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Objective: To develop evidence-informed recommendations to support the delivery of best practice therapeutic exercise for people with knee and/or hip osteoarthritis (OA). Design: A multi-stage, evidence-informed, international multi-disciplinary consensus process that included: 1) a narrative literature review to synthesise existing evidence; 2) generation of evidence-informed proposition statements about delivery of exercise for people with knee and/or hip OA by an international multi-disciplinary expert panel, with statements refined and analysed thematically; 3) an e-Delphi survey with the expert panel to gain consensus on the most important statements; 4) a final round of statement refinement and thematic analysis to group remaining statements into domains. Results: The expert panel included 318 members (academics, health care professionals and exercise providers, patient representatives) from 43 countries. Final recommendations comprised 54 specific proposition statements across 11 broad domains: 1) use an evidence-based approach; 2) consider exercise in the context of living with OA and pain; 3) undertake a comprehensive baseline assessment with follow-up; 4) set goals; 5) consider the type of exercise; 6) consider the dose of exercise; 7) modify and progress exercise; 8) individualise exercise; 9) optimise the delivery of exercise; 10) focus on exercise adherence; and 11) provide education about OA and the role of exercise. Conclusion: The breadth of issues identified as important by the international diverse expert panel highlights that delivering therapeutic exercise for OA is multi-dimensional and complex.

Objectives: (a) To identify whether differences exist in the pattern of recovery with respect to functional outcomes for acutely ruptured anterior cruciate ligament deficient (ACLD) copers, adapters, and non-copers. (b) To identify clinically relevant outcomes that could distinguish between three functional subgroups. Methods: A longitudinal study was used to measure gait variables and distance hop at regular intervals after injury using a digital camcorder and computer for quantitative analysis. A sample of 63 ACLD subjects entered the study; 42 subjects were measured at least three times. At 12–36 months after injury, subjects were classified as functional copers, adapters, or non-copers on the basis of which of their preinjury activities they had resumed. To determine the pattern of recovery, repeated measurements were analysed using a least squares fit of the data. Results: 17% of ACLD subjects were classified as functional copers, 45% as adapters, and 38% as non-copers. Only 5% of those who participated in high demand activities before injury returned to them. ACLD copers had recovered above the control mean for all gait variables by 40 days after the injury. Hopping distance did not recover to the control mean. Non-copers struggled to recover to control limits and remained borderline for all the gait variables. Conclusions: Distinctive patterns of functional recovery for three subgroups of ACLD subjects have been identified. Gait variables and activity level before injury were the most useful variables for distinguishing between the subgroups. If potential for recovery is identified early after injury, then appropriate treatment can be given.

DRASTIC––Database Resource for the Analysis of Signal Transduction In Cells (http://www.drastic.org.uk/) has been created as a first step towards a data-based approach for constructing signal transduction pathways. DRASTIC is a relational database of plant expressed sequence tags and genes up- or down-regulated in response to various pathogens, chemical exposure or other treatments such as drought, salt and low temperature. More than 17700 records have been obtained from 306 treatments affecting 73 plant species from 512 peer-reviewed publications with most emphasis being placed on data from Arabidopsis thaliana. DRASTIC has been developed by the Scottish Crop Research Institute and the University of Abertay Dundee and allows rapid identification of plant genes that are up- or down-regulated by multiple treatments and those that are regulated by a very limited (or perhaps a single) treatment. The INSIGHTS (INference of cell SIGnaling HypoTheseS) suite of web-based tools allows intelligent data mining and extraction of information from the DRASTIC database. Potential response pathways can be visualized and comparisons made between gene expression patterns in response to various treatments. The knowledge gained informs plant signalling pathways and systems biology investigations.

Expanded specific affinity theory specifies the advantages of small dry mass and dilute cytoarchitecture in impoverished systems. For marine samples, bacterioplankton mean dry mass, according to flow cytometry, was near 24.5 fg cell -1 . Conversion to volume with buoyant density gave a cell volume of 0.124 μm 3 . Total DNA was 2.9 fg cell -1 . This compared with the size of a single genome of a small extinction-culture isolate, Sphingomonas sp. RB2256, of 3.96 fg or 3.6 Mb. The genome size of such isolates and other cultures decreased with metabolic simplicity. It was found that bacterioplankton could be exposed to radiolabeled amino acids and then sorted for size and that the specific affinities of the fraction of small organisms were as great as the fraction of large cells. Size, DNA, and metabolic-complexity distributions were concordant with the concepts that cell volume approaches a minimum set by sufficient space for the smallest genome that can provide sufficient information for competitive dissolved-nutrient acquisition, and that space requirements are further alleviated by the expression of few cytoplasmic-enzyme molecules in each of the various pathways and a dilute cytoplasm. Bacterioplankton approached a minimum genome size of 1.7 Mb with a minimum cell volume of about 0.06 μm 3 and a DNA content of 16% dry weight. The property of small dry mass with a low DNA content was common in in situ bacteria but absent from cultivated representatives, which led to speculation that failure to grow in the laboratory is related to missing regulatory information.

