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.     

Growth-promoting Activity of Spent Sulfite Liquor for Sphaerotilus natans Growing in a Continuous-Flow Apparatus

The calcium in ammonia-base spent sulfite liquor, unlike that in calcium-base spent liquor, is not available for growth of Sphaerotilus natans. It has not been possible to obtain a mixed slime with Sphaerotilus as the dominant organism in a continuous-flow apparatus fed spent sulfite liquor.     

Attached Growth of Sphaerotilus natans in Continuous-Flow Apparatus and Its Growth Inhibition by 9-beta-d-Arabinofuranosyladenine

Sphaerotilus natans grew at the maximum specific growth rate (mu(max)) of 0.43/h when cultivated on PGY medium at 25 degrees C. The organism mainly grew attached to inside of the culture vessels when the culture medium was fed to the completely mixed continuous-flow apparatus at a dilution rate above mu(max) and the attached growth was directly related to the dilution rate. When a low concentration of the medium was supplied to the apparatus, almost all of the cells grown were filamentous and attached to the inside of the vessels. When a high concentration of the medium was fed, the organism grew as single cells or short chains and flowed out into the effluent. The attached growth of S. natans in the continuous-flow apparatus was inhibited by the minimal inhibitory concentration of 0.5 to 1.0 mug of 9-beta-d-arabinofuranosyladenine per ml. 9-beta-d-Arabinofuranosyladenine showed bacteriocidal activity against S. natans at a concentration of 50 to 100 mug/ml.     

Attached Growth of Sphaerotilus and Mixed Populations in a Continuous-flow Apparatus

The effects of NH(4)Cl concentration, organic nitrogen compounds, glucose concentration, dissolved oxygen concentration, and flow rate on the attached growth of pure cultures of Sphaerotilus natans and of a mixed population in a continuous-flow apparatus are described. Low concentrations of NH(4)Cl and oxygen, and high flow rates resulted in attached populations that were dominated by Sphaerotilus. The conditions that allowed maximal attached growth in pure culture did not correspond to the conditions that promoted attached growth of Sphaerotilus in a mixed population.     

Growth Rate of Sphaerotilus in a Thermally Polluted Environment

In situ growth of Sphaerotilus on microscope slides immersed in a thermally polluted stream was studied. Ultraviolet radiation was used to differentiate between passive attachment of organisms from the water and growth of the organisms on the slides. Colonization of the slides in this environment took place solely by means of swarmer cells. A generation time of 2.3 hr was obtained at a water temperature of 18-22 C.     

Calcium Nutrition and the Aging Process: A Review

Adequate supply of calcium is essential for proper maintenance of the skeletal system. Recent studies suggest that, in the United States, dietary calcium in most women is inadequate to maintain skeletal integrity. Additionally, postmenopausal women will lose bone mass more rapidly because of estrogen deficiency and possibly tack of exercise. Severe loss of bone mass can result in several serious skeletal diseases, the most frequent of which is osteoporosis. This disease is a major contributor (25–30%) to hip fractures and spinal compression fractures in elderly people. Indirect sequelae of these fractures are responsible for 12% to 15% mortality in these patients within three months. Recent research indicates that osteoporitic bone loss may directly affect alveolar bone and may, in fact, exacerbate existing periodontally-induced bone loss. This article will review the literature concerning physiological and biochemical parameters related to the absorption and metabolism of calcium with special emphasis on calcium alterations in the aging process. The following areas will be discussed: Ingestion, Absorption, Factors Effecting Absorption and Bone Maintenance, Calcium/Protein Interactions Affecting the Aging Process, Altered Bone States Associated with the Aging Person, Osteoporosis, Osteomalacia, Paget's Disease, Calcium Affecting Drug Action, and Calcium Affecting Alveolar Bone.     

Calcium Chloride in Neonatal Parenteral Nutrition: Compatibility Studies Using Laser Methodology

Introduction

We have previously reported results of precipitation studies for neonatal parenteral nutrition solutions containing calcium chloride and sodium phosphate using visual methods to determine compatibility. The purpose of this study was to do further testing of compatibility for solutions containing calcium chloride using more sensitive methods.

Methods

Solutions of Trophamine (Braun Medical Inc, Irvine, CA) and Premasol (Baxter Pharmaceuticals, Deerfield, IL) were compounded with calcium chloride and potassium phosphate. Controls contained no calcium or phosphate. After incubation at 37° for 24 hours solutions without visual precipitation were analyzed to determine mean particle size using dynamic light scattering from a laser light source.

