Investigations on theSphaerotilus-Leptothrix group

Thirty-fourSphaerotilus andLeptothrix strains were isolated from sewage, activated sludge and iron-containing ditch- and well-water, and their morphological and physiological characters studied. The organisms were grown under different conditions, e.g. on peptoneglucose agar and yeast-extract-manganous-carbonate agar, and in running ditch-water containing ferrous iron. Growth of these bacteria in synthetic media, with glucose as carbon source and aspartic and glutamic acids or inorganic nitrogen compounds as nitrogen source, required added vitamin B₁₂ unless nitrogen was supplied as hydrolyzed casein or as a mixture ofl-amino acids. Methionine was found to be responsible for this replacement of vitamin B₁₂.Five different types of sheath-forming bacteria were distinguished in the present study. Type I is the typical sewage organismSphaerotilus natans. It has large cells, grows well with relatively high concentrations of organic substrates, but cannot oxidize manganous compounds. In running ditch-water containing ferrous iron, ferric hydroxide may be deposited in and on its sheaths. AlthoughS. natans under such conditions may resemble the iron bacteriumLeptothrix ochracea, it has relatively long sheaths, partly filled with cells in contrast with the short and mostly empty sheaths of the latter.S. natans could be readily reisolated from its iron-bacterium cultures but very seldom from crude cultures ofL. ochracea; thus the two organisms are clearly different. Types II and III have relatively large cells, respond poorly to organic nutrients, but are able to oxidize manganous compounds. Type II forms fungus-like flocks in liquid media and resembles microscopicallyL. lopholea, with which it may be identical. Type III resemblesL. ochracea more closely than does any other type, but is probably not identical with it; the nameL. pseudo-ochracea sp.n. is proposed for this type. Type IV is intermediate between types I and V. In media with relatively high concentrations of organic nutrients it behaves like a sewage organism, but in poor media containing ferrous and manganous compounds, it behaves like an iron bacterium, depositing large amounts of ferric and manganic oxides in and on its sheaths; for this type the nameL. cholodnii sp.n. is proposed. Type V has small cells, grows poorly in all media tested, but actively oxidizes manganous compounds; the nameLeptothrix discophora is reserved for this type.The globular inclusions in the cells ofS. natans and other members of theSphaerotilus-Leptothrix group consist of poly--hydroxybutyrate.     

A Comparison of the Rates of Fe(III) Reduction in Synthetic and Bacteriogenic Iron Oxides by Shewanella putrefaciens CN32

The susceptibility of various bacteriogenic iron oxides (BIOS) to bacterial Fe(III) reduction was examined. Reduction resulted in complete dissolution of the iron mineral from the surfaces of the Fe-oxidizing consortium. Reduction rates were compared to that of synthetic ferrihydrite (HFO). The reduction rate of HFO (0.162 day^{? 1}) was significantly lower than that of Äspö (Gallionella dominated) BIOS (0.269 day^{? 1}). Two Canadian (Leptothrix dominated) BIOS samples showed statistically equivalent rates of reduction (0.541 day^?1 and 0.467 day^{? 1}), which were higher than both Äspö BIOS and HFO. BIOS produced by different iron-oxidizing genera have different susceptibilities to microbial reduction.     

Cytochrome oxidase activity associated with the inside of the cytoplasmic membrane in swarm cells and sheath-forming cells of Sphaerotilus natans: an ultrastructural study using 3,3′-diaminobenzidine (DAB)

Cytochrome oxidase activity has been demonstrated by electronmicroscopical cytochemical methods in swarm cells as well as in the sheathforming cells of Sphaerotilus natans. This enzyme is associated with the inside of the cytoplasmic membrane. The conditions for demonstrating this cytoplasmic membrane-associated cytochrome oxidase differ from those enabling the demonstration of the cytochrome oxidase in the polar organelle of this bacterial species. The location of this enzyme also contrasted to that generally known from other bacteria and from mitochondria.     