Dilutions of raw seawater produced a bacterial isolate capable of extended growth in unamended seawater. Its 2.9-Mb genome size and 40-fg dry mass were similar to values for many naturally occurring aquatic organotrophs, but water and DNA comprised a large portion of this small chemoheterotroph, as compared to Escherichia coli . The isolate used only a few aromatic hydrocarbons and acetate, and glucose and amino acid incorporation were entirely absent, although many membrane and cytoplasmic proteins were inducible; it was named Cycloclasticus oligotrophus . A general rate equation that incorporates saturation phenomena into specific affinity theory is derived. It is used to relate the kinetic constants for substrate uptake by the isolate to its cellular proteins. The affinity constant K A for toluene was low at 1.3 μg/liter under optimal conditions, similar to those measured in seawater, and the low value was ascribed to an unknown slow step such as limitation by a cytoplasmic enzyme; K A increased with increasing specific affinities. Specific affinities, a° s , were protocol sensitive, but under optimal conditions were 47.4 liters/mg of cells/h, the highest reported in the literature and a value sufficient for growth in seawater at concentrations sometimes found. Few rRNA operons, few cytoplasmic proteins, a small genome size, and a small cell size, coupled with a high a° s and a low solids content and the ability to grow without intentionally added substrate, are consistent with the isolation of a marine bacterium with properties typical of the bulk of those present.

When a heterotrophic (Rhodotorula rubra) and a phototrophic (Selenastrum capricornutum) plankton were grown together in dilute phosphate (Pi) continuous cultures, coexistence occurred only when the heterotroph was growthrate limited by organic carbon (C). Because of its higher affinity for Pi, and because C starvation does not affect the heterotrophic yeast's ability to transport Pi, the concentration of organic carbon indirectly controlled the biomass of the phototroph. The results support a threshold model of microbial growth.

We investigate a holographic description of k-strings in higher representations via D5 branes with worldvolume fluxes. The D-brane configurations are embedded in supergravity backgrounds dual to confining field theories in 3 and 4 dimensions. We compute the tensions and find qualitative agreement for the totally symmetric and totally anti-symmetric representations with the results of other methods such as lattice as well as the Hamiltonian approach of Karabali and Nair. A one-loop computation on the D-brane configurations yields the Luescher term and allows us to confirm a previously proposed universal expression following from holography., 32 pages, 2 figures, 1 table. v2 references added; comparison with other methods enhanced

In Lake Zurich, a deep subalpine mesotrophic lake, phosphate was low or limiting at 0.2 to 1 μM relative to combined nitrogen at 50 μM. Heterotrophic bacteria were responsible for 53% of the observed microbial wet biomass in our depth profile while phytoplankton, largely Planktothrix (Oscillatoria) rubescens , contributed most of the remainder. Most cell carbon was contributed by this carbon-sufficient cyanobacterium. A material balance indicated that most of the phosphate was sequestered by the bacteria due to a higher phosphate content and specific affinity for this nutrient. Size distributions of the heterotrophic bacteria were narrow; 90% of organisms were from 0.06 to 0.06 μ 3 in volume. Several subpopulations of bacteria were resolved by flow cytometry, and bivariate fluorescence (DAPI-DNA) and light scatter (cell-size) histogram profiles varied with depth. One or two of these subpopulations appeared to be bacteria with sufficient cytoplasmic constituents to produce a normal light-scatter signal but retained only a small amount of DNA; an apparent content of 200 kbp or 5% of a usual oligobacterial genome. These helped increase the oligobacterial population to 6 × 10 6 ml −1 . Application of published specific affinities and measured nutrient concentrations to formulations of system kinetics led to the conclusion that growth rates of the heterotrophic bacterial fraction were carbon limited with cell size, and thus populations were controlled by grazing. The depth profile indicated that phototrophs affected concentrations in a significant way. Considerations of nutrient uptake kinetics suggested that much potential capacity remained in the dissolved phosphate pool to support additional phytoplanktonic biomass. Computations led to the conclusion that, if phosphate is generally limiting in lakes, then additional mechanisms exist which limit populations of phytoplankton to sufficiently small values to allow phosphate accumulation to observed levels. Bacterial biomass then depends on the organic carbon from these phosphate-controlled organisms.

We compute the asymptotic symmetry group of the four dimensional near-extremal Kerr-Sen black hole within an AdS2/CFT1 correspondence. We do this by performing a Robinson-Wilczek two dimensional reduction and construct a quantum theory of the remaining field content. The resulting energy momentum tensor generates an asymptotic Virasoro algebra, to s-wave, with a calculable central extension. This center in conjunction with the proper regularized lowest Virasoro eigen-mode yields the near-extremal Kerr-Sen entropy via the statistical Cardy formula. Finally we analyze quantum holomorphic fluxes of the dual CFT giving rise to a finite Hawking temperature weighted by the central charge of the near-extremal Kerr-Sen metric., Comment: 16 pages, references added, revised for clarity. To appear in JHEP