Results

Particle sizes were similar for control solutions and those without visual precipitation and a mean particle size <1000 nm. Compatible solutions were defined as those with added calcium and phosphate with no visual evidence of precipitation and mean particle size <1000 nm. In solutions containing 2.5–3% amino acids and 10 mmol/L of calcium chloride the maximum amount of potassium phosphate that was compatible was 7.5 mmol/L.

Conclusion

Maximum amounts of phosphate that could be added to parenteral nutrition solutions containing Trophamine and calcium chloride were about 7.5–10 mmol/L less for a given concentration of calcium based upon laser methodology compared to visual techniques to determine compatibility. There were minor differences in compatibility when adding calcium chloride and potassium phosphate to Premasol versus Trophamine.     

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.     

The Effect of Calcium Nutrition of Ethylene-induced Abscission

The influence of calcium nutrition on ethylene-induced abscission was studied by growing cotton (Gossypium hirsutum L. cv. Stoneville 213) and bean (Phaseolus vulgaris L. cv. Resistant Black Valentine) plants for several weeks in nutrient solutions containing 2, 10 (normal level), 15, or 20 meq/l of calcium, and then treating the plants with ethylene. Increasing the calcium level of cotton from 2 to 20 meq/l resulted in a 9-fold increase in the calcium content of the abscission zone and a maximum reduction of 25% in the amount of leaf abscission induced by ethylene (9 μl/l). Bean plants grown on 10, 15, or 20 meq/l calcium solutions showed corresponding increases in the calcium content of the abscission zone but showed no significant differences in the rate of ethyleneinduced abscission. Only at the lowest calcium level of 2 meq/l, where deficiency symptoms became apparent, was a significant effect observed. These results suggest that under normal cultural practices calcium nutrition has little influence on the rate of ethylene-induced abscission.     

Identification of a sheath-associated protein involved in phosphate transport in Sphaerotilus natans

Summary Sphaerotilus natans was shown to have a fourfold lower K _mof phosphate transport when grown in medium containing 0.1 mm phosphate, compared to cells grown in 10.0 mm phosphate. Analysis of sheath proteins from cells grown at these two phosphate levels revealed a protein of 53 kDa present in the sheath of cells grown at a phosphate concentration of 0.1 mm. This sheath-associated, phosphate-regulated protein, designated SapP, was gel purified and used to raise a polyclonal antibody. Enzyme-linked immunosorbent assay was used to localize this protein to the surface of the sheathed cells. Phosphate uptake assays done in the presence of the antibody also showed a rise in the K _mof phosphate transport in cells grown in 0.1 mm phosphate, indicating that this protein is involved in high-affinity phosphate transport.Offprint requests to: C. F. Kulpa Jr     

The Role of Calcium Nutrition on Fusarium-Wilt Syndrome in Muskmelon

The influence of various calcium salts was evaluated on non-inoculated muskmelon plants and on plants inoculated with the Fusarium-wilt pathogen, and maintained in silica sand or vermiculite. Disease severity of inoculated seedlings maintained in vermiculite was markedly higher than that of seedlings grown in silica sand, and onset of wilt was recorded earlier in the former. These trends were more conspicuous in seedlings treated with Ca²⁺, especially when this cation was accompanied, by nitrate. In vermiculite–compared with silica sand, Mg and K contents in the shoot were higher while Ca values were lower. Irrespective of the form of calcium salt or the plant's growth medium, the Ca concentration was higher in Fusarium-inoculated shoots while K content was depleted, compared with non-inoculated plants.     

Role of iron deposition in Sphaerotilus discophorus.

Various physiological aspects of the process of iron deposition in Sphaerotilus discophorus were examined to elucidate its role. The values of iron/protein ratios suggested that a direct relationship existed between the iron concentration of the media and the magnitude of final iron deposition. Saturation of the organism's iron deposition system occurred at a 2.0 mM iron concentration, at a value of 0.6 mg of ferric ion per mg of cell protein. Laboratory data indicated that the strain's very low capacity for iron deposition observed at low external iron concentrations makes it unlikely that it is significant in limiting iron in the natural milieu. Under optimal iron concentrations, however, strain SS1 caused precipitation of iron (adsorbed to cellular material) in broth cultures, which was 10 to 100 times that mediated by some "non-iron" microorganisms. The strain's iron requirement, which was found to be between 0.003 and 0.02 mM, is commensurate with that of other microbes. One hundred micrograms of Mn(II) per ml and possibly 10 mug of either Co(II) or Ni(II) per ml could inhibit iron uptake in the deposition system. Sphaerotilus, when tested for its ability to withstand toxic concentrations of certain trace elements (Co, Cu, Mn, Ni, and Cd), demonstrated no exceptional resistance with respect to several other common microorganisms. Final cell yields were not affected by a varying iron concentration for Sphaerotilus growing under conditions of limiting carbon and nitrogen.     

