Release of Rhizobium spp. from Tropical Soils and Recovery for Immunofluorescence Enumeration

Limitations associated with immunofluorescence enumeration of bacteria in soil derive largely from the efficiency with which cells can be separated from soil particles and collected on membrane filters for staining. Many tropical soils fix added bacteria tightly, resulting in low recoveries. Eight soils, representative of three of the major soil orders found in the tropics (oxisols, vertisols, and inceptisols), were tested for recovery of added Rhizobium strains. All except one Hawaiian andept (Typic Eutrandept) yielded recoveries ranging from <1 to 13%. Recovery from the andept was 100%. In soil-sand mixtures, addition of only a small amount of soil caused a dramatic decrease in recovery of added rhizobia. Increasing the soil content of the mixture from 0% (10 g of sand) to 50% (5 g of soil-5 g of sand) reduced recoveries from >90 to <1%. Varying the ionic strength and pH of the extracting solution did not cause marked increases in recovery. Protein solutions, ethylenediaminetetraacetate, and NaHCO₃, on the other hand, improved release of bacteria. We report a modification to the usual membrane filter immunofluorescence procedure which yielded consistently high and reproducible recovery (coefficient of variation, 30%) of rhizobia from several tropical soils. In the modified procedure, partially hydrolyzed gelatin, diluted in ammonium phosphate, was used to suspend the soil. This caused dispersion of the soil and release of the bacteria from soil flocs. The efficiency of recovery of Rhizobium spp. from several tropical and two temperate soils remained high as the content of these soils in soil-sand mixtures was increased from 0 to 100%. The modified membrane filter immunofluorescence procedure was used to follow the growth of a strain of chickpea (Cicer arietinum) Rhizobium in a sterilized oxisol. The results showed a close agreement with viable counts at different stages during the growth cycle. Diluent for the hydrolyzed gelatin also had a marked effect on recovery. The efficiency of release of Rhizobium spp. from an oxisol was in the following order for the diluents used: 0.1 M (NH₄)₂HPO₄ > 0.1 M Na₂HPO₄ = 0.1 M sodium-phosphate-buffered saline (pH 7.2) > 0.2 M NH₄Cl > 0.2 KCl > NaCl = LiCl > water.     

Fertilizer nitrogen budget of Na15NO3 and (15NH4)2SO4 split-applied to winter wheat in microplots on a loam soil

Labelled fertilizer N applied to winter wheat as Na¹⁵NO₃ and (¹⁵NH₄)₂SO₄ at a total N dressing of 100kg ha⁻¹ was used in a microplot balance study to investigate the fate of each split fraction at three growth stages: end of tillering, heading and beginning of flowering. Results indicated that while the percentage utilization of the applied N by the grain and total crop increased considerably from the first to the third split application, these values diminished steadily in the straw. Grain recovery values for the first, second and third split applications were 34.2%, 51.5% and 55.7% for the NO₃ and 32.3%, 48.4% and 52.5% for the NH₄ carrier, respectively. The corresponding recovery values for the whole plant were 54.6%, 67.8% and 69.9% for the NO₃ and 51.7%, 63.5% and 66.1% for the NH₄ carrier. A greater proportion of the fertilizer N applied at the end of tillering stage was found in the vegetative plant components as compared with the grain. The reverse occurred for the N applied at the heading and at the beginning of the flowering stages. The residual fertilizer N found in the soil amounted to 18.0%, 10.4% and 11.6% of the applied NO₃−N and to 22.5%, 12.7% and 15.2% of the applied NH₄−N for the respective split applications. No differences were found for each split application between the two carriers as far as the unaccounted fertilizer N was concerned. The losses were 26.6%, 22.3% and 18.6% of the applied N for the three split applications, respectively. The application of fertilizer N did not lead to any increase in soil N uptake by the crop.     

