Noninvasive Quantitative Measurement of Bacterial Growth in Porous Media under Unsaturated-Flow Conditions

Glucose-dependent growth of the luxCDABE reporter bacterium Pseudomonas fluorescens HK44 was monitored noninvasively in quartz sand under unsaturated-flow conditions within a 45- by 56- by 1-cm two-dimensional light transmission chamber. The spatial and temporal development of growth were mapped daily over 7 days by quantifying salicylate-induced bioluminescence. A nonlinear model relating the rate of increase in light emission after salicylate exposure to microbial density successfully predicted growth over 4 orders of magnitude (r² = 0.95). Total model-predicted growth agreed with growth calculated from the mass balance of the system by using previously established growth parameters of HK44 (predicted, 1.2 × 10¹² cells; calculated, 1.7 × 10¹² cells). Colonization expanded in all directions from the inoculation region, including upward migration against the liquid flow. Both the daily rate of expansion of the colonized zone and the population density of the first day's growth in each newly colonized region remained relatively constant throughout the experiment. Nonetheless, substantial growth continued to occur on subsequent days in the older regions of the colonized zone. The proportion of daily potential growth that remained within the chamber declined progressively between days 2 and 7 (from 97 to 13%). A densely populated, anoxic region developed in the interior of the colonized zone even though the sand was unsaturated and fresh growth medium continued to flow through the colonized zone. These data illustrate the potential of a light transmission chamber, bioluminescent bacteria, and sensitive digital camera technology to noninvasively study real-time hydrology-microbiology interactions associated with unsaturated flow in porous media.     

A model that uses the induction phase of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 to quantify cell density in translucent porous media.

A cooled charge-coupled device (CCD) camera was used to follow the kinetics of induction of lux gene-dependent bioluminescence in Pseudomonas fluorescens HK44 held either in aqueous suspensions minus sand, saturated or unsaturated translucent sand (0.348 and 0.07 cm(3) H(2)O/cm(3) of sand, respectively), and at cell densities ranging between 1 x 10(6) and 8.5 x 10(8) cells/ml. Before O(2) availability became a limiting factor, the rate of light emission (L) increased with the square of time (t) and linearly with increasing cell density (c). A nonlinear model was developed that contains a "rate of increase in light emission" constant, B', which is determined directly from the slope of a plot of radical L/c against t. The model predicted the behavior of lux induction in HK44 under a variety of conditions. Similar B' values were determined [49.0-57.6 x 10(-10) light units/(cell min(2))] for cell suspensions held in aqueous medium minus sand, in saturated or unsaturated 40/50 grade sand (0.36 mm grain diameter) and in two other textural classes of translucent sand. Although both the growth phase, and the presence of glucose during lux induction affected the first detectable time (FDT) of bioluminescence by HK44 in sand, the kinetics of induction of light emission were similar among treatments (stationary phase cells plus glucose, B'=61.6+/-3.2, log phase cells plus glucose, B'=63.2+/-7.2). The potential exists to use a combination of a CCD camera system, an inducible lux gene containing bioluminescent bacterium, and a light transmission chamber to nonintrusively visualize and quantify in real time the interactions between bacterial growth and unsaturated flow of water and solutes in porous media.     

Insertion of IS1 into the rpsE gene for ribosomal protein S5 causes cold-sensitivity in Escherichia coli

Summary The nucleotide sequence of the rpsE gene for the ribosomal protein S5 of a mutant of E. coli showing cold-sensitive growth revealed the presence of an insertion sequence, IS1, near the 3-end of the gene. This mutant grows very slowly even at the permissive temperature (30°C). At temperatures below 20°C, its growth becomes negligible. It is likely that the presence of IS1 disturbs the continued translation of the polycistronic messenger RNA of the spc-operon from the point of IS1 insertion downwards, especially at the unfavorable temperatures.     

Stomata from growth-chamber-grown Vicia faba have an enhanced sensitivity to CO2

Abaxial stomata from Vicia faba leaves grown in a growth chamber under constant light, temperature and humidity showed an elaborate pattern of aperture changes over the course of a light cycle. These aperture changes were tightly correlated with changes in chamber CO2 concentration (r2=0.83). Changes in chamber [CO2] resulted, in turn, from substantial daily fluctuations in ambient [CO2], typical of the Los Angeles environment, with a constant offset caused by photosynthesis and respiration of the plants within the chamber. The dominant role of the stomatal response to CO2 in the control of aperture was confirmed by manipulation of chamber [CO2]. Fast (15 min) increases and decreases in [CO2] caused rapid decreases and increases in aperture, while constant [CO2] resulted in constant aperture. In contrast, aperture changes in comparable plants grown under greenhouse conditions were tightly correlated with changes in incident solar radiation (r2=0.80), and poorly correlated with changes in [CO2] (r2=0.09). Greenhouse-grown plants transferred to growth chamber conditions showed no apparent response to CO2. These data indicate that growth-chamber-grown V. faba leaves provide an experimental system optimally suited for the study of the stomatal response to CO2, and suggest that acclimation to environmental conditions alters the sensitivity of stomata to CO2.     

