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PubMed1995

Determination of strontium-90 in milk samples using a controlled precipitation clean-up step prior to ion-chromatography.

Strontium-90 may be determined by beta-counting its yttrium-90 daughter following separation by ion-chromatography, using a three column system comprising a chelating concentrator column, a cation-exchange column and an anion-exchange separator column. The column system has previously been applied to the determination of strontium-90 in water and urine samples. The applicability of the system to the analysis of milk is hampered by the large concentrations of calcium present, which significantly reduces the extraction of yttrium-90 by the concentrator column. A maximum of approximately 200 mg of calcium can be present for the successful extraction of yttrium-90, which greatly limits the quantity of milk that can be analysed. The quantity of milk analysed can be increased by the inclusion of a controlled precipitation step prior to the ion-chromatographic separation. The precipitation is carried out on acid digested milk samples by the addition of ammonia solution until the addition of one drop causes a reduction in pH resulting in the precipitation of calcium hydrogenphosphate. Under these conditions, approximately 20% of the calcium present in the original milk sample is precipitated, yttrium-90 is precipitated whereas strontium-90 is not precipitated. Dissolution of the precipitate, followed by separation of yttrium-90 using the ion-chromatography system facilitates the analysis of a litre of milk with recoveries of greater than 80%.

Animals↗

PubMed1991

Dye-promoted precipitation of serum proteins. Mechanism and application.

Immobilized dyes have been used primarily for purification of nucleotide dependent enzymes and proteins from plasma and other sources. Due to their low costs, high protein binding capacity and resistance to degradation dyes bear the potential as ligand for affinity separation of proteins on a large scale. In this paper dyes have been used for precipitation of proteins. Using albumin, prealbumin, alpha 1-acid glycoprotein and immunoglobulin G as model proteins we could demonstrate that dye-promoted precipitation depends on several factors which include the structure of the dye, the pH of the solution, the dye/protein molar ratio and the intrinsic properties of the proteins. It revealed that most of the dyes tested were endowed with the precipitating potential. The efficacy of precipitation was found to increase with the complexity of the dye structure. However, the amount of a dye required for total precipitation was found to be different for a given protein. Electrostatic as well as hydrophobic forces are involved in the mechanism of precipitation. It was demonstrated that by optimizing the conditions, mixtures of proteins can be resolved by dye-promoted precipitation. The high sensitivity of the reaction offers the possibility of using this method for rapid concentration of very diluted protein solutions.

Blood Proteins↗

PubMed1996

Effects of hexafluorosilicate on the precipitate composition and dentine tubule occlusion by calcium phosphate.

OBJECTIVES: To improve the calcium phosphate precipitation (CPP) method for the occlusion of dentine tubules with calcium phosphate, the addition of calcium hexafluorosilicate (CaSiF6) to CPP solution was evaluated in vitro with respect to its occluding capacity and the composition of the precipitate. METHODS: The occlusion of dentine tubules was evaluated by SEM observations and by measurements of dentine permeability. The composition of the precipitate was determined by measuring the calcium to phosphorus (Ca/P) ratio of the precipitate in dentine tubules by energy dispersive X-ray spectroscopy (EDS). RESULTS: The addition of CaSiF6 to the CPP solution resulted in an increase of the Ca/P ratio in the precipitate not only on the dentine surface but also inside the dentine tubules; indicating that the precipitate became more apatitic in nature. The addition of CaSiF6 had no effect on occluding capacity in terms of measurements of dentine permeability or SEM observations. Dentine permeability decreased to approximately 4% of pretreatment values and the dentine tubules were occluded for approximately 10-15 microns from the dentine surface. CONCLUSION: It is concluded that the addition of CaSiF6 to CPP solution was desirable, since it provided a more apatitic precipitate in the dentine tubules, not only on the dentine surface but also inside the tubules, and there were no drawbacks with respect to its occluding capacity.

Apatites↗

PubMed1989

Rapid method for the isolation of two purified subfractions of high density lipoproteins by differential dextran sulfate-magnesium chloride precipitation.