Light dependency of the photosynthetic recovery of Nostoc flagelliforme

PS II photochemical efficiency (F_v/F_m) of Nostoc flagelliforme was examined after rewetting in order to investigate the light-dependency of its photosynthetic recovery. F_v/F_m was not detected in the dark, but was immediately recognized in the light. Different levels of light irradiation (4, 40 and 400 μmol photon m² s^-1) displayed different effects on the recovery process of photosynthesis. The intermediate level led to the best recovery of photochemical efficiency; the low light required longer and the high light inhibited the extent of the recovered efficiency. It was concluded that the photosynthetic recovery of N. flagelliforme is both light-dependent and influenced by photon flux density. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sorption of phosphorus by the iron oxide-impregnated filter paper (Pi soil test) embedded in soils

Incubation experiments were carried out to evaluate the feasibility of extracting phosphorus from soil by embedding iron oxide-impregnanted filter paper strips (P_i strips) in soils having a wide range in pH, texture, and extractable-P contents. Under flooded conditions, the amount of P extracted by the P_i strips increased with the period of submergence and embedding time of the P_i strips. Under unsaturated conditions, the P_i strips were found to extract P from soils over a wide range in moisture conditions; however, keeping the soil at moisture level between saturation and field capacity was found to result in maximal sorption of P by the strips. An embedding time of 16 h was found to be adequate.Phosphorus extracted by embedding P_i strips in soil columns for 16 h at field capacity moisture level correlated significantly with P extracted by shaking the soil with 0.01 M CaCl₂ solution and a P_i strip for 16 h in the laboratory (r=0.94^**). The P extracted by embedding P_i strips correlated best with Bray 1 P in acid soils (r=0.97^**) and with Olsen P in alkaline and calcareous soils (r=0.96^**). The results of the studies demonstrate the feasibility of developing a nondestructive method of monitoring changes in plant-available P in situ under field conditions.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr_Z) and side-path electron donors (Car/Chl_Z/Cyt_b559) can be oxidized by P₆₈₀⁺ at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S₁Tyr_Z EPR signal were independent of the treatment of K₃Fe(CN)₆, whereas formation and decay of the Car⁺/Chl_Z⁺ EPR signal correlated with the reduction and recovery of the Fe³⁺ EPR signal of the non-heme iron in K₃Fe(CN)₆ pre-treated PSII, respectively. Based on the observed correlation between Car/Chl_Z oxidation and Fe³⁺ reduction, the oxidation of non-heme iron by K₃Fe(CN)₆ at 0 °C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe³⁺ EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr_Z oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr_Z oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K₃Fe(CN)₆ takes place only in inactive PSII, which implies that the Fe³⁺ state is probably not the intermediate species for the turnover of quinone reduction.     

A new method for yeast recovery in batch ethanol fermentations: Filter aid filtration followed by separation of yeast from filter aid using hydrocyclones

In the Melle-Boinot process for alcohol production, centrifuges are normally used for yeast recovery at the end of a batch fermentation. Centrifuges are expensive equipment and represent an impressive part of the equipment costs in alcohol industries. In the present work, an alternative method for yeast recovery using less expensive equipment was studied. Instead of using centrifuges, yeast was separated from the fermented broth by filter aid filtration, followed by separation of yeast from the filter aid using hydrocyclones. A stainless steel plate-and-frame filter of filtration area 1.14 m² and two 30 mm hydrocyclones, which followed the Bradley and Rietema recommended proportions, were used in this work. The filter aid was perlite. Tests of direct separation of yeast from the fermented broth using the Bradley hydrocyclone proved to be completely unfeasible, since the maximal reduced total efficiency obtained was only 1%. When the hydrocyclones were used to separate perlite from the resuspended filtration cake, the perlite total separation efficiency obtained in the underflow was as high as 95% when using the Bradley hydrocyclone with an underflow diameter of 3 mm. To show the feasibility of the proposed new method of yeast recovery, a complete cycle of experiments, which included fermentation, yeast separation, and new fermentation using the recycled cells, was performed with good results.     

