In situ bacterial selenate reduction in the agricultural drainage systems of western Nevada.

Dissimilatory in situ selenate reduction to elemental selenium in sediments from irrigated agricultural drainage regions of western Nevada was measured at ambient Se oxyanion concentrations. Selenate reduction was rapid, with turnover rate constants ranging from 0.04 to 1.8 h-1 at total Se concentrations in pore water of 13 to 455 nM. Estimates of removal rates of selenium oxyanions were 14.38, and 155 mumol m-2 day-1 for South Lead Lake, Massie Slough, and Hunter Drain, respectively.     

In situ stimulation of bacterial sulfate reduction in sulfate-limited freshwater lake sediments

Abstract By adding sulfate in the form of solid gypsum, it was possible to transform in situ a predominantly methanogenic sediment ecosystem into a sulfate-reducing one. The concentrations of sulfate, sulfide, methane, acetate, propionate, soluble iron, and manganese were determined in the porewater before and after the transition. Although sulfate was no longer limiting, acetate and propionate continued to accumulate and reached much higher concentrations than under sulfate-limited conditions. Metabolic activities of fermenting bacteria and of sulfate reducers, which belong to the group that incompletely oxidizes organic material, might be responsible for the increased production of volatile fatty acids. The elevated concentrations of soluble Fe(II)²⁺ and Mn(II)²⁺ observed in the porewater stem from iron and manganese compounds which may be reduced chemically by hydrogen sulfide and other microbially produced reducing agents or directly through increased activities of the iron and manganese reducing bacteria. In the horizon with high sulfate-reducing activities the methane concentrations in the porewater were lower than in non-stimulated sediment regions. The shape of the concentration depth profile indicates methane consumption through sulfate reducing processes. The in situ experiment demonstrates the response of a natural microbial ecosystem to fluctuations in the environmental conditions.     

In situ NMR systems

In situ NMR is becoming an established technology for applications in bioprocessing and metabolic engineering. The in situ NMR biosensor acts as a noninvasive pH, ion, and concentration meter, with 31P and 13C as the two main isotopes of study. A substantial data base now exists for phosphorus and carbon spectra of bacteria and yeast. In situ NMR can provide many of the state variables needed for modeling glycolytic pathway function. NMR micro-reactor technology has improved significantly in the last decade. Several designs for immobilized cell reactors have been tested, and in particular, considerable gains have been made in the feasibility of studying aerobic, chemostat cultures with in situ NMR. Acquisition of 31P spectra from cell suspensions of 3-5% v/v under controlled conditions can be made in 3-7 minute time resolution in several systems.     

Transformations of selenate and selenite by Stenotrophomonas maltophilia isolated from a seleniferous agricultural drainage pond sediment

A Gram-negative bacterium, identified as Stenotrophomonas maltophilia by fatty acid analysis and 16S rRNA sequencing, was isolated from a seleniferous agricultural evaporation pond sediment collected in the Tulare Lake Drainage District, California. In cultures exposed to the atmosphere, the organism reduces selenate (SeO4(2-)) and selenite (SeO3(2-)) to red amorphous elemental selenium (Se degrees ) only upon reaching stationary phase, when O2 levels are less than 0.1 mg l(-1). In 48 h, S. maltophilia removed 81.2% and 99.8% of added SeO4(2-) and SeO3(2-) (initial concentration of 0.5 mM), respectively, from solution. Anaerobic growth experiments revealed that the organism was incapable of using SeO4(2-), SeO3(2-), SO4(2-) or NO3- as a terminal electron acceptor. Transmission electron microscopy of cultures spiked with either Se oxyanion were found to contain spherical extracellular deposits. Analysis of the deposits by energy-dispersive X-ray spectroscopy revealed that they consist of Se. Furthermore, S. maltophilia was active in producing volatile alkylselenides when in the presence of SeO4(2-) and SeO3(2-). The volatile products were positively identified as dimethyl selenide (DMSe), dimethyl selenenyl sulphide (DMSeS) and dimethyl diselenide (DMDSe) by gas chromatography-mass spectrometry. Our findings suggest that this bacterium may contribute to the biogeochemical cycling of Se in seleniferous evaporation pond sediments and waters. This organism may also be potentially useful in a bioremediation scheme designed to treat seleniferous agricultural wastewater.     