Calcium Nutrition and Resistance of Alfalfa to Ditylenchus dipsaci

Stem nematode-susceptible ''Atlantic'' and resistant ''Lahontan'' alfalfa seedlings, grown in sand and watered with complete nutrient solutions containing 0.75, 1.5, 3.0, 6.0, or 12.0 mM Ca⁺⁺/liter, were inoculated with Ditylenchus dipsaci (the stem nematode) 5-6 days after emergence. Approximately equal numbers of nematodes entered the tissues of each variety/Ca⁺⁺ concentration within 2 days. Penetration was reduced at 12 mM Ca⁺⁺/liter. Reproduction during 21 days following inoculation yielded 3-fold, or greater, nematode increases in ''Atlantic'' buds at all Ca⁺⁺ concentrations, in ''Atlantic'' cotyledons at the four lower concentrations, in ''Lahontan'' buds at the lowest concentration and in ''Lahontan'' cotyledons at the two lowest concentrations. Reproduction was lower at the higher Ca⁺⁺ concentrations.Increased nutrient Ca⁺⁺ concentrations resulted in increased Ca⁺⁺ content, decreased Na⁺ and K⁺ content, and unchanged Mg⁺⁺ content of buds and cotyledons. Accordingly, increased nutrient Ca⁺⁺ resulted in increased divalent/monovalent cation ratios (Ca⁺⁺ + Mg⁺⁺/Na⁺ + K⁺ ). Resistance to reproduction was correlated more closely with the divalent/monovalent cation ratio than with Ca⁺⁺ content of tissue, At the four higher nutrient Ca⁺⁺ concentrations, ''Lahontan'' buds had higher ratios than ''Atlantic,'' and infected buds had higher ratios than noninfected buds. Although cation balance modifies disease expression, the basic resistance mechanism remains unknown.     

An Apparatus (Georgiou-Petri Dish) for Growing Fungi and other Microorganisms on Liquid Media in a Petri Dish

This work describes a new apparatus for growing fungi and other microorganisms on liquid nutrient media in a Petri dish. The apparatus is composed of a net supporting a cellophane membrane stretched between an outer and an inner ring that is placed inside a Petri dish. This modification of the standard Petri dish offers many advantages for studying growth, metabolism, differentiation, and other aspects of fungi in liquid cultures with minimal waste of expensive chemicals. Monitoring of excreted or absorbed substances by the fungi, the aseptic transfer of undisturbed fungal colonies from dish to dish, and harvesting are made easier, using this apparatus.     

Sphaerotilus natans,a Neutrophilic Iron-Related Sheath-Forming Bacterium: Perspectives for Metal Remediation Strategies

Sphaerotilus natans is a neutrophilic sheath-forming microorganism from the Sphaerotilus-Leptothrix group of iron-related bacteria, known to form bacteriogenic iron oxides (BIOS) frequently deposited on cell surfaces as well as on sheaths and extracellular polymeric substances. S. natans has been reported to be an excellent sorbent for inorganic pollutants, either due to direct sorption onto biological surfaces or due to sorption onto BIOS. However, its filaments can cause bulking problems in wastewater treatment plants. This article promotes the potential applications of Sphaerotilus natans in bioremediation by reviewing its physiology and the fundamental understanding of sheath-forming mechanisms as well as iron biomineralization processes.     