We describe a rapid and reliable three-step precipitation procedure for the isolation of large amounts of the two major components of high density lipoproteins (HDL) in human serum. Precipitation was accomplished by means of dextran sulfate (DS) of mol. wt. 500,000 and MgCl2. First, all apoB-associated lipoproteins of any density were selectively precipitated with critical concentrations of reagents. Secondly, a subfraction of HDL was differentially precipitated from the apoB-depleted serum by increasing the concentration of both reagents. Eventually, the bulk of the remainder of HDL was precipitated by lowering the pH to 5.4. According to the precipitation patterns and the density profiles, the DS-Mg procedure provides a clear differentiation between the two HDL components. According to the compositional criteria and the ultracentrifugal characteristics, the two polyanion-precipitated subclasses are very similar to, if not identical with, the two density subclasses, the lighter HDL2 and the heavier HDL3, isolated by preparative ultracentrifugation after apoB-containing lipoproteins had been removed.

Chemical Precipitation↗

PubMed1995

A protein complex from Choristoneura fumiferana gut-juice involved in the precipitation of delta-endotoxin from Bacillus thuringiensis subsp. sotto.

A 75 kDa protein from spruce budworm (Choristoneura fumiferana) gut-juice has been isolated and shown to cause a specific precipitation of the delta-endotoxin from Bacillus thuringiensis subsp. sotto. This 75 kDa protein, separated by either column chromatography or SDS-PAGE, caused precipitation of the sotto toxin in both agarose diffusion gels and the PAGE gels. The precipitation event leads to limited proteolysis of the toxin and loss of larval toxicity. SDS-PAGE analysis of the precipitated toxin indicates that proteolysis of the toxin is not a prerequisite for precipitation. The protein responsible for precipitation, exhibits elastase-like activity and appears to be a complex which partially dissociates during boiling in SDS-PAGE sample buffer. Gut-juice from gypsy moth, forest tent caterpillar and white mark tussock moth also precipitated delta-endotoxin, but silkworm gut-juice gave a much weaker response. These results provide further evidence that, in the larval gut, differential processing of delta-endotoxin may play a role in the expression of activity towards various insect larvae.

Amino Acid Sequence↗

PubMed2004

One-step immunoassay for measuring protein concentrations in plasma, based on precipitate-enhanced ellipsometry.

Standard enzyme immunoassays (EIAs) require washing steps to remove excess enzyme-antibody complexes. Such washing is laborious, lengthens assay time, and increases assay scatter. Recently, so-called precipitate-enhanced immunoassays (PEIAs) were introduced. Instead of color formation due to enzymatic conversion of a chromogenic substrate, this technique measures the rate of precipitate formation due to conversion of a substrate with a precipitating product. Such precipitation can be measured in the presence of active enzyme-antibody complexes in the buffer and no washing is required. In the present study this technique was used in a one-step PEIA, without washing steps, for the measurement of plasma concentrations of fatty-acid-binding protein. Horseradish peroxidase was used as tagging enzyme and diaminobenzidine as precipitating substrate. Precipitate formation was measured by ellipsometry. Assay time of the one-step PEIA was much shorter than that for an existing standard EIA. Test results can be obtained within minutes, depending on the sensitivity required. Assay precision of the one-step PEIA was better than that of the standard EIA. In the one-step assay, loss of surface-bound conjugate due to washing is prevented, which could explain part of the improved sensitivity compared to that of the two-step PEIA. More importantly, the presence of substrate-converting enzyme-antibody complexes in the buffer caused a large enhancement of precipitation.

Biomarkers↗

PubMed2006

Comparative investigation of N,N'-bis-(dithiocarboxy)piperazine and diethyldithiocarbamate as precipitants for Ni(II) in simulated wastewater.