Daily growth increments in otoliths of milkfish, Chanos chanos (Forsskål), larvae

The formation of daily growth increments of otoliths was studied in the reared larvae of milkfish. The first growth increment was formed during the yolk–sac reabsorption period c .2 days after hatching, and increment formation continued on a daily schedule regardless of growth rate. The initial incremental zone was of amorphous structure, and the subsequent incremental zone was of needle–shaped crystalline structure; the former structure was formed in the yolk–sac reabsorption period of the larva and the latter in the exogenous feeding period. Accordingly, ageing of milkfish larvae is possible by counting growth increments, and timings within the developmental stage of the larvae can be understood by examining the otolith microstructure.     

Plant-plant interactions vary with different mycorrhizal fungus species

Because different species of mycorrhizal fungi have different effects on the growth of particular plant species, variation in mycorrhizal fungus species composition could cause changes in the strength of plant-plant interactions. Results are presented from a growth chamber experiment that compared the strength of interactions among seedlings of ponderosa pine (Pinus ponderosa) when the pines were colonized by two different groups of ectomycorrhizal fungi in the genus Rhizopogon. Plant density effects differed between the two groups of mycorrhizal fungi: plant growth was low regardless of density when plants were colonized with pine-specific Rhizopogon species, while plant growth declined with plant density when plants were colonized by Rhizopogon species having a broader host range. This result parallels results from previous studies showing that plant interactions are more antagonistic with mycorrhizal fungi than without, implying that plant responsiveness to beneficial mycorrhizal fungi declines with increasing plant density. If such effects are prevalent in plant communities, then variation in mycorrhizal fungus community composition is predicted to have a density-dependent effect on plants.     

Changes of insulin binding in rat tissues after exposure to stress.

The effects of various stressors on insulin receptors in adipose, liver and skeletal muscle tissues were studied in rats exposed to acute or repeated stress. Adult male rats were exposed to immobilization (IMO) for 2.5 hours daily for 1, 7 and 42 days, or to hypokinesia (HK) for 1, 7 and 21 days. We determined the values of specific insulin binding (SIB) and insulin receptor binding capacity (IR) of plasma cell membranes from adipose, liver and muscle tissue (IMO groups), or insulin binding to isolated adipocytes and hepatocytes (HK groups). A significant decrease of SIB and IR was observed in rats exposed to acute stress (1x IMO) in muscle, adipose and liver tissues. However, in animals exposed to repeated stress (7x and 42x IMO), SIB and IR were diminished in the muscle tissue, whereas no significant changes were noted in the liver and adipose tissue. When tissue samples were collected 3-24 hours after exposure to IMO stress, no changes of SIB and IR were found in liver and adipose tissue, but insulin binding was lowered in skeletal muscles. In animals exposed to HK for one day, a decrease of SIB and IR was found in isolated adipocytes, but no changes in insulin binding were noted in the liver tissue. In rats exposed to HK for 7 and 21 days, values of IR were similar as in control group. Our results indicate a) the different changes of IR in the liver, fat and muscle tissues after exposure to stress situations, b) a long-term decrease of insulin binding in muscles of rats exposed to repeated IMO stress, and c) the return of reduced SIB and IR (induced by acute stress) to control values in the liver and adipose tissue after a short recovery period.     

Effects of the bryozoan Membranipora tuberculata (Bosc.) on the photosynthesis and growth of Gelidium rex Santelices et Abbott

Several species of Membranipora commonly occur on brown and red algae. It is currently believed that these bryozoans could affect growth of the host plant by modifying incident light. In the present study, we report on the changes that occur in both intensity and quality of light passing through colonies of M. tuberculata (Bosc.) and on the effects of bryozoans on growth of the host plant. M. tuberculata decreases photon-flux density by up to 56% of the incident light and slightly affects the quality of the incident light. Although the bryozoan reduces the photosynthetic rate of Gelidium rex Santelices et Abbott, it does not have a net effect on algal growth. It is concluded that the effects of epifauna on algal growth depend on the nature of the algal structure colonized. Therefore, it cannot be predicted a priori that encrusted thalli should grow at a slower rate than nonencrusted thalli.     

