulking sludge

Summary A new gram-negative filamentous and sheath-forming bacterium with hardly visible rod-shaped single cells was isolated from bulking sludge. Three strains of this type grow aerobically, produce carotenoid pigments especially, on rich media and exhibit gliding motility on solid surfaces. Even numbered straight-chain acid were predominating in total fatty acid patterns, whereas a branched-chain odd numbered fatty acids, although occurring in lower amounts, is discussed as an appropriate chemotaxonomical marker for this bacterium. As respiratory quinones only menaquinones could be detected. The whole cell proteins of the isolates showed identical electropherograms on polyacrylamide slabs. The G+C-content was found to be 59 mol%. Morphological, physiological, and chemotaxonomical features imply that the strains belong to a new genus of the gliding bacteria. Taxonomically they have to be placed near Herpetosiphon and Chloroflexus These results could be confirmed by the sequence analysis of the 16 S r-RNA performed by Ludwig and Stackebrandt (1984, personal communication).     

Organoarsenical tolerance in Sphingobacterium wenxiniae,a bacterium isolated from activated sludge

Organoarsenicals enter the environment from biogenic and anthropogenic sources. Trivalent inorganic arsenite (As(III)) is microbially methylated to more toxic methylarsenite (MAs(III)) and dimethylarsenite (DMAs(III)) that oxidize in air to MAs(V) and DMAs(V). Sources include the herbicide monosodium methylarsenate (MSMA or MAs(V)), which is microbially reduced to MAs(III), and the aromatic arsenical roxarsone (3-nitro-4-hydroxybenzenearsonic acid or Rox), an antimicrobial growth promoter for poultry and swine. Here we show that Sphingobacterium wenxiniae LQY-18^T, isolated from activated sludge, is resistant to trivalent MAs(III) and Rox(III). Sphingobacterium wenxiniae detoxifies MAs(III) and Rox(III) by oxidation to MAs(V) and Rox(V). Sphingobacterium wenxiniae has a novel chromosomal gene, termed arsU1. Expressed in Escherichia coli arsU1 confers resistance to MAs(III) and Rox(III) but not As(III) or pentavalent organoarsenicals. Purified ArsU1 catalyses oxidation of trivalent methylarsenite and roxarsone. ArsU1 has six conserved cysteine residues. The DNA sequence for the three C-terminal cysteines was deleted, and the other three were mutated to serines. Only C45S and C122S lost activity, suggesting that Cys45 and Cys122 play a role in ArsU1 function. ArsU1 requires neither FMN nor FAD for activity. These results demonstrate that ArsU1 is a novel MAs(III) oxidase that contributes to S. wenxiniae tolerance to organoarsenicals.     

Isolation and characterization of a phenol-degrading bacterium from an industrial activated sludge

This paper reports the successful isolation and characterization of a new phenol-degrading bacterium, strain EDP3, from activated sludge. Strain EDP3 is a nonmotile, strictly aerobic, Gram-negative, and short-rod or coccobacillary bacterium, which occurs singly, in pairs, or in clusters. 16S rRNA gene sequence analysis revealed that strain EDP3 belonged to the gamma group of Proteobacteria, with a 97.0% identity to 16S rRNA gene sequences of Acinetobacter calcoaceticus. Strain EDP3 could aerobically grow on a number of aromatic compounds, such as phenol, sodium benzoate, p-hydroxybenzoate, phenylacetate, benzene, ethylbenzene, benzylalcohol, and so on. In particular, it could mineralize up to 1,000 mg l⁻¹ phenol at room temperature (25°C). The growth kinetics of strain EDP3 on phenol as a sole carbon and energy source at 25°C can be described using the Haldane equation. It has a maximal specific growth rate (μ_max) of 0.28 h⁻¹, a half-saturation constant (K _S) of 1,167.1 mg l⁻¹, and a substrate inhibition constant (K _i) of 58.5 mg l⁻¹. Values of yield coefficient (Y _X/S) are between 0.4 and 0.6 mg dry cell (mg phenol)⁻¹. Strain EDP3 has high tolerance to the toxicity of phenol (up to 1,000 mg l⁻¹). It therefore could be an excellent candidate for the biotreatment of high-strength phenol-containing industrial wastewaters and for the in situ bioremediation of phenol-contaminated soils.     

Microbial growth on dichlorobiphenyls chlorinated on both rings as a sole carbon and energy source

We have isolated bacterial strains capable of aerobic growth on ortho-substituted dichlorobiphenyls as sole carbon and energy sources. During growth on 2,2'-dichlorobiphenyl and 2,4'-dichlorobiphenyl strain SK-4 produced stoichiometric amounts of 2-chlorobenzoate and 4-chlorobenzoate, respectively. Chlorobenzoates were not produced when strain SK-3 was grown on 2,4'-dichlorobiphenyl.     

