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 from activated sludge: growth on Tween 80 as carbon and energy source

Microthrix parvicella, cultivated in a medium with Tween 80 and Casamino acids, utilized only the oleic acid moiety of Tween 80 as carbon and energy source. The cell yield from Tween 80 was about 0.32 g dry weight of cells per g of Tween 80 consumed. As only the oleic acid moiety of Tween 80 was utilized, the cell yield from oleic acid was 1.3 g dry weight of cells per g oleic acid consumed. The amount of carbon produced as CO2 was less than 30% of the oleic acid-carbon and this low value was in agreement with the high cell yield. In batch culture M. parvicella stored large amounts of lipid material during the early growth phase. The fatty acids of the lipid globules were similar to the fatty acids supplied as carbon source. The percentage composition of the biomass changed to give C/N percentage ratios of about 15 during the early growth phase due to the high concentration of internal lipids and the low concentration of protein. The growth rate in batch culture was about 0.016 h-1 but was affected by the concentration of Casamino acids in the medium.     

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.     

"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".     

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 .     

Quantification of Microthrix parvicella in activated sludge bacterial communities by real-time PCR

AIMS: This study was to develop a simple and reliable method for quantifying Microthrix parvicella 16S rRNA gene copies and its application to activated sludge samples collected from wastewater treatment plants (WWTP) with and without foaming problems. METHODS AND RESULTS: The relative frequency of M. parvicella was determined by combining real-time PCR assays for quantification of total bacterial 16S rRNA gene copies and M. parvicella 16S rRNA gene copies. The developed method was applied to analyse 32 activated sludge samples obtained from German WWTP. The level of M. parvicella 16S rRNA gene copies in the 18 nonfoaming samples was below 3% of the total number of 16S rRNA gene copies and in the range of 0-18% for the 14 foaming samples. CONCLUSIONS: The described method allows reliable monitoring of the amount of M. parvicella in activated sludge samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The described method may become an important component of a warning system for forthcoming bulking and foaming episodes.     

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.     

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.     

Studies on the cultivation of mammalian cell lines in a serum-free,chemically defined medium

Summary An improved chemically defined, serum-free medium for the cultivation of a variety of continuous cell lines has been developed. Eight lines of human origin, three lines of nonhuman primate origin, and five lines of rodent origin have been cultured serially for as long as a year in the medium. Growth rates of several serial lines resulted in as much as 20- to 30-fold increases per week. Hormones such as insulin, cortisol, and thyroxine significantly improved growth of cultures in the defined medium. Vitamin B₁₂ and biotin were required for growth. Lipids such as oleic acid, lecithin, and cholesterol also promoted growth of several cell lines. Virtually all continuous cell lines tested grew well upon initial transfer into the serum-free defined medium. Most cell lines could be serially subcultured rapidly with little evidence that selection of rare cell types was necessary for growth in the defined medium. However, a few cell lines such as the BHK-21 (hamster) cell and the AKR (mouse embryo) cell required prolonged periods (4 to 8 weeks) of culturing before rapid growth occurred. Primary cell cultures and other diploid cells such as human fibroblast (strain WI-38) could not be subcultured successfully in the present medium.     

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 ).     

An aerobic formate-utilizing bacterium,Cupriavidus sp., isolated from activated sludge of wastewater treatment

An aerobic formate-assimilating bacterium, denoted as strain FAB, was newly isolated from activated sludge of wastewater treatment. Phylogenetic analysis based on 16S rDNA sequence assigned the isolate to the genus Cupriavidus. Scanning electron micrography revealed that this bacterium has a coccal morphology, and from some physiological assays, the bacterium was characterized to be Gram-negative, nitrate-reduction-positive and catalase-positive. In addition to formate, strain FAB was able to utilize fructose, acetate or pyruvate as a preferred carbon source. Compared with a close relative, Cupriavidus necator, our isolate exhibited a greater growth rate on formate under an aerobic condition.     

Degradation of 17beta-estradiol by a gram-negative bacterium isolated from activated sludge in a sewage treatment plant in Tokyo, Japan

A 17beta-estradiol (E2)-degrading bacterium was isolated from activated sludge in a sewage treatment plant in Tokyo, Japan. The isolate was suggested to be a new Novosphingobium species. Gas chromatography-mass spectrometry and (1)H nuclear magnetic resonance analyses of the metabolites of E2 degradation suggested that no toxic products accumulated in the culture medium.     

Embryonic stem cell development in a chemically defined medium

Vertebrate germ layer development is an intricately interwoven process with the organism operating as an integrated whole. To examine these processes we have used embryonic stem (ES) cell in vitro differentiation in a serum-free, chemically defined medium (CDM). In CDM, ES cells differentiate as embryoid bodies to neuroectoderm with upregulation of pax-6, without commensurate expression of Brachyury. In the presence of Activin A, pax-6 and Brachyury mRNAs are readily detectable, suggestive of both neuroectoderm and mesoderm formation, while in the presence of BMP-4 a process resembling primitive streak formation at the molecular level occurs. Neuroectoderm development in CDM alone is consistent with the view that this process can occur by default, as reported in Xenopus, due to the absence or sequestration of mesoderm-inducing factors. Additionally, these data show that BMP-4 alone is capable of instigating a process resembling primitive streak formation in ES cells and possibly in vivo.