Cryopreservation of primate embryonic stem cells with chemically-defined solution without Me2SO

Human embryonic stem (hES) cells are expected to be useful in the fields of regenerative medicine and tissue engineering due to their pluripotency. Therefore, it is necessary to establish highly efficient and reliable methods for the cryopreservation of hES cells. We have cryopreserved cynomolgus and human ES cells by the vitrification method, using a chemically-defined dimethyl sulfoxide (Me₂SO)-free and serum-free medium composed of Euro-Collins solution as a base medium and 40% (v/v) ethylene glycol (EG) and 10% (w/v) polyethylene glycol (PEG) as cryoprotectants. When the vitrification and the cryoprotectants were combined, the recovery ratio of hES cells was 22.9 ± 7.7%, compared to 0.4 ± 0.2% when the conventional slow-freezing method was used. After the cryopreservation and thawing cycle, hES cells were easily cultured and expressed undifferentiated cell markers such as Nanog, Oct-4, SSEA-4, and alkaline phosphatase activity after several subculturing steps. We also found that the pluripotency of hES cells was maintained, as demonstrated by teratoma formation of ES cells transplanted into severe combined immunodeficient (SCID) mice. Thus, we conclude that we have successfully cryopreserved primate ES cells with high efficiency using a Me₂SO-free, chemically-defined medium.     

Alcian Blue and Colloidal Iron Staining Methods Adapted to Filter Paper Electrophoresis

Filter paper moistened with solutions used in electro-chromatography was spotted with 0.5-1.0μl of solutions of mucopolysaccharides and allowed to air dry. Substances tested with respect to their staining reaction were as follows: (a) From commercial sources: hyaluronic acid, heparin, chondroitin sulfate, ovomucoid and gastric mucin, (b) From natural sources: blood serum, saliva, tears, vitreous filtrate and aqueous humor. Alcian blue was found to be a good general stain for mucopolysaccharides and for locating such material on filter paper, especially when more specific means were used subsequently for identifying the kind of mucopolysaccharide present. Staining by colloidal iron of materials on filter paper was similar to that by the periodic acid-Schiff reaction. However, heparin and chondroitin sulfate were not stained by iron when on filter paper but were stained when placed on glass slides.     

Radioimmunoassay of estrone conjugates from urine dried on filter paper

Hormonal analysis of urine from free-ranging primates has been limited due to the difficulty of preserving samples under field conditions. Drying urine on filter paper is an alternative for field preservation. This study describes a new laboratory method for eluting steroids from filter paper with methanol, along with a series of experiments used to develop and validate this method. The overall elution recovery of estrone sulfate (ES) from filter paper was 86.4%. Estrone conjugate (E1C) levels in humans and captive orangutans were analyzed by radioimmunoassay (RIA). Values from samples dried on filter paper were significantly correlated with values from matched frozen samples, with elution efficiencies ranging between 97.1% and 102.4%. Creatinine (Cr) measurements from frozen urine compared to urine dried on filter paper were also significantly correlated (r=0.96) with an elution efficiency of 101.7%. After the samples were stored for 2 years, the absolute values of E1C and Cr were significantly lower but were still significantly correlated with frozen urine values. These data demonstrate the effectiveness of filter paper as a medium for preserving urinary steroid samples, and the efficiency of methanol as a solvent for eluting E1C and Cr. This method thus provides a viable alternative to the traditional procedure of freezing urine for field studies, where freezers are not available.     

Scavenging of free radicals in gas-phase mainstream cigarette smoke by immobilized catalase at filter level

Catalase is well known as capable of inducing the decomposition of H₂O₂. In this study, a kind of immobilized catalase (entrapped in cross-linked chitosan beads) was dispersed in conventional acetate filter as an antioxidant additive. Quantitative estimation of the free radicals in mainstream cigarette smoke (MCS) was performed to address the effect of this modified filter. It was found that the levels of PBN adduct and NO^?/NO₂^? associated with the gas-phase mainstream cigarette smoke (GPCS) were efficiently decreased by ～40% through catalase filtering. Besides, the modified filter was found to lower the MCS-induced adverse biological effects including lipid peroxidation and mutagenicity. This was proved to be substantially attributed to the catalase-dependent breakdown of NO^?, which was stimulated by some of peroxides (most probably being H₂O₂), the dismutation products of tar particulate matters (TPM). These results highlighted a promising approach to reduce the smoking-associated health risks to passive smokers. Moreover, the mechanisms of catalase filtering may be helpful for the development of appropriate immobilized enzyme systems to be applied for reducing health risks associated with gaseous pollutants.     