Specific affinity theory is compared with traditional ways of understanding the nutrient concentration dependency of microbial growth. It is demonstrated that the Michaelis constant increases with the ratio of metabolic enzyme to membrane permease content of bacteria so that small values can reflect specialization for nutrient collection. When compared to the specific affinity, Kt gives a measure of oligotrophic capacity. Specific affinity, on the other hand, reflects nutrient collection ability directly, and increases with the number of permeases. It can be estimated, along with the other kinetic constant, Vmax, by use of isotopes in natural samples. Because of systematic errors in estimating Vmax, specific affinity is the preferred measure of substrate accumulation ability. The advantage of simultaneous collection of multiple substrates in dilute solution is demonstrated. The structural basis of this advantage is computed from collision frequency and recollision probability, computations that further show that multisubstrate usage is essential for bacterial growth under low-nutrient conditions. Computed growth rates from specific affinities require that several substrates be used simultaneously for growth at measured concentrations. Formulations anticipate that the surface of oligobacteria should be occupied by a diversity of transporter types, that each type of transporter should occupy only a small portion of the cell surface, and the number of cytoplasmic enzymes can be small, allowing small cell size to give a large surface-to-volume ratio for high specific affinity. The large number of substrate types that may be accumulated by a single oligobacterial species is consistent with extensive species diversity.

We show that the near horizon regime of a Kerr-Newman-$AdS$ (KN$AdS$) black hole, given by its two dimensional analogue $a la$ Robinson and Wilczek (2005 Phys. Rev. Lett. 95 011303), is asymptotically $AdS_2$ and dual to a one dimensional quantum conformal field theory (CFT). The s-wave contribution of the resulting CFT's energy-momentum-tensor together with the asymptotic symmetries, generate a centrally extended Virasoro algebra, whose central charge reproduces the Bekenstein-Hawking entropy via Cardy's Formula. Our derived central charge also agrees with the near extremal Kerr/CFT Correspondence (2009 Phys. Rev. D 80, 124008) in the appropriate limits. We also compute the Hawking temperature of the KN$AdS$ black hole by coupling its Robinson and Wilczek two dimensional analogue (RW2DA) to conformal matter., 15 pages. Major edits and revisions. Final version accepted for publication in IJMPA

Turnover times for toluene in Resurrection Bay after the Exxon Valdez grounding were determined to be decades, longer than expected considering that dissolved hydrocarbons were anticipated to drift with the current and stimulate development of additional hydrocarbon-utilizing capacity among the microflora in that downcurrent location. These turnover times were based on the recovery of 14CO2 from added [14C]toluene that was oxidized. The concentrations of toluene there, 0.1 to 0.2 microgram/liter, were similar to prespill values. Oxidation rates appeared to be enhanced upstream near islands in the wake of the wind-blown slick, and even more within the slick itself. Specific affinities of the water column bacteria for toluene were computed with the help of biomass data, as measured by high-resolution flow cytometry. They were a very low 0.3 to 3 liters/g of cells.h-1, indicating limited capacity to utilize this hydrocarbon. Since current-driven mixing rates exceeded those of oxidation, dissolved spill components such as toluene should enter the world-ocean pool of hydrocarbons rather than biooxidize in place. Some of the floating oil slick washed ashore and permeated a coarse gravel beach. A bacterial biomass of 2 to 14 mg/kg appeared in apparent response to the new carbon and energy source. This biomass was computed from that of the organisms and associated naphthalene oxidation activity washed from the gravel compared with the original suspension. These sediment organisms were very small at approximately 0.06 microns 3 in volume, low in DNA at approximately 5.5 g per cell, and unlike the aquatic bacteria obtained by enrichment culture but quite similar to the oligobacteria in the water column.(ABSTRACT TRUNCATED AT 250 WORDS)

Formulations are presented that describe the concentration dependency of nutrient-limited transport and growth in molecular terms. They relate the rate of transport at steady state through a two-sequence process, transport and metabolism, to ambient concentrations according to the amounts and kinetic characteristics of the two rate-limiting proteins in these sequences. Sequences are separated by a metabolic pool. A novel feature of these formulations is the translation coefficient, which relates the transport rate attained at given ambient nutrient concentrations and membrane transporter characteristics to the nutrient concentrations sustained in the metabolic pools. The formulations, termed janusian kinetics, show that hyperbolic kinetics are retained during independent changes in transporter and enzyme contents or characteristics. Specific affinity ( a ° A ) depends strongly on the amount and kinetic characteristics of the transporters; it is also mildly affected by the amount and characteristics of the rate-limiting enzyme. This kinetic constant best describes the ability to accumulate substrate from limiting concentrations. Maximal velocity ( V max ) describes uptake from concentrated solutions and can depend strongly on either limiting enzyme content or the associated content of transporters. The whole-cell Michaelis constant ( K T ), which depends on the ratio of rate-limiting enzyme to transporter, can be relatively independent of change in a ° A and is best used to describe the concentration at which saturation begins to occur. Theory specifies that good oligotrophs have a large a ° A for nutrient collection and a small V max for economy of enzyme, giving a small K T . The product of the two constants is universally rather constant so that oligotrophy is scaled on a plot of a ° A versus K T , with better oligotrophs toward one end. This idea is borne out by experimental data, and therefore typical small difficult-to-culture aquatic bacteria may be classified as oligobacteria.

