Modification and field testing of a portable gas chromatograph forin situ acetylene reduction activity assay of tropical soils

Summary Small modifications of the Mallard/Holfeld/LaRue GC allow indirect measurement of biological N₂-fixation in tropical soils even in remote locations. Examples of ARA from Philippine rice fields are given.     

In situ plant water balance studies using a portable NMR spectrometer

A portable ¹H NMR spectrometer has been applied to whole plantsin situ, i.e. in climate rooms and in a greenhouse, to studyplant water relations under these conditions. The spectrometerconsists of a 30 kg permanent magnet system of 0.235 T, modifiedBruker Minispec electronics, and a standard XT pc for spectrometercontrol. Unattended, the system automatically measures the xylemsap stream and tissue water content in a well-defined sectionof the plant stem. Because of the small size of the magnet thesemeasurements can be made at different positions on the plantstem or on different plants in succession. Magnetic field driftdue to the varying climates that occur in greenhouses was correctedby field locking. Results are presented for a single plant ina climate room, demonstrating a method for the study of plantwater hydraulics. In addition, a single plant in a greenhousecrop was measured, and the NMR results were compared with thewater uptake and transpiration rates under (natural) variationof light intensity and relative humidity, demonstrating thereliability of the portable NMR under realistic greenhouse conditions.Finally, the application for the measurement of the root wateruptake efficiency is demonstrated for a number of grafted cucumberplants under constant climatic conditions in a phytotron. Key words: Portable NMR system, plant water balance sensor, greenhouse applications, flow, water content     

In situ acetylene-ethylene assay of biological nitrogen fixation in lowland rice soils

Summary An in situ device for assaying biological nitrogen fixation in flooded rice soils, using the acetylene reduction method, was developed. Diurnal variations in acetylene reduction by an inoculated field plot and by laboratory-grown cultures of nitrogen-fixing algae showed a prominent single-peak pattern of nitrogenase activity. The peak occurred at mid-day for laboratory-grown algae and at late afternoon for the algae grown in the field plot. Some nitrogenase activity was noted during the night. Acetylene reduction studies in rice fields of Albay province, Philippines, showed an estimated fixation of 18.5 to 33.3 kg N/ha each cropping season for the fields of Puro soil and 2.3 to 5.7 kg N/ha each cropping season for the fields of Santo Domingo soil. re]19751202     

Automatic on-line fermentation headspace gas analysis using a computer-controlled gas chromatograph

A computer-controlled headspace gas chromatograph was used to monitor the progress of ethanol production from both aerobic batch and anaerobic continuous fermentations. Using an automatic, electropneumatic sampling system, aliquots of fermentation headspace gas were injected directly onto the column for quantitative ethanol determinations every six minutes. A sample volume of 1 mL permitted liquid ethanol concentrations from 2 to 100 g/L to be measured with better than 3% standard deviation on five repeated injections. Provided fermenter liquid temperature and ionic strength were maintained constant, the signal-tohyphen;concentration ratio remained linear to 80 g/L ethanol. This quantitative gas chromatographic (GC) method is suitable for accurate, precise analysis of multiple solvent fermentations, and is limited only by the elution rate and separating capacity of the GC column.     

In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity

We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.     

In situ fixation of grape berries

Summary Usual immersion protocols in aldehyde solutions fail to fully preserve the fine structure of both exocarp and mesocarp cells of grape berries, especially for theveraison (onset of ripening) and post-veraison stages. In exocarp cells, fixative diffusion is hampered by the thick polysaccharide cell walls. In mesocarp cells, plasma membrane and tonoplast are disrupted before aldehyde crosslinking occurs, owing to the high osmotic pressure and cell wall texture. The fixative was therefore injected under pressure as small droplets in the outer and inner parts of the fruit, with limited changes in the steady-state organization of fruit tissues. Compared to a selective range of immersion protocols, a striking improvement in cell preservation was observed for all berry tissues, allowing new information on various compartments of grape berry cells. The preservation of organ integrity and local concentration of aldehyde molecules are the most critical parameters of improved fixation. This technique may be applicable to a large array of fleshy fruits containing mainly cells comprising a high volumetric proportion of vacuoles accumulating large amounts of organic acids and sugars and bounded by thick-walled exocarp cells.     