Optimization of H2SO4-catalyzed hydrothermal pretreatment of rapeseed straw for bioconversion to ethanol: Focusing on pretreatment at high solids content

A central composite design of response surface method was used to optimize H₂SO₄-catalyzed hydrothermal pretreatment of rapeseed straw, in respect to acid concentration (0.5–2%), treatment time (5–20 min) and solid content (10–20%) at 180 °C. Enzymatic hydrolysis and fermentation were also measured to evaluate the optimal pretreatment conditions for maximizing ethanol production. The results showed that acid concentration and treatment time were more significant than solid content for optimization of xylose release and cellulose recovery. Pretreatment with 1% sulfuric acid and 20% solid content for 10 min at 180 °C was found to be the most optimal condition for pretreatment of rapeseed straw for ethanol production. After pretreatment at the optimal condition and enzymatic hydrolysis, 75.12% total xylan and 63.17% total glucan were converted to xylose and glucose, respectively. Finally, 66.79% of theoretical ethanol yielded after fermentation.     

A simple and rapid harvesting method for microalgae by in situ magnetic separation

A simple and rapid harvesting method by in situ magnetic separation with naked Fe₃O₄ nanoparticles has been developed for the microalgal recovery of Botryococcus braunii and Chlorella ellipsoidea. After adding the magnetic particles to the microalgal culture broth, the microalgal cells were adsorbed and then separated by an external magnetic field. The maximal recovery efficiency reached more than 98% for both microalgae at a stirring speed of 120 r/min within 1 min, and the maximal adsorption capacity of these Fe₃O₄ nanoparticles reached 55.9 mg-dry biomass/mg-particles for B. braunii and 5.83 mg-dry biomass/mg-particles for C. ellipsoidea. Appropriate pH value and high nanoparticle dose were favorable to the microalgae recovery, and the adsorption mechanism between the naked Fe₃O₄ nanoparticles and the microalgal cells was mainly due to the electrostatic attraction. The developed in situ magnetic separation technology provides a great potential for saving time and energy associated with improving microalgal harvesting.     

Third instar nymphs of Rhodnius prolixus exposed to alpha-cyanopyrethroids: from hyperactivity to death

The hyperactivity, incoordination, recovery, and mortality produced by four alpha-cyanopyrethroids usually used for Chagas disease vector control (beta-cypermethrin, beta-cyfluthrin, lambda-cyhalothrin, and deltamethrin) were evaluated on third instar nymphs of Rhodnius prolixus. All pyrethroids modified the locomotor activity of the nymphs, which increased linearly as a function of the log of insecticide concentration. lambda-Cyhalothrin showed the lowest values of Effective Concentration 50%, Lethal Concentration 50%, Effective Time 50%, and Lethal Time 50% when insecticides were applied by contact with treated filter papers. Recovery from incoordination was observed after topical application of the insecticides. The recovery was inhibited by the simultaneous application of piperonyl butoxide, suggesting that biotransformation by mixed-function microsomal oxidases is involved in the process of recovery.     

Wastewater treatment efficiency of a multi-media biological aerated filter (MBAF) containing clinoptilolite and bioceramsite in a brick-wall embedded design

A multi-media biological aerated filter (MBAF) with clinoptilolite media was used to treat synthetic wastewater. Coal ash bioceramsite with supplemental metallic iron was added to the clinoptilolite media of MBAFs in a brick-wall embedded design. Performance parameters, such as hydraulic, organic, N and P loading capacity and microbial community composition were studied for different quantity of supplemental metallic iron contained in three MBAFs. The MBAFs with more metallic iron were found to have superior hydraulic and organic loading, and higher N and P capacities. COD, NH₃-N and TP removal dropped by 7-10%, 6-7% and 4-5%, respectively, with when hydraulic loading was raised from 2.8 to 7.5 m³ m⁻² d⁻¹. NH₃-N removal also decreased 8-9% when ammonia loading was elevated from 0.078 to 0.156 kg NH₃-N m⁻³ d⁻¹. Real-time PCR revealed a relatively stable bacterial community composed primarily of eubacteria that formed after an initial 120 d operational period. Doubling the amount of metallic iron in the bioceramsite media resulted in a twofold increase of eubacteria in the MBAF, but a decrease in the ratio of anaerobic ammonia-oxidizing bacteria to total bacteria.     