Growth of Sessile Sphaerotilus natans in a Tubular Recycle System

The growth of sessile Sphaerotilus natans was measured in a continuous-flow recycle system. Four methods were used to confirm that the growth of the biofilm was a linear function that increased with time. Of the methods used, one monitored in situ biofouling (increased fluid frictional resistance), two indicated biomass (ATP and DNA per square centimeter), and one allowed the direct observation of replaceable test surfaces by scanning electron microscopy. The filamentous growth of S. natans caused an increase in the fluid frictional resistance. The increase in biofouling was directly proportional to the increase in biomass.     

Preliminary characterization and biological reduction of putative biogenic iron oxides (BIOS) from the Tonga-Kermadec Arc, southwest Pacific Ocean

Sediment samples were obtained from areas of diffuse hydrothermal venting along the seabed in the Tonga sector of the Tonga‐Kermadec Arc, southwest Pacific Ocean. Sediments from Volcano 1 and Volcano 19 were analyzed by X‐ray diffraction (XRD) and found to be composed primarily of the iron oxyhydroxide mineral, two‐line ferrihydrite. XRD also suggested the possible presence of minor amounts of more ordered iron (hydr)oxides (including six‐line ferrihydrite, goethite/lepidocrocite and magnetite) in the biogenic iron oxides (BIOS) from Volcano 1; however, Mössbauer spectroscopy failed to detect any mineral phases more crystalline than two‐line ferrihydrite. The minerals were precipitated on the surfaces of abundant filamentous microbial structures. Morphologically, some of these structures were similar in appearance to the known iron‐oxidizing genus Mariprofundus spp., suggesting that the sediments are composed of biogenic iron oxides. At Volcano 19, an areally extensive, active vent field, the microbial cells appeared to be responsible for the formation of cohesive chimney‐like structures of iron oxyhydroxide, 2–3 m in height, whereas at Volcano 1, an older vent field, no chimney‐like structures were apparent. Iron reduction of the sediment material (i.e. BIOS) by Shewanella putrefaciens CN32 was measured, in vitro, as the ratio of [total Fe(II)]:[total Fe]. From this parameter, reduction rates were calculated for Volcano 1 BIOS (0.0521 day^?1), Volcano 19 BIOS (0.0473 day^?1), and hydrous ferric oxide, a synthetic two‐line ferrihydrite (0.0224 day^?1). Sediments from both BIOS sites were more easily reduced than synthetic ferrihydrite, which suggests that the decrease in effective surface area of the minerals within the sediments (due to the presence of the organic component) does not inhibit subsequent microbial reduction. These results indicate that natural, marine BIOS are easily reduced in the presence of dissimilatory iron‐reducing bacteria, and that the use of common synthetic iron minerals to model their reduction may lead to a significant underestimation of their biological reactivity.     

ATPase and cytochrome oxidase activities at the polar organelle in swarm cells of Sphaerotilus natans: an ultrastructural study

The polar organelle of bacteria presumably is part of the flagellar apparatus. In order to characterize this structure, cytochemical studies on Sphaerotilus natans have been performed.Marked ATPase activity is associated with the inner boundary layer and central layer of this organelle. The spaces between the boundary layers and the central layer of the polar organelle which are traversed by fine fibrilles are positive for reactions with diaminobenzidine. This indicates cytochrome oxidase activity. S. natans possesses a ribbon-like, helically shaped polar organelle which is divided concomitantly with cell fission, possibly explaining inheritance of this structure and of the flagellar apparatus.Dedicated to Prof. G. Drews on occasion of his 60th birthday     

Effect of different pH,temperatures and Fe2+ concentration on Sphaerotilus natans growth and clarity performance index

Summary An investigation was made of the effect of various environmental conditions of Sphaerotilus natans, an organism which causes sludge bulking, during the application of Fe²⁺ as inhibitor for bacterial growth. The ratio between non-settleable bacterial aggregates (turbidity) and bacterial plate count was regarded as representing the Clarity Performance Index (CPI).The results showed that ideal clarity was obtained at pH 6.5 and 30°C when 15 mg 1^-1 of Fe²⁺ applied. Under conditions, the percentage of inhibition of S. natans, the highest percentage of Fe²⁺ removal, and the CPI, compared to a control, were 87.50%, 29.58 and 0.962 respectively.     