The performances of a coordination polymerization precipitant, N,N'-bis-(dithiocarboxy)piperazine (BDP) were compared with the widely used heavy metal precipitant, diethyldithiocarbamate (DDTC), through the treatment of three kinds of nickel-containing wastewaters, NiSO(4), nickel citrate (NiCA) and Ni(2+)-dye. Results indicated that both BDP and DDTC at their stiochiometric doses could reduce 50.00 mg l(-1) free nickel(II) to lower than 1.0 mg l(-1) (discharge limit of nickel ions in China) and 10% precipitants doses increase could treat NiCA containing 50.00 mg l(-1) Ni(2+) to meet the discharge limit. But [NiBDP](n) coordination polymerization precipitates had more rapid settling speed than Ni(DDTC)(2) precipitates. It was also observed that BDP could still partly remove Reactive Brilliant Red X-3B from Ni(II)-dye integrated wastewater through the adsorption of the precipitates [NiBDP](n), in addition to precipitating Ni(II). A 1:1 stiochiometric dose of BDP/Ni(II) could decrease Ni(II) from 50.00 to 0.87 mg l(-1), and simultaneously reduce the dye from 30.00 to 19.52 mg l(-1).

Adsorption↗

PubMed2006

Kinetics of struvite to newberyite transformation in the precipitation system MgCl2-NH4H2PO4NaOH-H2O.

The influence of the initial reactant concentrations on the composition of the solid phases formed in the precipitation system MgCl(2)-NH(4)H(2)PO(4)-NaOH-H(2)O was investigated. The precipitation diagram constructed shows the approximate concentration regions within which struvite, newberyite, and their mixtures exist at 25 degrees C and an aging time of 60 min. It was found that immediately after mixing the reactant solutions, struvite (MgNH(4)PO(4).6H(2)O) precipitated in nearly the whole concentration area, while newberyite (MgHPO(4).3H(2)O) appeared mostly within the region of the excess of magnesium concentration. It was also found that after aging time of 60 min the precipitation domain of struvite alone is much broader than that of newberyite or the domain of their coexistence, and shows that struvite is more abundant in the systems in which the initial concentration of ammonium phosphate is higher than that of magnesium. The kinetics of struvite to newberyite transformation (conversion) was systematically studied under the conditions of different initial reactant concentrations and different initial pH in the systems in which a mixture of both phases precipitated spontaneously. The struvite to newberyite conversion period was found to be strongly related to the ratio of initial supersaturations, S(N)/S(S), rather than to the any particular physical quantity that can describe and predict the behavior of the precipitation system. Experimental data suggest a solution-mediated process as a most possible transformation mechanism. Along with a continuous monitoring of the changes in the liquid phase, the content of struvite in the solid phase was estimated by means of a Fourier transform infrared (FT-IR) method, developed for this particular precipitation system.

Chemical Precipitation↗

PubMed2005

Which seizure-precipitating factors do patients with epilepsy most frequently report?

When treating patients with epilepsy, dealing with seizure-precipitating factors is a partly neglected and underestimated supplement to more traditional therapies. The aim of this study was to investigate the incidence of seizure precipitants in a large epilepsy population and to determine which precipitants patients most often reported. Study participants included twins and their family members ascertained from the Norwegian Twin Panel (NTP), the Danish Twin Registry (DTR), and the Mid-Atlantic Twin Registry (MATR). One thousand six hundred seventy-seven patients with epilepsy were identified and were asked about seizure precipitants using a closed-ended questionnaire. Fifty-three percent reported at least one seizure-precipitating factor, while 30% claimed to have experienced two or more such factors. Emotional stress, sleep deprivation, and tiredness were the three most frequently reported precipitants. Patients with generalized seizures seemed to be more sensitive to sleep deprivation and flickering light than those with partial seizures, while women with partial seizures appeared to be more prone to seizures during menstruation than women with generalized seizures. Knowledge of seizure precipitants has practical implications, not only in patient treatment and counseling, but also for diagnosis, in that it may be helpful in facilitating the appearance of interictal epileptiform discharges in EEG and ictal EEG recordings.

Adult↗

PubMed2002

Solvent-dependent precipitation of prion protein.