Inhibition of metastasis of syngeneic murine melanoma in vivo and vasculogenesis in vitro by monoclonal antibody C11C1 targeted to domain 5 of high molecular weight kininogen

Metastasis of malignant tumors is a major cause of morbidity and mortality. Inhibition of tumor growth in distant organs is of clinical importance. We have demonstrated that C11C1, a murine monoclonal antibody to the light chain region of high molecular weight kininogen (HK), reduces growth of murine multiple myeloma in normal mice and human colon cancer in nude mice. C11C1 inhibits angiogenesis by reducing tumor microvascular density by blocking binding of HK to endothelial cells. We now evaluate the anti-metastatic effect of C11C1 on C57BL/6 mouse lung metastatic model using B16F10 melanoma cells. The tail veins of mice were injected with 0.5 × 10⁶ cells of melanoma B16F10. One group received C11C1 and the other received saline (control) intraperitoneally. When mice were killed at 28 days, 6 of 10 control mice had detectable metastatic pulmonary nodules which stained positive with an antibody against S-100 protein, a tumor antigen present in malignant melanoma cells. In the C11C1 groups, none of the mice showed metastatic foci in their lungs. We showed that C11C1 inhibits endothelial cell tube formation in a 3-D collagen fibrinogen gel model by inhibiting the rate of cleavage of HK by plasma kallikrein without changing the binding affinity for HK. These studies demonstrate that a monoclonal antibody to HK has the potential to prevent metastasis with minimal side effects.     

Two-stage cultures for the production of astaxanthin from Haematococcus pluvialis

A two-stage culture system was established for the production of astaxanthin from Haematococcus pluvialis. In a first stage green vegetative cells were produced in semicontinuous cultures maintained with daily renewal rates between 10 and 40%. The steady-state cell density decreased with increasing renewal rates. Highest cell productivity, 64 x 10(6) cells l(-1) day(-1) was obtained with a daily renewal rate of 20%. In a second stage the harvested cultures were submitted to high light (240 micromol photon m(-2) s(-1)) under batch conditions for 15 days in order to stimulate the transition to the aplanospore stage and the accumulation of astaxanthin. No decrease in cell density was recorded during the induction period in any of the cultures. Cultures obtained at high renewal rates continued growing during the induction period and no astaxanthin was accumulated until all nitrogen in the media had been consumed. The final concentration of astaxanthin was inversely correlated to the growth rate at which first-stage cultures were maintained. Optimal renewal rate for maximal astaxanthin production depended on the duration of the induction period. After a 12-day induction period the highest astaxanthin production, 5.8 mg l(-1) of semi-continuous culture day -1, was obtained with cultures maintained at a renewal rate of 20%. When the induction period was increased to 15 days maximal astaxanthin productivity, 9.6 mg l(-1) of semi-continuous culture day -1, was obtained from cultures maintained at a renewal rate of 40% despite the much lower astaxanthin concentration achieved in these cultures. Results demonstrate the feasibility of semi-continuous cultivation of H. pluvialis for the two-stage production of astaxanthin.     

Growth rate,photosynthetic activity,and leaf development of Brazil pine seedlings (Araucaria angustifolia[Bert.] O. Ktze.)

Seedlings of Brazil pine, a large-seeded South American conifer, were grown in a climate chamber to investigate vertical growth pattern and the time course of leaf development. We examined shoot growth, photosynthetic performance and markers of leaf maturation such as contents of soluble sugars and activities of sucrose-phosphate synthase (SPS), neutral invertase (nI) and sucrose synthase (Susy). The daily increment of shoot length showed an optimum curve during the first 70 days after germination. The low growth rate during the first 20 days of development correlated with net CO² emission of the seedling. Analyses of leaf maturation markers in older seedlings revealed low sucrose/hexose ratios and high activities of nI and Susy in the uppermost leaves. Although the SPS/Susy ratio was low in these leaves the extractable SPS activity did not change significantly among leaves of different age. The photosynthetic light compensation points of young leaves were about 2-fold higher than those of mature leaves and their photosynthetic capacity was only 50% as high. Our results indicate that a rapid maturation of leaves of Brazil pine seedlings may reduce the respiratory loss of carbohydrates and that the mobilisation of seed storage compounds supports initial shoot growth under light-limiting conditions which may occur in the forest-grassland succession zone.     