Characterization of biogenic iron oxides collected by the newly designed liquid culture method using diffusion chambers

We designed a new culture method for neutrophilic iron‐oxidizing bacteria using liquid medium (i) to study the formation and mineralogical characteristics of biogenic iron oxides (BIOS) and (ii) to apply BIOS to various scientific and engineering applications. An iron‐oxidizing bacterium, Mariprofundus ferrooxydans PV‐1^T (ATCC, BAA–1020), was cultured using a set of diffusion chambers to prepare a broad anoxic–oxic interface, upon which BIOS formation is typically observed in natural environments. Iron oxide precipitates were generated in parallel with bacterial growth. A scanning electron microscopy analysis indicated that the morphological features of the iron oxide precipitates in the medium (in vitro BIOS) were similar to those of BIOS collected from natural deep‐sea hydrothermal environments in the Northwest Eifuku Seamount field in the northern Mariana Arc (in situ BIOS). Further chemical speciation of both the in vitro and in situ BIOS was examined with X‐ray absorption fine structure (XAFS). A bulk XAFS analysis showed that the minerals in both BIOS were mainly ferrihydrite and oligomeric stages of amorphous iron oxyhydroxides with edge‐sharing octahedral linkages. The amount of in vitro BIOS produced with the diffusion‐chamber method was greater than those produced previously with other culture methods, such as gel‐stabilized gradient and batch liquid culture methods. The larger yields of BIOS produced with the new culture method will allow us to clarify in the future the mineralization mechanisms during bacterial growth and to examine the physicochemical properties of BIOS, such as their adsorption to and coprecipitation with various elements and substances.     

Transient behavior and time aspects of intermittently and continuously fed bacterial cultures with regard to filamentous bulking of activated sludge

Summary Pure culture transient experiments with Arthrobacter globiformis and Sphaerotilus natans revealed that the floc-forming species A. globiformis can adapt better to intermittent feeding (I-feeding) than the filamentous species S. natans. The floc-forming bacterium showed a larger overcapacity for substrate uptake, a larger accumulation of reserves (polysaccharides and poly--hydroxybutyric acid) and a more efficient mobilization of these polymers. As a consequence A. globiformis became dominant in an I-fed dual culture of S. natans and A. globiformis. The transient behaviour of filamentous continuously fed (C-fed) sludge was similar to the response of S. natans. Consequently, I-feeding of activated sludge could prevent the excessive growth of filamentous bacteria. I-fed sludge, showed a higher overcapacity, the accumulation of more reserves and a shorter lag phase in protein synthesis than C-fed activated sludge, during the transient response, after a pulse dose of substrate. However, to be effective in the control of bulking, the frequency of I-feeding should allow for a sufficiently long endogenous phase. In addition the available fraction of the COD is important in the optimization of I-feeding as a control strategy for filamentous bulking.     

A Novel Gene Encoding an Enzyme That Degrades a Polysaccharide from the Sheath of Sphaerotilus natans

A gene encoding an enzyme that is able to depolymerize the basic polysaccharide prepared from the sheath of Sphaerotilus natans was identified in a sheath-degrading bacterium, Paenibacillus koleovorans. The gene was constructed from 2217 bp coding for 738 amino acids, including the signal sequence of 34 amino acids. No closely related protein or gene was indicated by a homology search. The gene was expressed in Escherichia coli as a glutathione S-transferase fusion protein. The fusion protein depolymerized the sheath polysaccharide into an oligosaccharide, introducing an unsaturated sugar residue, suggesting that the gene codes for a polysaccharide lyase acting on a basic polysaccharide.     