In situ nutrient enrichment experiments with periphyton in agricultural streams

The abundance, composition and seasonal distribution of planktonic protozoans and physical and chemical variables such as temperature, dissolved oxygen content, pH, and chlorophyll a concentration were analyzed monthly from February 1985 to January 1986 in eutrophic Rio Grande Reservoir. Analyses were performed on water samples collected at the surface and bottom (0.5 m above the sediment) in three stations, and individual distribution profiles were constructed for the major species. Dominant protozoan taxa included Codonella cratera, Coleps amphacantus, Coleps hirtus, Halteria grandinella, Mesodinium pulex, Tintinnidium sp., Urotricha spp., Vorticella spp., and Difflugia sp.. The distribution of some species was probably related to temperature and dissolved oxygen content.     

In situ evidence for microdomains in the polymer matrix of bacterial microcolonies

Confocal laser scanning microscopy and fluorescent lectin-binding analyses (FLBA) were used to study the form, arrangement, and composition of exopolymeric substances (EPS) surrounding naturally occurring microcolonies in biofilms. FLBA, using multiple lectin staining and multichannel imaging, indicated that the EPS of many microcolonies exhibit distinct multiple binding regions. A common pattern in the microcolonies is a three zone arrangement with cell-associated, intercellular, and an outer layer of EPS covering the exterior of the colony. Differential binding of lectins suggests that there are differences in the glycoconjugate composition or their arrangement in the EPS of microcolonies. The combination of FLBA with fluorescent in situ hybridization (FISH) indicates that the colonies consist of the major groups, alpha- and beta-Proteobacteria. It is suggested that the EPS arrangement observed provides a physical structuring mechanism that can segregate extracellular activities at the microscale.     

In situ assay for identifying inhibitors of bacterial transglycosylase

An in situ transglycosylase assay has been developed using endogenously synthesized lipid II. The assay involves the preferential synthesis and accumulation of lipid II in a reaction mixture containing the cell wall membrane material isolated from Escherichia coli, exogenously supplied UDP-MurNAc-pentapeptide, and radiolabeled UDP-GlcNAc. In the presence of Triton X-100, the radiolabeled product formed is almost exclusively lipid II, while the subsequent formation of peptidoglycan is inhibited. Removal of the detergent resulted in the synthesis of peptidoglycan (25% incorporation of radiolabeled material) from the accumulated lipid II. This reaction was inhibited by moenomycin, a known transglycosylase inhibitor. In addition, tunicamycin, which affects an earlier step of the pathway by inhibiting MraY, had no effect on the formation of peptidoglycan in this assay, as expected. Similarly, ampicillin and bacitracin did not inhibit the formation of peptidoglycan under the conditions established.     

In situ corrosion control in industrial water systems

The main objective of this study was to evaluate the efficacy of a biocide and a corrosion inhibitor against the corrosion of a circulating pipe in a cooling tower. Isothiazolone was tested as the industrial biocide. The results showed that the biocide by itself or combined with a dispersant was not efficient to control corrosion in the industrial system. Corrosion rates of 0.324 mm/year were recorded in both the presence and absence of the biocide. Corrosion control was successfully accomplished by using a corrosion inhibitor. In the latter case the maximum corrosion rate of 0.024 mm/year were obtained.     

In situ measurement of archaeal and bacterial specific growth rates in two stage high rate anaerobic treatment systems

The thymidine assay is applied and evaluated as a direct measure of archaeal and bacterial volumetric growth rate in the first and second stages of a full scale High Rate Anaerobic Treatment system (HRAT). By coupling the method to an epifluorescent microscopic technique we show how to measure microbial specific growth rates in situ. In the first stage reactor, the acidogenic (hydrolytic-fermentative) apparent bacterial specific growth rate measurements (2.2 d –1 ) agreed with that predicted by the hydraulic residence time. For the methanogenic archaeal populations in the second stage upflow anaerobic sludge blanket (UASB) reactor the measurement was the same as published values. The results also showed that in the UASB reactor the archaeal growth dynamics in the liquid phase and 'sludge' granules were the same.     