The misfolded isoform of the prion protein (PrP(Sc)) possesses many unusual physiochemical properties. Previously, we and others reported on the differential partitioning of PrP(Sc) from plasma derived therapeutic proteins during their purification processes. To understand the driving force behind these partitioning differences, we investigated the effects of various solvent conditions on the precipitation of PrP(Sc). In a physiological buffer, PrP(Sc) remained in the supernatant after low speed centrifugation. At pH 5, PrP(Sc) precipitation was nearly complete regardless of the salt content. PrP(Sc) could also be precipitated at pH 8 by adding ethanol, but this precipitation was salt dependent. Based on these observations, an empirical mathematical model was constructed in which the PrP(Sc) precipitation trends were fully described as a function of solvent pH, salt, and ethanol concentration. This model consistently predicted PrP(Sc) partitioning during cold ethanol precipitation steps used in plasma protein purification processes, as shown by experimentally determined distributions of PrP(Sc) and transmissible spongiform encephalopathy (TSE) infectivity. These results indicate that pH, salt, and ethanol content are the major solvent factors determining the precipitation of the infectious PrP(Sc) in these processes and may provide a useful tool for assessing the differential partitioning of PrP(Sc) in a given solvent environment.

Algorithms↗

PubMed2000

Aluminium phosphate adjuvants prepared by precipitation at constant pH. Part I: composition and structure.

Aluminium phosphate adjuvant was precipitated under constant pH conditions in an effort to characterize materials formed at defined precipitation conditions. A reaction vessel was designed to provide a continuous steady-state process. An aqueous solution containing aluminium chloride and sodium dihydrogen phosphate was pumped into the reaction vessel at a constant rate. A second pump infused a sodium hydroxide solution at the rate required to maintain the desired pH. Precipitations were performed between pH 3.0 and 7.5, at intervals of pH 0.5. The adjuvants were characterized using 27Al NMR, FTIR, Raman and X-ray diffraction methods along with elemental analysis. The results of this study indicate that a continuum of amorphous aluminium hydroxyphosphates were formed having properties that changed as a continuous function of the precipitation pH. The phosphate content decreased as the pH of precipitation increased. 27Al NMR spectra revealed that the majority of the aluminium was octahedrally coordinated, with a small percentage of tetrahedrally coordinated aluminium. The density of the adjuvants was directly related to the pH of precipitation. The most prominent feature of the IR and Raman spectra is the P-O stretching vibration of the structural PO(4) groups. The positions of these bands decreased linearly as the precipitation pH increased. The results of selective deuteration FTIR experiments are consistent with high surface area materials as most of the OH groups were exposed near the surface of the adjuvant.

Adjuvants, Immunologic↗

PubMed2000

Aluminium phosphate adjuvants prepared by precipitation at constant pH. Part II: physicochemical properties.

The impact of the pH of precipitation on the physicochemical properties of aluminium phosphate adjuvants was investigated by precipitating aluminium phosphate adjuvants under constant pH conditions at pH values from 3.0 to 7.5 at intervals of 0.5. The pH of precipitation did not affect the morphology, but the point of zero charge (PZC) and rate of acid neutralization varied directly with pH of precipitation. Aggregation and protein adsorption capacity exhibited a parabolic relationship to the pH of precipitation. Minimum protein adsorption and maximum aggregation were observed at pH 5.5. In contrast to adjuvants precipitated from the same reactants but under uncontrolled pH conditions, the pH of all of the adjuvants precipitated under constant pH conditions remained constant for a 3-month aging period at room temperature.

Adjuvants, Immunologic↗

PubMed1999

Evaluation of post-treatment solutions for clinical use with the calcium phosphate precipitation method.