In situ nitrogen fixation associated with seagrasses in the Gulf of Elat (Red Sea)

In situ nitrogen fixation associated with the seagrass Halophila stipulacea, at the northern Gulf of Elat (Red Sea), is eight to ten times higher than that of nearby plant-free areas. A daily cycle of nitrogen fixation is evident, with rates during the day being seven times greater than during the night. Removal of seagrass leaves only from a patch within a seagrass bed gradually decreases nitrogen fixation activity, reaching the rates of plant-free areas after ten hours. A method devised for the in situ measurement of nitrogen fixation rates using belljars is described in detail. Nitrogen fixation rates in situ are higher than in the laboratory and lack the lag period typical to laboratory measurements. In laboratory experiments using intact plant samples, glucose enhances nitrogen fixation rates both in light and dark. Photosystem II inhibitor (3-3,4-dichloro-phenyl-1,1-dimethylurea) doubles nitrogen fixation rates in light. Both field and laboratory results indicate that light is essential for nitrogen fixation activity in the H. stipulacea bed possibly through its effect on cyanobacterial population that occupy the aerobic niches of the phyllosphere and on photosynthetic Rhodospirillacean bacteria that inhabit the anaerobic ones. Nitrogen fixation rates evident in H. stipulacea beds in situ account for a considerable portion of the biomass production by the seagrasses. The dependence of high nitrogenase activity by the diazotrophs on the presence of the seagrasses indicates the great importance of the seagrass community to the nitrogen cycle in its highly oligotrophic surroundings of the Gulf of Elat.     

A portable flow chamber for in situ determination of benthic metabolism

1. Many stream ecologists are interested in determining the metabolic rates of benthic organisms, particularly those of production and respiration. It is often necessary to make these measurements on fresh material in the field at remote sites. Recirculating chambers are commonly used for this purpose.
2. A broad variety of recirculating chambers are described in the literature, but each design has inherent limitations. The most common are inability to control flow in the chamber and match it with external flow rates, and a lack of the power required to do this for extended periods. Alteration of spectral irradiance, temperature rise and elevated internal chamber pressures are additional limitations that have received little attention.
3. We have designed and constructed a flow chamber that eliminates some of these problems. The chamber utilizes a DC motor-driven propeller as an efficient recirculator (axial impeller), minimizing power requirements and it is constructed of UVB transparent acrylic to allow a full spectral complement of solar irradiance in the interior. Modular components allow the chamber to be taken apart quickly for cleaning and replacement of parts, making it more functional than some previous designs.
4. The axial impeller chamber was compared to a similar sized conventional chamber that had a small diameter return line and a high capacity centrifugal pump. The axial impeller chamber had less of a temperature rise during field incubations, lower power consumption and less internal pressure in the return line when producing equivalent water velocities.
5. The reported axial impeller design had relatively homogeneous velocity across the working section relative to other chambers and was capable of water velocities in excess of 1 m s^–1.     

Salt stress sensitivity of nitrogen fixation in Enterobacter agglomerans strains

Two strains 333 to 339 of Enterobacter agglomerans were selected in the present study to evaluate the response of increasing concentrations of NaCl on growth, N(2)-fixation, and nitrogenase activity/synthesis. E. agglomerans strains 333 and 339 showed optimum growth and acetylene-reducing activity with 0.5 to 1.0% NaCl in a nitrogen-free minimal medium (NFDM) with glucose, respectively, in 28 h incubation, although both strains displayed better growth and acetylene-reducing activity with 3.0% and 2.0% NaCl after 52 h and 100 h incubation periods than the 28 h culture did. Our experiments with shiftings of salt concentrations in NFDM medium indicated that a synthesis of nitrogenase enzyme was generally more sensitive to higher concentrations of NaCl than nitrogenase activity was.     