In situ protein folding and activation in bacterial inclusion bodies

Recent observations indicate that bacterial inclusion bodies formed in absence of the main chaperone DnaK result largely enriched in functional, properly folded recombinant proteins. Unfortunately, the molecular basis of this intriguing fact, with obvious biotechnological interest, remains unsolved. We have explored here two non-excluding physiological mechanisms that could account for this observation, namely selective removal of inactive polypeptides from inclusion bodies or in situ functional activation of the embedded proteins. By combining structural and functional analysis, we have not observed any preferential selection of inactive and misfolded protein species by the dissagregating machinery during inclusion body disintegration. Instead, our data strongly support that folding intermediates aggregated as inclusion bodies could complete their natural folding process once deposited in protein clusters, which conduces to significant functional activation. In addition, in situ folding and protein activation in inclusion bodies is negatively regulated by the chaperone DnaK.     

In situ morphology of nitrifying-like bacteria in aquaculture systems

[This corrects the article on p. 423 in vol. 31.].     

Electrocatalytic reduction of nitrate and selenate by NapAB

Bacterial cellular metabolism is renowned for its metabolic diversity and adaptability. However, certain environments present particular challenges. Aerobic metabolism of highly reduced carbon substrates by soil bacteria such as Paracoccus pantotrophus presents one such challenge since it may result in excessive electron delivery to the respiratory redox chain when compared with the availability of terminal oxidant, O2. The level of a periplasmic ubiquinol-dependent nitrate reductase, NAP, is up-regulated in the presence of highly reduced carbon substrates. NAP oxidizes ubiquinol at the periplasmic face of the cytoplasmic membrane and reduces nitrate in the periplasm. Thus its activity counteracts the accumulation of excess reducing equivalents in ubiquinol, thereby maintaining the redox poise of the ubiquinone/ubiquinol pool without contributing to the protonmotive force across the cytoplasmic membrane. Although P. pantotrophus NapAB shows a high level of substrate specificity towards nitrate, the enzyme has also been reported to reduce selenate in spectrophotometric solution assays. This transaction draws on our current knowledge concerning the bacterial respiratory nitrate reductases and extends the application of PFE (protein film electrochemistry) to resolve and quantify the selenate reductase activity of NapAB.     

In situ observations of a possible skate nursery off the western Antarctic Peninsula

A dense aggregation of skate egg cases was imaged during a photographic survey of the sea floor along the western Antarctic Peninsula in November 2013. Egg cases were noted in a narrow band between 394 and 443 m depth. Although some skate species in other oceans are known to utilize restricted areas to deposit eggs in great numbers, such nurseries have not been described in the Southern Ocean.     

In situ synthesis of CdS nanoparticles on bacterial cellulose nanofibers

CdS nanoparticles have been synthesized and stabilized on unique bacterial cellulose (BC) nanofibers in situ. The obtained nanocomposite material have been characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), fourier transformed infrared (FTIR), thermogravimetric analysis (TGA), ultraviolet–visible (UV–Vis) and photoluminescence (PL) spectroscopy. The results indicated that CdS nanoparticles of about 30 nm diameter deposited on BC nanofibres are well-dispersed in the BC nanofibre-network and the uniform spherical CdS nanoparticles are comprised of nano-sized CdS crystal. Moreover, the crystallite sizes of CdS crystals are about 8 nm. The nanocomposites would have potential application as photocatalyst, novel luminescence and photoelectron transfer devices.     

In situ morphology of nitrifying-like bacteria in aquaculture systems.

The in situ microbiota from several aquaculture facilities with active nitrification was examined by transmission electron microscopy of thin sections for the presence of bacteria that contained intracytoplasmic membranes characteristic of the nitrifying bacteria. Colonies of bacteria with the cellular morphology of a species of Nitrosomonas were found to be present in both the culture water and in the biological filter slime of a freshwater chinook salmon (Oncorhynchus tshawytscha) culture system. bacteria in the water possessed the normal nitrosomonas type of ultrastructure, whereas similar bacteria in the slime had an aberrant morphology due to multiple invaginations of the cell wall and cyto-membranes and a significantly greater number of ribosomes. These nitrosomonas-like bacteria lysed during enrichment in commonly used media. Bacteria with the morphology of species of Nitrosomonas and Nitrosococcus were also observed in colonies in the surface slimes of marine culture systems for striped bass (Morone saxatilis) and quahaug (Mercenaria mercenaria).     