Hydrogen peroxide as a supplemental oxygen source for activated sludge: Microbiological investigations

Summary Parallel bench-scale activated sludge systems were operated using air or hydrogen peroxide as oxygen source. The use of H₂O₂ resulted in a temporary decrease of COD reduction, an increase of the catalase activity of the activated sludge, a depression of the nitrification, and a marked decrease of some filamentous organisms. Enumeration of some microbiologic groups indicated that the counts of enterobacteria, coliforms, staphylococci, and streptococci were lower in the H₂O₂ unit than in the parallel air unit. Also the use of H₂O₂ did not induce the selection of bacterial species that are more resistant to H₂O₂. The increase in catalase activity after H₂O₂ addition might be the result of a stimulation of catalase synthesis in catalase positive microorganisms.List of Abbreviation COD chemical oxygen demand, mg O₂/1 - COD_eff chemical oxygen demand of the effluent, mg O₂/1 - DO dissolved oxygen, mg O₂/1 - MLSS mixed liquor suspended solids, g dry weight/1 - SVI sludge volume index, ml settled sludge per liter/MLSS (ml/g) - F:M sludge loading factor or the Food to Microorganisms ratio, g COD/g MLSS.day     

Screening and characterization of an aerobic nitrifying-denitrifying bacterium from activated sludge

Taking advantage of the good biocompatibility and high efficiency of nitrogen removal with microbes, nitrifying and denitrifying bacteria, are becoming increasingly more widely used for wastewater treatment and prevention of eutrophication. In this research, an aerobic nitrifying-denitrifying bacterium was successfully screened from activated sludge and identified as Pseudomonas sp. (CCTCC No M2010209) by the 16S rDNA sequence. The activity verification confirmed its nitrifying-denitrifying capability of removing ammonium, nitrate and nitrite nitrogen. The types of carbon sources and carbon-nitrogen ratio greatly influenced the removal efficiency of NH₄ ⁺-N and NO₃ ⁻-N. When the initial concentrations of NH₄ ⁺-N and NO₃ ⁻-N in synthetic wastewater were less than 70 and 50 mg/L, the nitrogen removal rates reached 94 and 90% in 9 h, respectively. Preliminary comparisons of nitrogen removal capacity between this isolate and other commercial preparations in the treatment of synthetic wastewater revealed its promising potential to be used in the actual wastewater treatment.     

Comamonas badia sp. nov., a floc-forming bacterium isolated from activated sludge

Strain IAM 14839, isolated from activated sludge in Japan, forms a visible floc and grows in the flocculated state. This bacterium is Gram-negative, rod-shaped, strictly aerobic and highly motile with a single polar flagellum. Both oxidase and catalase activities are positive. No growth was observed on sugars. The strain can grow at 20 degrees C, but does not grow at 37 degrees C. The G+C content of DNA is 66.3 mol% and Q-8 is the major quinone. The major cellular fatty acids are 16:1omega7c, 16:0, 18:1omega7c, 2OH 16:0, 3OH 10:0. The 16S rDNA sequence analysis indicated that the bacterium clustered within the genus Comamonas. On the basis of the phylogenetic analysis and phenotypic properties, it is proposed that the strain IAM 14839T be classified in a novel species of the genus Comamonas, Comamonas badia sp. nov. The type strain is IAM 14839T (=KCTC 12244T ).     

Selective enrichment and molecular characterization of a previously uncultured Nitrospira-like bacterium from activated sludge

Previously uncultured nitrite-oxidizing bacteria affiliated to the genus Nitrospira have for the first time been successfully enriched from activated sludge from a municipal wastewater treatment plant. During the enrichment procedure, the abundance of the Nitrospira-like bacteria increased to approximately 86% of the total bacterial population. This high degree of purification was achieved by a novel enrichment protocol, which exploits physiological features of Nitrospira-like bacteria and includes the selective repression of coexisting Nitrobacter cells and heterotrophic contaminants by application of ampicillin in a final concentration of 50 microg ml(-1). The enrichment process was monitored by electron microscopy, fluorescence in situ hybridization (FISH) with rRNA-targeted probes and fatty acid profiling. Phylogenetic analysis of 16S rRNA gene sequences revealed that the enriched bacteria represent a novel Nitrospira species closely related to uncultured Nitrospira-like bacteria previously found in wastewater treatment plants and nitrifying bioreactors. The enriched strain is provisionally classified as 'Candidatus Nitrospira defluvii'.     

Growth of an aerobic bacterium with trichloroacetic acid as the sole source of energy and carbon

The aerobic microbial decomposition of trichloroacetic acid (TCA) was studied. A TCA-decomposing culture was enriched in continuous-flow and batch experiments on a medium containing TCA as the only organic component. Pure cultures of TCA degraders were isolated from the enrichment on TCA agar plates. Characterization of several isolates proved them all to be representatives of the same bacterium, a Gram-negative, catalase-positive and cytochrome C-oxidase-positive, non-motile, somewhat irregular rod. The bacterium could not be identified on the basis of its carbon-source-utilization pattern, but a partial sequencing of the 16S rDNA gene showed the isolate to belong to the gamma sub-group of Proteobacteria, and to be phylogenetically close to Acinetobacter calcoaceticus. The isolated bacterium grew exponentially with TCA as the sole source of energy and carbon. The maximum growth rate (µ_max) and the growth yield on TCA (Y _X/S ) were determined to be 0.027 h^–1 and 0.027 g biomass/g TCA, respectively. The bacterium was not able to grow on mono- or dichloroacetic acid, but it could grow on acetate.