Relationship between availability indices and plant uptake of nitrogen and phosphorus from organic products

A better knowledge of the plant-availability of nitrogen (N) and phosphorus (P) in organic products may help to improve the efficient use of these products as fertilizers. In the present study, availability indices for N and P of nine widely differing organic products obtained by different fractionation methods were compared with the plant uptake of N and P from these products. The fractionation methods included CaCl₂ extraction, thermal fractionation (heating of organic products), and pepsin extraction, for N, and extraction with diluted sulphuric acid, P-Bray-I, P-Olsen, and extraction using an iron oxide coated filter paper, for P. The results of pot experiments with ryegrass using a double-pot technique (Janssen, 1990) over 62 (N experiment) and 93 days (P experiment) were used as reference for plant-availabe N and P. The 0.01 M CaCl₂ extractable inorganic N reasonably predicted plant-available N only in organic products with a high inorganic N fraction. Thermal fractionation and pepsin extraction provided a reasonable index for mineralizable N in organic products having a high fraction of mineralizable N. Of the P fractionation methods, the extraction using iron oxide coated filter paper was the best indicator of plant-available P in the products.     

Assessment of Methods for Detection of Infectious Cryptosporidium Oocysts and Giardia Cysts in Reclaimed Effluents

This study evaluates the occurrence of Cryptosporidium oocysts and Giardia cysts in reclaimed effluents if method 1623 with the Envirochek capsule filters (standard and high-volume [HV] filters) and a modified version of the Information Collection Rule method (ICR) with the polypropylene yarn-wound cartridge filter are used. The recovery efficiency of the analytical methods was evaluated with samples of reagent, tap, and reclaimed water by using flow cytometer-sorted spike suspensions. (Oo)cyst recovery efficiency determined filter performance and method reproducibility in the water matrix tested. Method 1623 with the Envirochek HV capsule filter generated significantly higher recovery rates than did the standard Envirochek filter and the modified ICR method. Notwithstanding, large variations in recovery rates (>80%) occurred with samples of reclaimed water, and none of the water quality parameters analyzed in the reclaimed effluents could explain such variability. The highest concentrations of indigenous oocysts were detected by method 1623 with the HV filter, which provided a sufficient number of oocysts for further confirmation of infectious potential. Confirmation of species and potential infectivity for all positive protozoan samples was made by using a nested PCR restriction fragment polymorphism assay and the focus detection method most-probable-number assay, respectively. The methodology and results described in the present investigation provide useful information for the establishment of pathogen numeric standards for reclaimed effluents used for unrestricted irrigation.     

The effect of manganese oxides and manganese ion on growth and siderophore production by Azotobacter vinelandii

The addition of manganese oxides to iron-limited medium promoted the formation of the pyoverdin siderophore azotobactin by Azotobacter vinelandii. When active-MnO₂ was used, there was greatly decreased iron uptake into the cells, hyperproduction of azotobactin and the abiotic, chemical destruction or adsorbtion of the catechol siderophores azotochelin and aminochelin by this strong oxidizing agent. Although the iron content of the cells was the same as iron-limited cells, the growth of cells in medium with active-MnO₂ was increased 1.5- to 2.5-fold over iron-limited controls. This growth promotion was not caused by iron contaminating the oxide or by manganese solubilized from the oxide. Soluble 0.05–4 mm Mn²⁺ inhibited the growth of iron-limited cells and had a minimal effect on iron uptake, but caused hyperproduction of azotobactin. This later effect was caused by the inhibition of soluble ferric reductase, in a manner identical to that previously observed for Zn²⁺. These results suggest that active-MnO₂ may interfere with a surface-localized iron uptake site, possibly another ferric reductase. The reason for the growth promotion by active-MnO₂ remains unknown, but is most likely related to decreased oxygen toxicity.     