Effects of Light and Nitrogen Nutrition on the Organization of the Photosynthetic Apparatus in Spinach

Nitrogen budgets of fully expanded young leaves of Spinaciaoleracea L. grown under three growth irradiances at four nitrateconcentrations, were compared in relation to photosynthesis.The proportion of nitrogen allocated to thylakoid membraneswas 24% of total leaf nitrogen irrespective of the growth conditions.The composition of the photosynthetic components in thylakoidmembranes was affected by growth irradiance but unaffected bynitrogen levels. The proportion of total leaf nitrogen allocatedto soluble protein and RuBP carboxylase (RuBPCase) increasedwith the increases in nitrogen and in irradiance levels. Someultrastructural properties of chloroplasts and their intra-leafgradients were also compared. The results suggest that nitrogennutrition affects the amount of thyalkoids per unit leaf areabut neither the properties of thyalkoids nor their intra-leafgradient. Growth irradiance, however, controls both the propertiesand the amount of thylakoids. The ratio of in vitro RuBPCase activity to electron transport/photophosphorylationactivity increased with the increase in nitrogen level, butdecreased with the increase in growth irradiance. The changein the ratio of in vitro activities may serve to balance thein vivo activities, given that the in vivo efficiency of RuBPCasedeclines with the increase in volume of a chloroplast due tothe increased liquid phase resistance to CO₂ diffusion. ³Present address: Plant Environmental Biology Group, ResearchSchool of Biological Sciences, The Australian National University,P.O. Box 475, Canberra, A.C.T. 2601, Australia. (Received July 29, 1987; Accepted November 2, 1987)     

Treatment of Fungal Bioaerosols by a High-Temperature,Short-Time Process in a Continuous-Flow System