OBJECTIVE: We have proposed a calcium phosphate precipitation (CPP) method that occludes dentine tubules with apatitic minerals for the treatment of dentine hypersensitivity. The current CPP method uses 1 mol/l NaOH as the post-treatment solution. However, its high pH is not desirable for clinical use. The aim of this study was to evaluate several solutions, especially buffer solutions, as post-treatment solutions for the CPP method. METHODS: The CPP solution was mixed with several post-treatment solutions at various mixing ratios in test tubes. The precipitates were collected, and freeze-dried in a vacuum. Their weights were measured to evaluate the feasibility of buffer solution for the post-treatment solutions. Among the solutions, we selected one buffer solution and have done further evaluation using human dentine disks. The degree of occlusion of dentine tubules was evaluated by scanning electron microscopic observation and measurement of dentine permeability. Also, the composition of the precipitate was analyzed using energy-dispersive X-ray microanalysis and powder X-ray diffraction. RESULTS: Among the solutions, 1 mol/l NaHCO3 gave sufficient amounts of precipitate at a relatively mild pH, and thus was considered to be a good candidate for a post-treatment solution. Scanning electron microscopic observation showed that dentine tubules were occluded with the precipitate to a depth of approximately 10 microns from the surface when the CPP treatment was done twice, and dentine permeability was reduced to 98.8%. The precipitate was dicalcium phosphate dihydrate (DCPD; CaHPO4-2H2O) when the post-treatment solution was free of NaF. In contrast, the precipitate was an apatitic mineral with CaF2 as a by-product when the post-treatment solution contained NaF. CONCLUSIONS: We concluded that 1 mol/l NaHCO3 containing 0.3 mol/l NaF would be suitable as the post-treatment solution for the CPP method as it has a relatively mild pH and occludes dentinal tubules well.

Adult↗

PubMed2001

Chronopotentiometry and Faradaic impedance spectroscopy as signal transduction methods for the biocatalytic precipitation of an insoluble product on electrode supports: routes for enzyme sensors, immunosensors and DNA sensors.

The biocatalyzed precipitation of an insoluble product produced on electrode supports is used as an amplification path for biosensing. Enzyme-based electrodes, immunosensors and DNA sensors are developed using this biocatalytic precipitation route. Faradaic impedance spectroscopy and chronopotentiometry are used as transduction methods to follow the precipitation processes. While Faradaic impedance spectroscopy leads to the characterization of the electron-transfer resistance at the electrode, chronopotentiometry provides the total resistance at the interfaces of the modified electrodes. A horseradish peroxidase, HRP, monolayer-functionalized electrode is used to sense H(2)O(2) by the biocatalyzed oxidation of 4-chloro-1-naphthol (1), to the insoluble product benzo-4-chlorohexadienone (2). An antigen monolayer electrode is used to sense the dinitrophenyl antibody, DNP-Ab, applying an anti-antibody-HRP conjugate as a biocatalyst for the oxidative precipitation of 1 by H(2)O(2) to yield the insoluble product 2. An oligonucleotide (3) functionalized monolayer electrode is used to sense the DNA-analyte (4), that is one of the Tay-Sachs genetic disorder mutants. Association of a biotin-labeled oligonucleotide to the sensing interface, followed by the association of the avidin-HRP conjugate and the biocatalyzed precipitation of 2 leads to the amplified sensing of 4. The amount of the precipitate accumulated on the conductive support is controlled by the concentration of the respective analytes and the time intervals employed for the biocatalytic precipitation of 2. The electron-transfer resistances of the electrodes covered by the insoluble product (2) are derived from Faradaic impedance measurements, whereas the total electrode resistances are extracted from chronopotentiometric experiments. A good correlation between the total electrode resistances and the electron-transfer resistances at the conducting supports are found. Chronopotentiometry is suggested as a rapid transduction means (a few seconds). The precautions needed to apply chronopotentiometry in biosensors are discussed.

Animals↗

PubMed1998

Subjective perception of seizure precipitants: results of a questionnaire study.

We evaluated self-perception of seizure precipitants in 149 adult subjects with epilepsy: 71% of the subjects reported at least one factor that, according to the perception, increased the risk of suffering from a seizure. The subjects most often reported psychological stress, change of weather and sleep deprivation. Among the disease-related factors, seizure frequency and the state of consciousness at the onset of the seizures influenced perception of precipitants. Furthermore the perception of some precipitants was dependent on such social variables as rural versus urban surroundings. It is argued that the perception of seizure precipitants is the result of a combination of physiologically based temporal and causal correlations and of beliefs of the patient about such relationships. Hence reports of seizure precipitants are determined by somatic as well as psychological factors. Efforts to clarify the relationship between possible precipitants and the occurrence of seizures should be intensified. In addition the great importance of seizure precipitants in lay theories of epilepsies should be considered in counselling patients with epilepsies.