Isolation of Sphaerotilus natans-lysing microorganism from activated sludge

Sphaerotilus natans is thought to be a causative agent for bulking of activated sludge. Four microorganisms lysing S. natans were isolated from sewage activated sludge. One of the isolates, K-01, was investigated further. K-01 formed plaques 3–5 d after plating together with S. natans on PYG agar plates. The plaques enlarged gradually into surrounding S. natans cells and finally covered the entire surface of the plates. The isolate could not be filtered through a membrane filter with a pore size of 0.2 μm. Electron microscopic observation revealed that K-01 was long-rod shaped, Gram negative microorganism. K-01 could not be cultured on PYG agar medium without S. natans, but it was able to form small colonies on diluted bouillon agar medium without the host cell. K-01 strongly suppressed the growth of S. natans in liquid culture.     

Mechanism of malate dehydrogenase isoform formation in <Emphasis Type="Italic">Sphaerotilus natans</Emphasis> 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.     

Isolation and characterization of filamentous bacteria present in bulking activated sludge

Summary Several types of filamentous microorganisms were observed and identified in samples of poorly settling (bulking) activated sludge. The major types encountered and the frequency (percentage) of appearance in the total of all treatment plants sampled were: Eikelboom type 0041 (60), type 1701 (45), Haliscomenobacter hydrossis (35), type 021N (30), Thiothrix spp. (20), and Sphaerotilus natans (20). Isolation techniques and culture media were developed and used to recover 42 axenic strains of filamentous bacteria from sludge samples collected. The isolates were identified as strains of Thiothrix, Beggiatoa, S. natans, and Eikelboom types 021N, 1701, 0041, and 0803. Nutritional and differential characterization of the bacteria was important to the differentiation of groups which could not be easily distinguished on the basis of morphology. Although certain treatment plant operating parameters (organic loading) seemed associated with the presence of specific filamentous organism types, possible interaction among factors precluded definite establishment of a cause and effect relationship for most of the treatment plant characteristics and organisms observed.     

Composition of an Extracellular Polysaccharide Produced by Sphaerotilus natans

The capsular polysaccharide of Sphaerotilus natans has been isolated, purified, and analyzed. Chromatographic and chemical analyses performed on acid hydrolyzates of the purified material have shown that the major components are fucose, galactose, glucose, and glucuronic acid in approximately equimolar amounts. Glucose and glucuronic acid are believed to occur as an aldobiuronic acid unit.     

Germination Requirement of Vallisneria natans Seeds: Implications for Restoration in Chinese Lakes

Due to eutrophication submerged macrophytes have disappeared from many Chinese lakes. This is unfortunate as submerged macrophytes are important to improve water quality, and its re-establishment is therefore desirable. For this purpose a potential method to use is re-seeding, this being particularly attractive due to the high seed productivity of V. natans. We conducted laboratory studies to investigate the effects of five environmental variables (temperature, substratum, oxygen, light availability, and burial depth) on the seed germination of V. natans. Our results showed that a wide temperature range (25–35 °C) was favorable for germination; that seeds germinated well under both gravel and silt; that anaerobic condition proved to accelerate seed germination although the final germination percentage did not rise; and that light and burial acted as limiting factors. These results suggest that V. natans is a potential candidate for successful restoration of vegetation in lakes recovering from eutrophication.     