In situ nitrogen removal in phase-separate bioreactor landfill

The feasibility of in situ nitrogen removal in phase-separate bioreactor landfill was investigated. In the experiment, two sets of bioreactor landfill systems, namely conventional two-phase and in situ nitrogen removal bioreactor landfills, were operated. The in situ nitrogen removal bioreactor landfill (NBL) was comprised of a fresh-refuse filled reactor (NBLF), a methanogenic reactor (NBLM) and a nitrifying reactor (NBLN), while the two-phase bioreactor landfill (BL) used as control was comprised of a fresh-refuse filled reactor (BLF) and a methanogenic reactor (BLM). Furthermore, the methanogenic and nitrifying reactors used aged refuse as bulk agents. The results showed that in situ nitrogen removal was viable by phase-separation in the bioreactor landfill. In total 75.8 and 47.5 g of nitrogen were, respectively, removed from the NBL and the BL throughout the experiment. The methanogenic reactor used the aged refuse as medium was highly effective in removing organic matter from the fresh leachate. Furthermore, the aged refuse was also suitable to use as in situ nitrification medium. The degradation of fresh refuse was accelerated by denitrification in the initial stage (namely the initial hydrolyzing stage) despite being delayed by denitrification in a long-term operation.     

Accurate estimation of nitrogen concentration in deciduous tree leaves in a field study using a portable non-destructive nitrogen detector

A non-destructive nitrogen (N) detector [Agriexpert PPW-3000 (PPW-3000)] is a useful device for rapid and non-destructive measurement of leaf N content. However, some studies find a poor correlation between the PPW-3000 reading and the actual leaf N content; the R2 value of the approximate equation was low. To improve the accuracy of N estimation, we determined the approximate equation taking into account the leaf development stage (maturing and mature leaves) and leaf flush type (early and late leaves). For the leaf development stage, we determined approximate equations for maturing leaves (Ya), mature leaves (Yb), and "maturing+mature" leaves (Yc) in species having simultaneous leaf emergence. The resulting accuracy of Ya, Yb, and Yc was quite high. For leaf flush species, we determined approximate equations for early leaves (Y1), late leaves (Y2), and "early+late" leaves (Y3) in species having heterophyllous leaf emergence. The accuracy of Y1 and Y2 was relatively high, but that of Y3 was low. We conclude that, when using a PPW-3000, we can determine an approximate equation for maturing and mature leaves jointly, but should treat early and late leaves separately.     

Diurnal variation in algal acetylene reduction (nitrogen fixation) in situ

Diurnal variation in algal nitrogen fixation was studied in Lake Mendota, Wisconsin, during the summers of 1971 to 1973. Approximately two-thirds of the daily acetylene reduction in the surface decimeter occurred before noon. The decline in acetylene reduction (nmoles/liter·hr) near midday was partially because the algae relocated themselves at greater depths. However, acetylene reducing activity (nmoles per A₆₆₃ unit chlorophyll a per hour) also decreased as midday approached. Occasionally algae would resurface near the end of the day. On average, acetylene reduction (nmoles per liter per hour) was maximum at about 0900 Central standard time in the top decimeter, and acetylene reduction between 0830 and 0930 Central standard time represented 13% of the total daily acetylene reduction. Furthermore, acetylene reduction in the top decimeter, on average, represented 3.6% of the total acetylene reduction in the column. Calculation of the contribution by nitrogen fixation to a lake's fixed nitrogen budget is discussed.     