A theory for solute uptake by whole cells was derived with a focus on the ability of oligobacteria to sequester nutrients. It provided a general relationship that was used to obtain the kinetic constants for in situ marine populations in the presence of naturally occurring substrates. In situ affinities of 0.9 to 400 liters g of cells −1 h −1 found were up to 10 3 times smaller than those from a “ Marinobacter arcticus ” isolate, but springtime values were greatly increased by warming. Affinities of the isolate for usual polar substrates but not for hydrocarbons were diminished by ionophores. A kinetic curve or Monod plot was constructed from the best available data for cytoarchitectural components of the isolate by using the theory together with concepts and calculations from first principles. The order of effect of these components on specific affinity was membrane potential > cytoplasmic enzyme concentration > cytoplasmic enzyme affinity > permease concentration > area of the permease site > translation coefficient > porin concentration. Component balance was influential as well; a small increase in cytoplasmic enzyme concentration gave a large increase in the effect of permease concentration. The effect of permease concentration on specific affinity was large, while the effect on K m was small. These results are in contrast to the Michaelis-Menten theory as applied by Monod that has uptake kinetics dependent on the quality of the permease molecules, with K m as an independent measure of affinity. Calculations demonstrated that most oligobacteria in the environment must use multiple substrates simultaneously to attain sufficient energy and material for growth, a requirement consistent with communities largely comprising few species.

The distribution of DNA among bacterioplankton and bacterial isolates was determined by flow cytometry of DAPI (4′,6′-diamidino-2-phenylindole)-stained organisms. Conditions were optimized to minimize error from nonspecific staining, AT bias, DNA packing, changes in ionic strength, and differences in cell permeability. The sensitivity was sufficient to characterize the small 1- to 2-Mb-genome organisms in freshwater and seawater, as well as low-DNA cells (“dims”). The dims could be formed from laboratory cultivars; their apparent DNA content was 0.1 Mb and similar to that of many particles in seawater. Preservation with formaldehyde stabilized samples until analysis. Further permeabilization with Triton X-100 facilitated the penetration of stain into stain-resistant lithotrophs. The amount of DNA per cell determined by flow cytometry agreed with mean values obtained from spectrophotometric analyses of cultures. Correction for the DNA AT bias of the stain was made for bacterial isolates with known G+C contents. The number of chromosome copies per cell was determined with pure cultures, which allowed growth rate analyses based on cell cycle theory. The chromosome ratio was empirically related to the rate of growth, and the rate of growth was related to nutrient concentration through specific affinity theory to obtain a probe for nutrient kinetics. The chromosome size of a Marinobacter arcticus isolate was determined to be 3.0 Mb by this method. In a typical seawater sample the distribution of bacterial DNA revealed two major populations based on DNA content that were not necessarily similar to populations determined by using other stains or protocols. A mean value of 2.5 fg of DNA cell −1 was obtained for a typical seawater sample, and 90% of the population contained more than 1.1 fg of DNA cell −1 .

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus , for which dry weight was determined by CHN analysis and 14 C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4′,6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 μg/liter. The lowest value for mean dry mass, 0.027 ± 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli . The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10 3 cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.

The far-ranging distribution of genes for aromatic hydrocarbon catabolism, predominantly studied in soil pseudomonads, is extended to a marine oligobacterium by finding five homologous sequences in a 5.7-kb chromosomal DNA from a new isolate, Cycloclasticus oligotrophus RB1. RB1 is capable of growth in unamended seawater or mineral salts media supplemented with a variety of aromatic compounds, including toluene, o-, m-, or p-xylenes, as sole carbon sources. The five open reading frames, designated xylM, K, G, C1, and C2, are 57% A+T-rich. XylM is predicted to be an integral membrane protein; XylK and XylG possess glutathione S-transferase (GST) and 2-hydroxy-5methyl-6-oxohexa2,4-dienoate dehydrogenase activities, respectively; XylC1C2 are homologs of the large and small subunits of the iron sulfur protein component of the biphenyl dioxygenase (e.g., BphA1A2).