Biogeochemical Properties of Bacteriogenic Iron Oxides

Bacteriogenic iron oxides (BIOS) are composite materials that consist of intact and partly degraded remains of bacterial cells intermixed with variable amounts of poorly ordered hydrous ferric oxide (HFO) minerals. They form in response to chemical or bacterial oxidation of Fe²⁺, which gives rise to Fe³⁺. Once formed, Fe³⁺ tends to undergo hydrolysis to precipitate in association with bacterial cells. In acidic systems where the chemical oxidation of Fe²⁺ is slow, bacteria are capable of accelerating the reaction by several orders of magnitude. At circumneutral pH, the chemical oxidation of Fe²⁺ is fast. This requires Fe²⁺ oxidizing bacteria to exploit steep redox gradients where low pO₂ slows the abiotic reaction enough to allow the bacteria to compete kinetically. Because of their reactive surface properties, BIOS behave as potent sorbents of dissolved metal ions. Strong enrichments of Al, Cu, Cr, Mn, Sr, and Zn in the solid versus aqueous phase (log 10 K_d values range from 1.9 to 4.2) are common; however, the metal sorption properties of BIOS are not additive owing to surface chemical interactions between the constituent HFO and bacteria. These interactions have been investigated using acid-base tritrations, which show that the concentration of high pK_a sites is reduced in BIOS compared to HFO. At the same time, hydroxylamine insoluble material (i.e., residual bacterial fraction) is enriched in low pK_a sites relative to both BIOS and HFO. These differences indicate that low pK_a or acidic sites associated with bacteria in BIOS interact specifically with high pK_a or basic sites on intermixed HFO.     

Utilization of Inorganic Nitrogen Compounds by Sphaerotilus natans Growing in a Continuous-Flow Apparatus

Sphaerotilus natans was grown, attached, in a continuous-flow apparatus with inorganic nitrogen compounds (NH₄Cl, NaNO₂, or NaNO₃) as the only sources of nitrogen. The growth rate with NH₄⁺-containing medium did not differ from that with media containing glutamate or Casitone as the nitrogen source.     

Modification of benthic insect communities in polluted streams: combined effects of sedimentation and nutrient enrichment

Responses of the benthic insect community of a southern Appalachian trout stream to inorganic sedimentation and nutrient enrichment were monitored over a period of eight months. Entry of pollutants from point sources established differentially polluted zones, allowing an assessment of impacts due to sedimentation alone and in association with elevated nutrient levels. Input of sediment resulted in a significant increase in bed load and decrease of pH at the substrate-water interface (P < 0.05). The zone receiving nutrient runoff from livestock pasture exhibited elevated levels of nitrate and phosphate, but available data indicated such concentrations to be quite low. Species richness, diversity, and total biomass of filter feeding Trichoptera and Diptera, predaceous Plecoptera, and certain Ephemeroptera were significantly reduced in the polluted zones. Inorganic sedimentation, operating indirectly through disruption of feeding and filling of interstitial spaces, was considered to be the primary factor affecting filter feeding taxa. Decomposition of compounds associated with materials in the bed load may depress pH and eliminate acid sensitive species of Plecoptera and Ephemeroptera. Such processes of acidification may be particularly important to Appalachian streams since the pH of regional surface waters is characteristically acidic prior to sedimentation. Accumulation of particles on body surfaces and respiratory structures, perhaps as a function of wax and mucous secretion or surface electrical properties, appears to be the major direct effect of inorganic sedimentation on stream insects. Growths of the filamentous bacterium Sphaerotilus natans were also frequently associated with silted individuals in the zone receiving nutrient addition. Distribution of the bacterium suggested that silted substrates, perhaps as related to the presence of iron compounds, are required for colonization in dilute nutrient solutions. The primary effect of Sphaerotilus colonies appears to be augmentation of particle accumulation through net formation by bacterial filaments. Data indicate that inorganic sedimentation and nutrient addition operate synergistically, eliminating a significantly greater number of taxa than exposure to one pollutant alone.     

Structural and physiological characteristics of some sheathed bacteria

The fine structure and growth of two gram-negative and two gram-positive strains of sheathed bacteria, isolated from activated sludge, have been studied. Their anatomy is quite different fromSphaerotilus natans. The cells are rectangular, tightly joined together, non-motile and enclosed by a sheath which is thin in the gram-negative strains and rather electron-dense in the two larger gram-positive strains. The four strains grow slowly and form pink filamentous flocs in liquid media.