Adolescent↗

PubMedDate not supplied

Affinity precipitation of proteins by surfactant-solubilized, ligand-modified phospholipids.

The use of ligand-modified phospholipids solubilized in aqueous solution by nonionic surfactant for affinity precipitation of proteins is described. Avidin was precipitated by contact with solutions in which dimyristoylphosphatidylethanolamine (DMPE) functionalized with biotin (DMPE-B) was solubilized in octaethylene glycol mono-n-dodecyl ether (C12E8) solutions. The nonionic surfactant solubilizes the phospholipid in micelles above its critical micelle concentration (CMC) and in small submicellar aggregates below this concentration. At C12E8 concentrations significantly exceeding its CMC, determined to be about 100 microM, precipitation of avidin by solubilized DMPE-B is not observed. In this regime, binding of protein by DMPE-B was monitored by a hyperchroic shift in the protein's UV-visible spectrum at 231.5 nm. The data were analyzed using a model that considers the four binding sites on the protein to be independent and identical in binding strength for DMPE-B. Below the CMC of C12E8, precipitation is observed and is monitored by increasing turbidity of the solution. The kinetics of precipitation and the aggregate size measured by quasielastic light scattering were analyzed using Smoluchowski kinetics and the Mie scattering theory. These results help establish more completely the factors that influence the precipitation of proteins by ligand-modified phospholipids, and they are helpful in specifying conditions for the precipitation of other proteins.

Avidin↗

PubMed2005

Toxaphene deposition to Lake Ontario via precipitation, 1994-1998.

Precipitation samples collected continuously at Point Petre on Lake Ontario from November 1994 through December 1998 were analyzed for total toxaphene (=sum of hexa-, hepta-, octa-, and nonachloro bornanes) and chlorobornane congeners (1997-98 only). Composite triplicate samples were collected during 4-week intervals throughoutthe 4-year study using heated wet-only samplers. These results represent the first detailed data for toxaphene in Great Lakes precipitation. Seasonal volume-weighted mean concentrations for total toxaphene in precipitation ranged from 0.25 to 1.5 ng/L. Highest concentrations were found during the four spring (March-May) periods at roughly twice the annual means. The pattern for hexathrough nona-homologues over the 4 years did not vary appreciably with average ratios (relative to hepta-) of 0.08: 1.0:1.3:0.2. The volume-weighted mean concentrations for individual chlorobornane congeners were consistent in their season pattern with maximums seen in the spring. The major chlorobornane in precipitation, B8-2229 (Parlar 44), which was present at concentrations ranging from 0.016 to 0.079 ng/L, constituted 28 and 29% of the congener sum for 1997 and 1998, respectively. Lakewide loadings of toxaphene for Lake Ontario via precipitation were estimated to be 12, 17, 12, and 13 kg/year for 1995-1998, respectively. Previous toxaphene loading estimates were calculated for the individual Great Lakes on the basis of the only concentration data available, a single precipitation estimate of 0.2 ng/L from early work in northwestern Ontario. The loading estimates in this study indicate that precipitation inputs of toxaphene are 3-4 times higher than previously reported for Lake Ontario. The 1998 estimates of Lake Ontario wet deposition flux are 50% of the estimated gas deposition flux. However, wet flux values from this study exceed the net gas-phase mass transfer of toxaphene across the air-water interface.

Air↗

PubMed2006

Column experiments to assess the effects of electron donors on the efficiency of in situ precipitation of Zn, Cd, Co and Ni in contaminated groundwater applying the biological sulfate removal technology.