Marine bacteria in Resurrection Bay near Seward, Alaska, and in the central North Sea off the Dutch coast were cultured in filtered autoclaved seawater following dilution to extinction. The populations present before dilution varied from 0.11 × 10 9 to 1.07 × 10 9 cells per liter. The mean cell volume varied between 0.042 and 0.074 μm 3 , and the mean apparent DNA content of the cells ranged from 2.5 to 4.7 fg of DNA per cell. All three parameters were determined by high-resolution flow cytometry. All 37 strains that were obtained from very high dilutions of Resurrection Bay and North Sea samples represented facultatively oligotrophic bacteria. However, 15 of these isolates were eventually obtained from dilution cultures that could initially be cultured only on very low-nutrient media and that could initially not form visible colonies on any of the agar media tested, indicating that these cultures contained obligately oligotrophic bacteria. It was concluded that the cells in these 15 dilution cultures had adapted to growth under laboratory conditions after several months of nutrient deprivation prior to isolation. From the North Sea experiment, it was concluded that the contribution of facultative oligotrophs and eutrophs to the total population was less than 1% and that while more than half of the population behaved as obligately oligotrophic bacteria upon first cultivation in the dilution culture media, around 50% could not be cultured at all. During one of the Resurrection Bay experiments, 53% of the dilution cultures obtained from samples diluted more than 2.5 × 10 5 times consisted of such obligate oligotrophs. These cultures invariably harbored a small rod-shaped bacterium with a mean cell volume of 0.05 to 0.06 μm 3 and an apparent DNA content of 1 to 1.5 fg per cell. This cell type had the dimensions of ultramicrobacteria. Isolates of these ultramicrobacterial cultures that were eventually obtained on relatively high-nutrient agar plates were, with respect to cell volume and apparent DNA content, identical to the cells in the initially obligately oligotrophic bacterial dilution culture. Determination of kinetic parameters from one of these small rod-shaped strains revealed a high specific affinity for the uptake of mixed amino acids ( a° A , 1,860 liters/g of cells per h), but not for glucose or alanine as the sole source of carbon and energy ( a° A , ± 200 liters/g of cells per h). The ultramicrobial strains obtained are potentially a very important part of picoplankton biomass in the areas investigated.

Dilution culture, a method for growing the typical small bacteria from natural aquatic assemblages, has been developed. Each of 11 experimental trials of the technique was successful. Populations are measured, diluted to a small and known number of cells, inoculated into unamended sterilized seawater, and examined three times for the presence of 10 4 or more cells per ml over a 9-week interval. Mean viability for assemblage members is obtained from the frequency of growth, and many of the cultures produced are pure. Statistical formulations for determining viability and the frequency of pure culture production are derived. Formulations for associated errors are derived as well. Computer simulations of experiments agreed with computed values within the expected error, which verified the formulations. These led to strategies for optimizing viability determinations and pure culture production. Viabilities were usually between 2 and 60% and decreased with >5 mg of amino acids per liter as carbon. In view of difficulties in growing marine oligobacteria, these high values are noteworthy. Significant differences in population characteristics during growth, observed by high-resolution flow cytometry, suggested substantial population diversity. Growth of total populations as well as of cytometry-resolved subpopulations sometimes were truncated at levels of near 10 4 cells per ml, showing that viable cells could escape detection. Viability is therefore defined as the ability to grow to that population; true viabilities could be even higher. Doubling times, based on whole populations as well as individual subpopulations, were in the 1-day to 1-week range. Data were examined for changes in viability with dilution suggesting cell-cell interactions, but none could be confirmed. The frequency of pure culture production can be adjusted by inoculum size if the viability is known. These apparently pure cultures produced retained the size and apparent DNA-content characteristic of the bulk of the organisms in the parent seawater. Three cultures are now available, two of which have been carried for 3 years. The method is thus seen as a useful step for improving our understanding of typical aquatic organisms.

The steady-state growth rate of a marine isolate was related to the concentrations of several carbon and energy source substrates when these substrates limited growth simultaneously in continuous culture. Glucose limitation was characterized by a threshold of 0.21 mg/liter for growth, a half-maximal growth rate at 0.48 mg/liter, U-shaped curves in extractable pool concentration-versus-growth velocity plots, and slow maximal growth rates. Arginine addition reduced the glucose threshold to 0.008 mg/liter, more than doubled the maximal growth rate, and stabilized pool concentrations at low growth rates. Addition of a third substrate, glutamate, caused further reduction of the glucose concentration a steady state. Maximal reduction of the glucose concentration was effected by adding a mixture of 20 amino acids. Steady-state limiting nutrient concentration was dependent on the specific identity of the auxiliary nutrients and on the concentration ratio at which they were supplied. When glucose was supplemented with an equal quantity of an amino acid mixture, the external steady-state glucose remained below 10 mug/liter. When 1 mug of glucose was added to a 2.5-mg/liter amino acid mixture, at least 70% of it was consumed at steady state in spite of the threshold observed. Lack of crossover between metabolic pathways, suggested by the absence of glucose carbon in pool glutamate of arginine-glucose-grown cells, may have been partly responsible for the mixed carbon source stimulation of nutrient accumulation observed. The affinity observed is sufficient to account for normal growth at a total organic substrate concentration of only 0.11 mg/liter when supplied from a suitable mixture.

Flow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high-intensity UV excitation, condensing the laser beam onto a small area, using blemish-free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high-transmittance optical filters, collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 micron 3 as calibrated by Coulter impedance. Plastic spheres down to 0.014 micron 3, 0.3 micron in diameter, were resolved. Aquatic bacteria 0.05 micron 3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA-dependent because DNase treatment eliminated most fluorescence. These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA-free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.