A novel double-layered photobioreactor for simultaneous Haematococcus pluvialis cell growth and astaxanthin accumulation

This study proposes a novel double-region photobioreactor to simplify the commercial two-stage process of astaxanthin production by the cultivation of Haematococcus pluvialis. The feasibility of the double-region photobioreactor has been investigated and found to achieve high biomass yield in the inner core region and simultaneous astaxanthin accumulation in the outer jacket region. Among many environmental factors, light condition and nitrate level were manipulated for selective cell growth and astaxanthin production. In the outer jacket region, efficient astaxanthin production was accomplished by excessive irradiation (770+/-20 microE m(-2)s(-1)) and nitrate starvation, resulting in a dramatic increase of astaxanthin productivity (357 mg l(-1)). Meanwhile, attenuated light energy (40+/-3 microE m(-2)s(-1)) and sufficient nitrates were supplied to the vegetative cells in the inner core region, which continued to grow to a high cell concentration of 4.0 x 10(5) cells ml(-1). The sequential batch run was performed by utilizing the high-density vegetative cells as inoculum for the next batch run. The cultivation results exhibited similar trends as the previous run, reaching high cell density (4.3 x 10(5) cells ml(-1)) in the inner core region and high astaxanthin content (5.79% on a dry weight basis) in the outer jacket region. The present study indicates that the double-region photobioreactor and its method of operation possess a good potential for commercial production of astaxanthin by H. pluvialis.     

Establishment of Vesicular-Arbuscular Mycorrhizal Fungi and Other Microorganisms on a Beach Replenishment Site in Florida

Beach replenishment is a widely used method of controlling coastal erosion. To reduce erosional losses from wind, beach grasses are often planted on the replenishment sands. However, there is little information on the microbial populations in this material that may affect plant establishment and growth. The objectives of this research were to document changes in the populations of vesicular-arbuscular mycorrhizal (VAM) fungi and other soil microorganisms in replenishment materials and to determine whether roots of transplanted beach grasses become colonized by beneficial microbes. The study was conducted over a 2-year period on a replenishment project in northeastern Florida. Three sampling locations were established at 1-km intervals along the beach. Each location consisted of three plots: an established dune, replenishment sand planted with Uniola paniculata and Panicum sp., and replenishment sand left unplanted. Fungal and bacterial populations increased rapidly in the rhizosphere of beach grasses in the planted plots. However, no bacteria were recovered that could fix significant amounts of N₂. The VAM fungi established slowly on the transplanted grasses. Even after two growing seasons, levels of root colonization and sporulation were significantly below those found in the established dune. There was a shift in the dominant VAM fungi found in the planted zone with respect to those in the established dunes. The most abundant species recovered from the established dunes were Glomus deserticola, followed by Acaulospora scrobiculata and Scutellospora weresubiae. The VAM fungi that colonized the planted zone most rapidly were Glomus globiferum, followed by G. deserticola and Glomus aggregatum.     

Juvenile growth of Acipenser ruthenus and 4 different sturgeon hybrids

Juvenile growth of A. ruthenus and four different sturgeon hybrids ( Huso huso x A. ruthenus, A. baerii x A. ruthenus, A. gueldenstaedtii x A. ruthenus, A. gueldenstaedtii x H. huso ) was studied for 71 days applying two feeding regimes, offering a fry diet (3.5 % daily rate) for 26 days for the hybrids and 41 days for A. ruthenus and a fattening diet (3 % daily rate) for 45 days and 30 days respectively. Significant growth differences occured at the end of experiment: The hybrid A. gueldenstaedtii x H. huso showed the highest, A. ruthenus the lowest growth.     

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.     

The effect of a day at low irradiance of a maize crop. II. Photosynthesis, transpiration and respiration

The kinetics of daily photosynthesis, transpiration and dark respiration of shoots of maize plants ( Zea mays L.) were studied in a growth chamber. After one or two days of reduced light intensity, the photosynthesis and the transpiration returned immediately to a level higher than expected. Respiration, which was reduced by at least 50%, returned slowly for over 2 days towards the initial level. These results suggest the existence of reserves of assimilation products which are used up while maintaining the growth of the aerial part in spite of the lack of assimilation. The prolonged reduction of the respiration shows that it is not strongly correlated to photosyntheis or to growth but rather to the amount of available reserves of assimilate.     