BACKGROUND, AIMS AND SCOPE: In a previous study, we explored the use of acetate, lactate, molasses, Hydrogen Release Compound (HRC, which is based on a biodegradable poly-lactate ester), methanol and ethanol as carbon source and electron donor to promote bacterial sulfate reduction in batch experiments, this with regards to applying an in situ metal precipitation (ISMP) process as a remediation tool to treat heavy metal contaminated groundwater at the site of a nonferrous metal work company. Based on the results of these batch tests, column experiments were conducted with lactate, molasses and HRCI as the next step in our preliminary study for a go-no go decision for dimensioning an on site application of the ISMP process that applies the activity of the endogenous population of sulfate-reducing bacteria (SRB). Special attention was given to the sustainability of the metal precipitation process under circumstances of changing chemical oxygen demand (COD) to [SO4(2-)] ratios or disrupted substrate supply. METHODS: To optimize the ISMP process, an insight is needed in the composition and activity of the indigenous SRB community, as well as information on the way its composition and activity are affected by process conditions such as the added type of C-source/ electron donor, or the presence of other prokaryotes (e.g. fermenting bacteria, methane producing Archaea, acetogens). Therefore, the biological sulfate reduction process in the column experiments was evaluated by combining classical analytical methods [measuring heavy metal concentration, SO4(2-)-concentration, pH, dissolved organic carbon (DOC)] with molecular methods [denaturing gradient gel electrophoresis (DGGE) fingerprinting and phylogenetic sequence analysis] based on either the 16S rRNA-gene or the dsr (dissimilatory sulfite reductase) gene, the latter being a specific biomarker for SRB. RESULTS AND DISCUSSION: All carbon sources tested promoted SRB activity, which resulted within 8 weeks in a drastic reduction of the sulfate and heavy metal contents in the column effluents. However, unexpected temporal decreases in the efficiency of the ISMP process, accompanied by the release of precipitated metals, were observed for most conditions tested. The most dramatic observation of the failing ISMP process was observed within 12 weeks for the molasses amended column. Subsequent lowering the COD/ SO4(2-) ratio from 1.9 to 0.4 did not alter the outcome of sulfate reduction and metal precipitation efficiency in this set-up. Remarkably, after 6 months of inactivity, bacterial sulfate reduction was recovered in the molasses set up when the original COD/ SO4(2-) ratio of 1.9 was applied again. Intentional disruption of the lactate and HRC supplies resulted in an immediate stagnation of the ISMP processes and in a rapid release of precipitated metals into the column effluents. However, the ISMP process could be restored after substrate amendment. 16S rDNA-based DGGE analysis revealed that the SRB population, in accordance with the results of the previously performed batch experiments, consisted exclusively of members of the genus Desulfosporosinus. The community of Archaea was characterized by sequencing amplicons of archaeal and methanogen-specific PCR reactions. This approach only revealed the presence of non-thermophilic Crenarchaeota, a novel group of organisms which is only distantly related to methane producing Euryarchaeota. DGGE on the dsrB genes was successfully used to link the results of the ISMP process to the community composition of the sulfate reducing bacteria. CONCLUSIONS: In the case of an intentional disruption of substrate supply, the ISMP process failed most likely because the growth and activity of the indigenous SRB community stopped due to a lack of a carbon and electron donor. On the other hand, the cause of the sudden temporal shortcomings of the ISMP process in the presence of different substrates was not immediately clear. It was first thought to be the result of competition between methanogenic prokaryotes (MP) and sulfate reducers, since the formation of small amounts of CH4 (0.01-0.03 ppm ml(-1) was detected. However, the results of molecular analyzes indicate that methanogens do not constitute a major fraction of the microbial communities that were enriched in the column experiments. Therefore, we postulate that the SRB population becomes inhibited by the formed metal sulfides. RECOMMENDATION AND PERSPECTIVE: Our results indicate that the ISMP process is highly dependent on SRB-stimulation by substrate amendments and suggest that this remedial approach might not be viable for long-term application unless substrate amendments are continued and environmental conditions are strictly controlled. This will include the removal of affected aquifer material from the metal precipitation zone at the end of the remediation process, or removal of metal precipitates when the microbial activity decreases. Additional tests are necessary to investigate what will happen when clear groundwater passes through the reactive zone while no more C-sources are amended and all indigenous carbon is consumed. Also, the effects of dramatic increases in sulfate- or HM-concentrations on the SRB-community and the concomitant ISMP process need to be studied in more detail.

Bacteria↗