A method having sufficient sensitivity to resolve the kinetic constants for dissolved nonpolar substrate metabolism, together with the related rate constants in natural waters, is presented. The method is based on the rate of 14 CO 2 recovery from radioactive dissolved substrate. Sensitivity is enhanced by using large seawater volumes, high-specific-activity isotopes, and by reducing background radioactivity. Before use, commercial isotopes are purified by mild alkaline hydrolysis followed by sublimation from base to remove 14 CO 2 as well as interfering polar 14 C-substrates. During sample analysis, chilled Tenax resin is used to remove volatile 14 C-substrate from the nitrogen stream containing 14 CO 2 recovered from substrate oxidation. Chromatographic evidence of purity, shown to be insufficient, is augmented by kinetic data from toluene utilization by mixed cultures and by rates in induced versus noninduced pure cultures. Accuracy is enhanced by using short (

Analysis of continuous culture methodology suggests that this potentially powerful tool for kinetic analysis can be improved by minimizing several inherent shortcomings. Medium background substrates - organic carbon, phosphate, and manganese - were shown to dominate kinetic observations at concentrations below chemical detection methods. Reactor wall growth, culture size distribution changes, sample removal-induced steady state perturbations, and limiting substrate leakage from organisms are treated in terms of kinetic measurement errors. Large variations in maximal growth rates and substrate uptake rates found are attributed to experimental protocol-induced transient states. Relationships are presented for correcting limiting substrate concentrations for lability during sampling, contamination with unreacted medium, and background substrate effects. Analytical procedures are discussed for improved measurement of limiting substrate kinetics involving enzymes, isotopes, and material balance manipulation. Relaxation methods as applied to continuous culture are introduced as a means for isolating separate rate constants describing net substrate transport and for evaluating cellular metabolite leakage. Low velocity growth, multiple substrate metabolism, and endogenous metabolism are discussed along with measurements showing that 1-month generation times for aquatic microorganisms can be quite normal and that the kinetics are compatible withμg/liter limiting substrate concentrations. The concept of regarding growth kinetics as the sum of several net accumulation processes is suggested.

The continuous addition of toluene as a solute of treated ballast water from oil tankers into a well-defined estuary facilitated the study of the dynamics of dissolved hydrocarbon metabolism in seawater. Most rates of toluene oxidation were in the range of 1 to 30 pg/liter per h at 0.5 μg of toluene per liter. Near the ballast water injection point, a layer of warm ballast water, rich in bacteria, that was trapped below the less-dense fresh surface water was located. Toluene residence times were approximately 2 weeks in this layer, 2 years elsewhere in Port Valdez, and 2 decades in the surface water of a more oceanic receiving estuary adjacent. Mixing was adequate for a steady-state treatment which showed that 98% of the toluene was flushed from Port Valdez before metabolism and gave a steady-state concentration of 0.18 μg/liter. Total bacterial biomass from direct counts and organism size data was usually near 0.1 mg/liter, but ranged up to 0.8 mg/liter in the bacteria-rich layer. The origin of bacteria in this layer was traced to growth in oil tanker ballast during shipments. The biomass of toluene oxidizers in water samples was estimated from the average affinity of pure-culture isolates for toluene (28 liters per g of cells per h) and observed toluene oxidation kinetics. Values ranged from nearly all of the total bacterial biomass within the bacteria-rich layer down to 0.2% at points far removed. Because the population of toluene oxidizers was large with respect to the amount of toluene consumed and because water from a nearby nonpolluted estuary was equally active in facilitating toluene metabolism, we searched for an additional hydrocarbon source. It was found that terpenes could be washed from spruce trees by simulated rainfall, which suggested that riparian conifers provide an additional and significant hydrocarbon source to seawater.

The metabolism of toluene by natural populations of marine bacteria, stored to enhance their activity, gave hyperbolic kinetics with saturation at only K/sub t/ = 0.6 to 3.4 ..mu..g liter/sup -1/. Similarly K/sub t/ in fresh seawater was 0.26 ..mu..g toluene liter/sup -1/. Freshly collected populations could be moderately active toward toluene. These moderate affinities taken together with the small K/sub t/ values give an explanation for the failure of most marine bacteria to grow at the expense of a single hydrocarbon. In two experiments there was a significant first-order region in toluene in the 10-50 ..mu..g liter/sup -1/ range. A large widespread population of bacteria with inducible capacity to metabolize hydrocarbons is indicated. Observed turnover times for toluene were 0.2-40 years with growth rates of 10/sup -6/h/sup -1/ from toluene at 1 ..mu..g liter/sup -1/.