In situ determination of sulfide turnover rates in a meromictic alpine lake

A push-pull method, previously used in groundwater analyses, was successfully adapted for measuring sulfide turnover rates in situ at different depths in the meromictic Lake Cadagno. In the layer of phototrophic bacteria at about 12 m in depth net sulfide consumption was observed during the day, indicating active bacterial photosynthesis. During the night the sulfide turnover rates were positive, indicating a net sulfide production from the reduction of more-oxidized sulfur compounds. Because of lack of light, no photosynthesis takes place in the monimolimnion; thus, only sulfide formation is observed both during the day and the night. Sulfide turnover rates in the oxic mixolimnion were always positive as sulfide is spontaneously oxidized by oxygen and as the rates of sulfide oxidation depend on the oxygen concentrations present. Sulfide oxidation by chemolithotrophic bacteria may occur at the oxicline, but this cannot be distinguished from spontaneous chemical oxidation.     

Repression of nitrogen fixation in Klebsiella pneumoniae at high temperature

Clostridium perfringens 11268 CDR (Rifr Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C. perfringens 11268 (Rifr Tcr). High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid. The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 micrograms/ml) and 0.4 M sucrose. After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants. Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation. Transformation of L-phase variants of C. perfringens 11268 CDR (Rifr Tcs) was mediated by polyethylene glycol. Transformation frequency is a nonlinear function of DNA concentration. Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied. Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.     

Soybean genotypic differences in sensitivity of symbiotic nitrogen fixation to soil dehydration

Nitrogen fixation activity by soybean (Glycine max (L.) Merr.) nodules has been shown to be especially sensitive to soil dehydration. Specifically, nitrogen fixation rates have been found to decrease in response to soil dehydration preceding alterations in plant gas exchange rates. The objective of this research was to investigate possible genetic variation in the sensitivity of soybean cultivars for nitrogen fixation rates in response to soil drying. Field tests showed substantial variation among cultivars with Jackson and CNS showing the least sensitivity in nitrogen accumulation to soil drying. Glasshouse experiments confirmed a large divergence among cultivars in the nitrogen fixation response to drought. Nitrogen fixation in Jackson was again found to be tolerant of soil drying, but the other five cultivars tested, including CNS, were found to be intolerant. Experiments with CNS which induced localized soil drying around the nodules did not result in decreases in nitrogen fixation rates, but rather nitrogen fixation responded to drying of the entire rooting volume. The osmotic potential of nodules was found to decrease markedly upon soil drying. However, the decrease in nodule osmotic potential occurred after significant decreases in nitrogen fixation rates had already been observed. Overall, the results of this study indicate that important genetic variations for sensitivity of nitrogen fixation to soil drying exist in soybean, and that the variation may be useful in physiology and breeding studies.     

In situ oxygen microelectrode measurements of bottom-ice algal production in McMurdo Sound, Antarctica

In-situ estimates of fast-ice algal productivity at Cape Evans, McMurdo Sound, in 1999 were lower than at the same site in previous years. Under-ice irradiance was between 0 and 8 µmol photons m^-2 s^-1; the ice was between 1.9 and 2.0 m thick and the algal biomass averaged 150 mg chl a m^-2, although values as high as 378 mg chl a m^-2 were recorded. Production on 11 and 12 November was between 0.053 and 1.474 mg C m^-2 h^-1. When the data from 11 November were fitted to a hyperbolic tangent function, a multilinear regression gave estimates for P_max of 0.571 nmol O₂ cm^-2 s^-1, an ! of 0.167 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1 and an E_k of 3.419 µmol photons m^-2 s^-1. A P_max of 2.674 nmol O₂ cm^-2 s^-1, an ! of 0.275 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1, r of 0.305 nmol O₂ cm^-2 s^-1 and an E_k of 9.724 µmol^-1 photons m^-2 s^-1 were estimated from the 12 November data. The sea-ice algal community was principally comprised of Nitzschia stellata, Entomoneis kjellmanii and Berkeleya adeliensis. Other taxa present included N. lecointei, Fragilariopsis spp., Navicula glaciei, Pleurosigma spp. and Amphora spp. Variations in the method for estimating the thickness of the diffusive boundary layer were not found to significantly affect the measurements of oxygen flux. However, the inability to accurately measure fine-scale variations in biomass is thought to contribute to the scatter of the P versus E data.