Corn stover pretreatment by inorganic salts and its effects on hemicellulose and cellulose degradation

Inorganic salts, NaCl, KCl, CaCl₂, MgCl₂, FeCl₂, FeSO₄, FeCl₃, and Fe₂(SO₄)₃, were studied as catalysts for the degradation of hemicellulose in corn stover. FeCl₃ significantly increased the hemicellulose degradation in aqueous solutions heated between 140 and 200 °C with high xylose recovery and low cellulose removal, amounting to 90% and <10%, respectively. Hemicellulose removal increased 11-fold when the corn stover was pretreated with 0.1 M FeCl₃ compared to pretreatment with hot water under otherwise the same conditions, which was also 6-fold greater than pretreatment with dilute sulfuric acid at the same pH. Optimum pretreatment conditions were found where the corn stover was pretreated with 0.1 M FeCl₃ at 140 °C for 20 min. Under such conditions, 91% of hemicellulose was removed, and the recovery of monomeric and oligomeric xylose in liquid fraction achieved 89%, meanwhile, only 9% of cellulose was removed.     

A kinetic study on iron stimulation of the xanthine oxidase dependent oxidation of ascorbate

Xanthine oxidase reduces molecular oxygen to H₂O₂ and superoxide radicals during its catalytic action on xanthine, hypoxanthine or acetaldehyde. Ascorbate is catalytically oxidized by the superoxide radicals generated, when present in the reaction solution (Nishikimi 1975). The present study shows that iron ions markedly stimulate the enzyme dependent ascorbate oxidation, by acting as a red/ox-cycling intermediate between the oxidase and ascorbate. An apparent K_m-value of 10.8 M characterized the iron stimulatory effect on the reaction at pH 6.0. Reduced transition-state metals can be oxidized by H₂O₂ through a Fenton-type reaction. Catalase was found to reduce the effect of iron on the enzyme dependent ascorbate oxidation, strongly suggesting that H₂O₂, produced during catalysis, is involved in the oxidation of ferrous ions.     

Iron-induced changes in light harvesting and photochemical energy conversion processes in eukaryotic marine algae

The role of iron in regulating light harvesting and photochemical energy conversion processes was examined in the marine unicellular chlorophyte Dunaliella tertiolecta and the marine diatom Phaeodactylum tricornutum. In both species, iron limitation led to a reduction in cellular chlorophyll concentrations, but an increase in the in vivo, chlorophyll-specific, optical absorption cross-sections. Moreover, the absorption cross-section of photosystem II, a measure of the photon target area of the traps, was higher in iron-limited cells and decreased rapidly following iron addition. Iron-limited cells exhibited reduced variable/maximum fluorescence ratios and a reduced fluorescence per unit absorption at all wave-lengths between 400 and 575 nm. Following iron addition, variable/maximum fluorescence ratios increased rapidly, reaching 90% of the maximum within 18 to 25 h. Thus, although more light was absorbed per unit of chlorophyll, iron limitation reduced the transfer efficiency of excitation energy in photosystem II. The half-time for the oxidation of primary electron acceptor of photosystem II, calculated from the kinetics of decay of variable maximum fluorescence, increased 2-fold under iron limitation. Quantitative analysis of western blots revealed that cytochrome f and subunit IV (the plastoquinone-docking protein) of the cytochrome b₆/f complex were also significantly reduced by lack of iron; recovery from iron limitation was completely inhibited by either cycloheximide or chloramphenicol. The recovery of maximum photosynthetic energy conversion efficiency occurs in three stages: (a) a rapid (3-5 h) increase in electron transfer rates on the acceptor side of photosystem II correlated with de novo synthesis of the cytochrome b₆/f complex; (b) an increase (10-15 h) in the quantum efficiency correlated with an increase in D1 accumulation; and (c) a slow (>18 h) increase in chlorophyll levels accompanied by an increase in the efficiency of energy transfer from the light-harvesting chlorophyll proteins to the reaction centers.