The pink yeast Rhodotorula rubra of marine origin was found to be capable of extended growth at very low phosphate concentrations ( K 0.5 = 10.8 n m ). Average intracellular phosphate concentrations, based on isotope exchange techniques, were 15 to 200 n m , giving concentration gradients across the cell envelope of about 10 6 . Sensitivity to metabolic inhibitors occurred at micromolar concentrations. Inability of the phosphate transport system, K s = 0.5 to 2.8 μ m , V max = 55 μmoles per g of cells per min, to discriminate against arsenate transport led to arsenate toxicity at 1 to 10 n m , whereas environmental arsenate levels are reportedly much higher. Phosphate competitively prevented arsenate toxicity. The K i for phosphate inhibition of arsenate uptake was 0.7 to 1.2 μ m . Phosphate uptake experiments showed that maximal growth rates could be achieved with approximately 4% of the total phosphate-arsenate transport system. Organisms adapted to a range both of concentration of NaCl and of p H. Maximal affinity for phosphate occurred at p H 4 and at low concentrations of NaCl; however, V max for phosphate transport was little affected. Maximal specific growth rates on minimal medium were consistent in batch culture but gradually increased to the much higher rates found with yeast extract media when the population was subjected to long-term continuous culture with gradually increasing dilution rates. Phosphate initial uptake rates that were in agreement with the steady-state flux in continuous culture were obtained by using organisms and medium directly from continuous culture. This procedure resulted in rates about 500 times greater than one in which harvested batch-grown cells were used. Discrepancies between values found and those reported in the literature for other organisms were even larger. Growth could not be sustained below a threshold phosphate concentration of 3.4 n m . Such thresholds are explained in terms of a system where growth rate is set by intracellular nutrient concentrations. Threshold concentrations occur in response to nutrient sinks not related to growth, such as efflux and endogenous metabolism. Equations are presented for evaluation of growth rate-limiting substrate concentrations in the presence of background substrate and for evaluating low inhibitor concentration inhibition mechanisms by substrate prevention of inhibitor flux.

SUMMARY: A small single-phase continuous fermentor system for convenient longterm operation at steady state with small population densities is described. A method by which the effect of extracellular limiting nutrient on growth rate can be evaluated from the resulting data is discussed. This method was used for Torulopsis utilis to determine the Michaelis constants for growth with oxygen and glycerol; these were found to be 1·4×10-5 m and 4·9×10-5 m. Size of organisms and yields were found to decrease with growth rate. Average volume of organisms decreased from 60 to 41 μ3 and the yield decreased from 1·8 to 1·2 g. dry wt./organism/g. oxygen consumed. An endogenous metabolism rate constant determined from yields at growth rates above 30% maximum was 0·056 g. oxygen used/g. dry wt. organism/hr.

The quantum effects in the degenerate valence bands of germanium have been observed as multiple cyclotron resonance absorption lines at a temperature of 165°K. Additional lines were resolved at 40°K. The combination of high intensity magnetic fields up to 180 000 gauss and the output power of a cyanide submillimeter laser (0.337 millimeters) was used. The construction and operation of a simple resonance spectrometer is described. Cryogenic techniques and the choice of detectors are discussed. The possible use of molecular gas laser spectrometers for EPR, ferrimagnetic resonance, and antiferromagnetic resonance at submillimeter wavelengths is considered.

The effect of clays on the availability of small organic molecules to microorganisms was determined. Recovered suspended sedimentary material and Bentonite was tested for its ability to compete with yeast and bacteria for thiamine or glucose in continuous culture systems at low organism concentrations. The added clays do not render the organics tested unavailable to microorganisms or remove them from solution to a detectable or significant extent. The addition of clays can, however, effect a perturbation to the steady system, possibly by altering the inorganic chemistry of the dissolved phase.

SUMMARY The concentration/growth rate relationship of thiamine-requiring Cryptococcus albidus was examined. Data suggest a diffusion-limited mechanism characterized by an apparent Michaelis constant for growth of 4.7 × 10-13 M. This relationship was obtained from continuous culture at steady state and tested by a non-steady state procedure and in batch growth. It was concluded that most natural water systems have sufficient thiamine to support some thiamine-requiring micro-organisms at above rate limiting concentrations.

A slow feed batch fermentation is described for the production of glycerol from sugar. The conversion efficiency was approximately 1 mole of glycerol produced per mole of glucose utilized after the cell growth phase. The glycerol production phase was extended several-fold by periodic glucose addition. The yeast cell count remained constant during this time as limited by phosphate, a deficiency required for an efficient glycerol fermentation. A small amount of phosphate was supplied during the extended fermentation, maintaining an active culture, by the normal autolysis of spent cells. Interfering or inhibitory by-products did not accumulate, and the osmophilic yeasts are tolerant of high glycerol concentrations. These factors combined to allow a particularly efficient fermentation well suited to product enrichment by supplying large quantities of substrate over an extended period.

The kinetics of concentration-dependent toluene metabolism were examined by evaluating each term in the second-order rate equation. Marine and freshwater pseudomonads were used. Uptake for Pseudomonas sp. strain T2 was characterized by a completely saturatable system with small transport constant ( K t = 44 μg/liter) and large specific affinity. Kinetics for Pseudomonas putida PpF1 were similar. Induction had little effect on K t , but it caused the specific affinity to increase from about 0.03 to 320 liters/g of cells per h. The level of induction depended on the time of exposure, the concentration of inducer, and the initial level of induction. If loss of the inducible system was not severe, toluene caused a linear increase in specific affinity with time, and the maximal value achieved at intermediate times (1 to 3 days) was hyperbolic with concentration when K ind was 96 μg/liter (A. T. Law and D. K. Button, Appl. Environ. Microbiol. 51:469-476, 1986). As repression became complete, specific affinities were greatly reduced. Then induction required higher toluene concentrations and longer times, and the shape of the specific-affinity curve became sigmoidal with concentration. Cell yields (0.10 to 0.17 g of cells per g of toluene used) were low owing to liberation of organic products: 2-hydroxy-6-oxohepta-2,4-dienoic acid, toluene dihydrodiol, 3-methylcatechol, acetate, formate, and possibly pyruvate, which in turn caused lower rates of growth. Michaelis constants for the reaccumulation of products exceeded those for toluene, but specific affinities were lower and maximal velocities were higher, so that recycling was favored in cultures with high toluene concentration. Although these kinetics predict deviation from the linear relationship between uptake rate and biomass, we could detect none. Effects of saturation and induction were incorporated into the basic specific-affinity relationship. The result appears to be an improvement in the equation used for describing the kinetics of uptake and growth.