Airborne fungi, termed fungal bioaerosols, have received attention due to the association with public health problems and the effects on living organisms in nature. There are growing concerns that fungal bioaerosols are relevant to the occurrence of allergies, opportunistic diseases in hospitals, and outbreaks of plant diseases. The search for ways of preventing and curing the harmful effects of fungal bioaerosols has created a high demand for the study and development of an efficient method of controlling bioaerosols. However, almost all modern microbiological studies and theories have focused on microorganisms in liquid and solid phases. We investigated the thermal heating effects on fungal bioaerosols in a continuous-flow environment. Although the thermal heating process has long been a traditional method of controlling microorganisms, the effect of a continuous high-temperature, short-time (HTST) process on airborne microorganisms has not been quantitatively investigated in terms of various aerosol properties. Our experimental results show that the geometric mean diameter of the tested fungal bioaerosols decreased when they were exposed to increases in the surrounding temperature. The HTST process produced a significant decline in the (1→3)-β-d-glucan concentration of fungal bioaerosols. More than 99% of the Aspergillus versicolor and Cladosporium cladosporioides bioaerosols lost their culturability in about 0.2 s when the surrounding temperature exceeded 350°C and 400°C, respectively. The instantaneous exposure to high temperature significantly changed the surface morphology of the fungal bioaerosols.Fungi are omnipresent in indoor and outdoor environments (2, 28, 39). Most fungi are dispersed through the release of spores into the air, a phenomenon known to be driven by two kinds of energy (17): the energy provided by the fungus itself and the energy provided by external sources, such as air currents, rain, gravity, or changes in temperature and nutritional sources. Of these various mechanisms of fungal particle release, dispersal by air currents is the most prevalent mechanism for indoor fungal particles (19, 31). These airborne fungal spores, termed fungal bioaerosols, are resistant to environmental stresses and are adapted to airborne transport.Fungal bioaerosols constitute the major component of ambient airborne microorganisms (23, 50, 51). Several studies have reported that the concentration of fungal bioaerosols is relevant to the occurrence of human diseases and public health problems associated with acute toxic effects, allergies (3, 18), and asthma (4, 5, 13, 48). Fungal bioaerosols are of particular concern in healthcare facilities, where they can cause major infectious complications as opportunistic pathogens in patients with an immunodeficiency (9). For instance, invasive mycoses can affect patients undergoing high-dose chemotherapy for hematological malignancies associated with a prolonged period of neutropenia; they can also affect solid-organ transplant recipients. Despite all diagnostic and therapeutic efforts, the outcome of an invasive fungal infection is often fatal (with a mortality rate of around 50% for aspergillosis) (37). The main fungal genera responsible for these infections are as follows: Aspergillus spp., Fusarium spp., Scedosporium spp., and Mucorales spp. (10, 12, 20). However, virtually any filamentous fungus can be a pathogen (22, 41). In the hospital environment, possible sources of airborne nosocomial infection include ventilation or air-conditioning systems, decaying organic material, dust, water, food, ornamental plants, and building materials in and around hospitals (1).One of the major bioaerosols of concern is (1→3)-β-d-glucans, which comprises up to 60% of the cell wall of most fungal organisms. The (1→3)-β-d-glucans are glucose polymers with a variable molecular weight and a degree of branching (49). The results of several studies about the exposure of subjects to airborne (1→3)-β-d-glucans suggest that these agents play a role in bioaerosol-induced inflammatory responses and resulting respiratory symptoms, such as a dry cough, phlegmy cough, hoarseness, and atopy (11, 44). In addition, given that many epidemiological studies have reported that (1→3)-β-d-glucan has strong immuno-modulating effects (42, 47), (1→3)-β-d-glucan is an important parameter for exposure assessment by itself and as a surrogate component for fungi (16).To prevent the adverse health effects of fungal bioaerosols, we must ensure that control methods for airborne fungal spores are studied and developed. However, despite the necessity of controlling fungal bioaerosols, few studies have focused on such control mechanisms. The most common control methods are UV irradiation and electric ion emission. Given that UV irradiation is known to have a germicidal effect, several studies have examined how UV irradiation affects the viability of bioaerosols (35, 42). However, although UV irradiation can be easily applied by simply installing and turning on a UV lamp, the 254-nm-wavelength UV light produces ozone and radicals, which cause harmful effects to surrounding humans. Electric ion emission has also been studied as a means of controlling bioaerosols (21, 27). When the efficiency of the filter is increased, the efficacy of respiratory protection devices against bioaerosols can be enhanced. Although electric ions decrease the viability of airborne bacteria (25), the generation of the ions produces ozone, a pollutant, and also causes electric charges to accumulate on surrounding surfaces.Recently, heat treatment of indoor air using thermal processes has been considered a safe, effective, and environment-friendly method; it does not produce ozone or use ion or filter media. A thermal heating process has long been considered a suitable and reliable method for controlling microorganisms. Two types of heat are generally used, moist heat and dry heat. Moist heat utilizes steam under pressure, whereas dry heat involves high-temperature exposure without additional moisture. Several types of heat treatment are currently used for killing microorganisms. The treatments include incineration, Tyndallization, pasteurization, and autoclaving (32). However, most of these technologies were originally limited to controlling microorganisms in liquid or on material surfaces. In addition, they may not be adequate for controlling bioaerosols because the continuous surrounding environment of bioaerosols is significantly different from the conditions in liquid and on solid surfaces. Therefore, it is necessary to find adequate and practical conditions for controlling bioaerosols. Thus far, several investigations regarding the use of thermal processes against bioaerosols have been reported. Some of these studies have targeted airborne bacteria spores widely used as surrogates for biological warfare agents (8, 34), while others have focused on environmental parameters for the culture and survival of various vegetative cells (14, 29, 46). However, in these studies novel techniques for aerosols, such as measuring and analyzing aerosol particle size, distributions, and concentrations, were not utilized. In addition, to the best of our knowledge, there has been no study on the use of a thermal process for controlling fungal bioaerosols in continuous airflow. Fungal bioaerosols were found to be very resistant to a thermal environment in previous studies.In this study, we investigated the thermal heating effects on the physical, chemical, and biological properties of fungal bioaerosols using a high-temperature, short-time (HTST) sterilization process. The HTST process, a type of thermal heating process, is based on high-temperature stresses for very short periods. Although this thermal process has been used for the microbial decontamination of seeds and dried, powdered products, such as pharmaceuticals and heat-sensitive drink and food, it can be also applied to the control of an airborne microorganism in a continuous-flow system, such as a heating, ventilation, and air-conditioning system (15, 33, 38). When the fungal bioaerosol was passed through a thermal electric heating system, the fungal spores were exposed to various temperatures for short periods. Then, we examined the bioaerosol and aerosol characteristics, including aerosol size distribution, culturability, (1→3)-β-d-glucan production, and surface morphology, using a novel technique for sampling and measuring aerosols.     

Distribution of Iron in Sphaerotilus and the Associated Inhibition

The distribution of iron between the sheaths and the cells of iron-inhibited Sphaerotilus cultures was determined. The experiment was conducted with different soluble iron forms as inhibitors. The growth inhibition was found to be related to the iron sorbed by the cells rather than by the sheaths. At the 90% inhibition level, iron sorbed by the cells ranged from 13 to 15 mg/g of organism for all three inhibitors tested. For 50% inhibition, the iron sorbed by the cells ranged from 7 to 8 mg/g of organism. The iron sorbed by the sheaths varied widely, ranging from 23 to 118 mg/g of organism at the 90% inhibition level and from 11 to 61 mg/g at the 50% inhibition level. The degree of inhibition is closely related to the amount of iron sorbed by the cells, which in turn is a function of the type of iron compound or complex used. The solubility of the iron is a major consideration.