Growth and Deposition of Inorganic Nutrient Elements in Developing Leaves of Zea mays L

Spatial distributions of growth and of the concentration of some inorganic nutrient elements were analyzed in developing leaves of maize (Zea mays L.). Growth was analyzed by pinprick experiments with numerical analysis to characterize fields of velocity and relative elemental elongation rate. Inductively coupled plasma and atomic emission spectroscopy were used to measure nutrients extracted from segments of leaf tissue collected by position. Leaves 7 and 8, both elongating 3 millimeters per hour had maximum relative elemental growth rates of 0.06 to 0.08 millimeters per hour with maximum rates 20 to 50 millimeters from the node and cessation of growth by 90 millimeters from the node. Spatial distribution of dry weight density revealed that the rate of biomass deposition was maximum in the most rapidly expanding region and continued beyond the elongation zone. The nutrient elements K, Cl, Ca, Mg, and P showed different distribution patterns of ion density (on a dry weight basis). K and Cl had minimal density in the leaf tips; K density was maximum in the growing region, whereas Cl density was maximum at the region of growth cessation. Ca, Mg, and P had relatively high densities at the base of the elongation zone near the node and also in the tip regions. Near the node, P and Mg densities were higher in the young, growing leaves, whereas Ca density near the node was higher in older leaves that had completed elongation. Deposition rates of all nutrients were greatest in the region of maximum elongation rate.     

Real time monitoring of biofilm development under flow conditions in porous media

Biofilm growth can impact the effectiveness of industrial processes that involve porous media. To better understand and characterize how biofilms develop and affect hydraulic properties in porous media, both spatial and temporal development of biofilms under flow conditions was investigated in a translucent porous medium by using Pseudomonas fluorescens HK44, a bacterial strain genetically engineered to luminesce in the presence of an induction agent. Real-time visualization of luminescent biofilm growth patterns under constant pressure conditions was captured using a CCD camera. Images obtained over 8 days revealed that variations in bioluminescence intensity could be correlated to biofilm cell density and hydraulic conductivity. These results were used to develop a real-time imaging method to study the dynamic behavior of biofilm evolution in a porous medium, thereby providing a new tool to investigate the impact of biological fouling in porous media under flow conditions.     

Development and replenishment of the P-depletion zone around the primary root of maize during the vegetation period

The dynamics of the development and replenishment of P-depletion zones around the primary root of maize (Zea mays L. cv ‘Garbo’) was studied during a vegetation period (80 days) under greenhouse conditions in a loamy sand of low P-availability. A recently described freeze-cutting technique was used to determine radial diffusion of labelled phosphate to the primary root. The development of the depletion zone was biphasic. In the initial phase after two days of growth of the primary root in a soil layer labelled with³³P a minimum of isotopically exchangeable P (EP) was observed which had decreased to about 30% of its original amount at the root surface. At that time the corresponding P-concentration in the soil solution was calculated to be as low as 5×10⁻⁷ M. The depletion zone had already spread 0.4 mm from the root surface. During the second phase, between the 10th and 20th day of plant growth the concentration of EP at the root surface increased slowly but did not change markedly. However, the depletion zone continued to spread and after the 20th day of growth reached its maximal diameter (1.07 mm from the root surface) but remained completely within the root hair cyclinder; the single root hairs never exceeded 1.14 mm in length. The biphasic growth of the depletion zone was probably caused by proton extrusion of the root tip. Acidification of the soil solution from pH 5.8 to about 3.9 results in an about 3-fold rise of the concentration of desorbed phosphate and might also have activated acidophilic P-translocators of the root during the initial phase. Anion over cation uptake normally prevailing during the later stage of root development might resulted in a rise of the soil pH within the root hair zone. Consequently P-availability, as well as P-uptake capacity declined, but P-uptake by the seminal root still continued until the 20th day. Subsequently, the P-concentration within the depletion zone increased again while simultaneously its extent was reduced until it was almost completely replenished after 60 days indicating a loss of P-uptake capacity of the primary root. Within the root tissue³³P was accumulated to about twice the concentration of that in the undepleted soils. This accumulation corresponded to periods of high uptake due to the development of root laterals. In the root cortex a high P-content was observed during the first 30 days of growth. At the onset of the reproductive stage of the plant the P-content of the shoot and especially in the developing seeds rose considerably at the cost of phosphate stored in the root cortex. The accumulation of³³P in the root tissue indicated that nutrient gain was mainly achieved during the early stages of plant development and that P was temporarily stored to some extent within the root system.