Trace (microgram liter −1 ) quantities of either toluene or benzene injected into an amino-acid-limited continuous culture of Pseudomonas sp. strain T2 were utilized immediately with affinities of 2.6 and 6.8 liters g of cells −1 h −1 , respectively, and yielded large amounts of organic products, carbon dioxide, and cells. The immediate utilization of hydrocarbons by hydrocarbon-deprived organisms helps to establish the nutritional value of nonpolar substrates in the environment. The observation of small Michaelis constants for toluene transport led to tests of metabolic competition between hydrocarbons; however, competitive inhibition of toluene metabolism was not found for benzene, naphthalene, xylene, dodecane, or amino acids. Benzene and terpenes were inhibitory at milligram liter −1 concentrations. Toluene was metabolized by a strongly inducible system when compared with benzene. The capacity of toluene to effect larger affinity values increased with exposure time and concentration. The kinetics of induction suggested saturation phenomena, resulting in an induction constant, K ind , of 96 μg of toluene liter −1 . Maximal induction of amino-acid-grown cells required about 80 h, with the affinity reaching 317 liters g of cells −1 h −1 .

The phosphate-limited growth kinetics of Rhodotorula rubra, a small yeast of marine origin, were examined by analysis of 32P distributions in continuous cultures. Isotope relaxation procedures were used to identify unidirectional flows of Pi and organic phosphate among compartments modeled during growth. The concentrations of phosphates in these compartments at various growth rates were used, together with attendant flows, to produce a mathematical model of growth. Both Pi and phosphate-containing metabolic intermediates leaked from cells during growth. Total leakage ranged from 4 to 10% of influx and was comprised mostly of Pi. Transport capacity was at least 10 times that required for growth at saturating Pi concentrations, so that influx was linear with concentration during growth. This led to the realization that the curvature of Monod plots (Kmu = 12 nM mumax = 0.18/h, and the threshold At = 2.5 nM) is due to change in yield with growth rate. Growth rate related to Pi by the affinity, aA (= 0.43 liter/mg of cells.h) of cells for Pi and the growth rate-dependent yield. It was also specified by a series of kinetic constants that specified flow among the various compartments and equilibrium compartment concentrations as they were set by extracellular Pi. The importance of leakage by healthy cells to the organic chemistry of aquatic systems is noted.

A mixture of 14 C-terpenes was prepared from conifer seedlings and introduced into fresh seawater samples taken near Seward, Alaska. Initial rates of oxidation by the indigenous bacteria were linear and faster than the rates of toluene oxidation. Turnover times were 4 to 19 days. Autoradiographic measurements with 3 H-terpenes indicated that at least 10% of the 0.6 � 10 9 to 2.7 � 10 9 bacteria per liter present could catabolize terpenes. The rate of terpene oxidation, 24 μg of terpenes per g of cells per h with 3 μg of terpenes added per liter, was a constant function of bacterial biomass. The specific affinity of the process was estimated to be between 8.1 and 81 liters/g of cells per h, indicating a high state of induction and the probable presence of terpenes. Terpene-oxidizing bacteria were grown on [ 14 C]alanine and added to fresh seawater samples. Transfer of the bacterial radioactivity into larger particles at a rate of 146 pg/liter per h from the 2.3 � 10 9 organisms added indicated that any terpenes present would participate in the food chain.

The two primary kinetic constants for describing the concentration dependency of nutrient uptake by microorganisms are shown to be maximal rate of substrate uptake and, rather than the Michaelis constant for transport, specific affinity. Of the two, the specific affinity is more important for describing natural aquatic microbial processes because it can be used independently at small substrate concentrations. Flow cytometry was used to evaluate specific affinities in natural populations of aquatic bacteria because it gives a convenient measure of biomass, which is an essential measurement in the specific-affinity approach to microbial kinetics. Total biomass, biomass in various filter fractions, and the specific affinity of the bacteria in each fraction were determined in samples from a near-arctic lake. The partial growth rate of the pelagic bacteria from the 25 micrograms/liter of dissolved amino acids present (growth rate from the amino acid fraction alone) was determined to be 0.78 per day. By measuring activity in screened and whole-system populations, the biomass of the bacteria associated with particles was computed to be 427 micrograms/liter.