Characterization of the reduction of selenate and tellurite by nitrate reductases.

Preliminary studies showed that the periplasmic nitrate reductase (Nap) of Rhodobacter sphaeroides and the membrane-bound nitrate reductases of Escherichia coli are able to reduce selenate and tellurite in vitro with benzyl viologen as an electron donor. In the present study, we found that this is a general feature of denitrifiers. Both the periplasmic and membrane-bound nitrate reductases of Ralstonia eutropha, Paracoccus denitrificans, and Paracoccus pantotrophus can utilize potassium selenate and potassium tellurite as electron acceptors. In order to characterize these reactions, the periplasmic nitrate reductase of R. sphaeroides f. sp. denitrificans IL106 was histidine tagged and purified. The V(max) and K(m) were determined for nitrate, tellurite, and selenate. For nitrate, values of 39 micromol x min(-1) x mg(-1) and 0.12 mM were obtained for V(max) and K(m), respectively, whereas the V(max) values for tellurite and selenate were 40- and 140-fold lower, respectively. These low activities can explain the observation that depletion of the nitrate reductase in R. sphaeroides does not modify the MIC of tellurite for this organism.     

Removal of selenate from sulfate-containing media by sulfate-reducing bacterial biofilms

A biofilm-selected strain of a Desulfomicrobium sp. removed selenate from solution to sub-micromolar concentrations during growth on lactate (or hydrogen) and sulfate. Under sulfate-limited growth conditions, selenium was enzymatically reduced to selenide. Under excess sulfate conditions, selenate removal was primarily by enzymatic reduction to elemental selenium. Sequestration by biofilms was greater under the latter condition. Experiments with washed cell suspensions showed that high sulfate concentrations inhibited cell-specific selenate reduction, but when growing cells were exposed to selenate, the biomass increase achieved during incubations with abundant sulfate resulted in more rapid selenate removal. The addition of small amounts of sulfite, or thiosulfate, ameliorated this inhibition. Nitrate also inhibited selenate reduction in washed cell suspensions, apparently due to a general oxidizing effect. These results suggest that where biofilm-based sulfate-reducing bacteria (SRB) bioreactors are considered for the treatment of mixed metalliferous wastes that contain selenium oxyanions, adequate selenate removal should be achievable under a range of environmental conditions. The form and fate of the precipitated product will, however, be influenced by the dominant reduction pathway, which is controlled by environmental variables.     

In situ optical spectroscopy of some systems of biological interest

Monitoring the optical absorption or emission spectrum of a condensed phase sample offers information about the supramolecular assembly, packing effects and other features characteristic of the phase that would be missed when one studies solution-state spectra. We have used the technique of photoacoustic spectroscopy to study intact biological specimens, such as algae, parasite cells and the eye lens. Such a study has offered information about the status of endogenous hemin inPlasmodium cells and the mode of interaction of antimalarial drugs of the chloroquine class therein. We have also attempted to doin situ fluorescence spectroscopy on isolated intact eye lenses, which has enabled us to follow the photochemistry and the status of the photoproduct of the oxidation of the trp residues of the crystallins of the lens.     

In situ biochemical and bacterial variation of sediments enriched with mussel biodeposits

In order to estimate the qualitative variation of sedimented mussel deposits, biochemical and microbial measurements were undertaken after a sediment enrichment with fresh faeces and pseudo-faeces collected in a mussel farming area (Carteau, Gulf of Fos).During two months, cores sampled were investigated at three stations: a first station enriched with mussel deposits, a second considered as a reference station without mussel deposits and occasionally a third corresponding to a continuously enriched sediment under a rope culture.Carbohydrate, organic carbon and nitrogen contents in the sediments gave evidence of a short term variation after sedimentation.Bacterial production increased rapidly at the enriched station and returned to its initial level 8 days later. At this station, exoglucosidasic activity of bacteria, low at the beginning, presented a maximum two weeks after enrichment whereas exoproteolytic activity, which was high in the biodeposits, decreased in the course of the first week. These exoenzymatic activities were significantly higher at the enriched station than at the reference stations. Carbohydrate measurements were in agreement with these results.Degradation rate of biodeposits is discussed on the basis of exoenzymatic activity, organic carbon, nitrogen and free amino acid content at the three stations.