Assessing the environmental impact of ashes used in a landfill cover construction.

Large amounts of construction materials will be needed in Europe in anticipation for capping landfills that will be closed due to the tightening up of landfill legislation. This study was conducted to assess the potential environmental impacts of using refuse derived fuel (RDF) and municipal solid waste incineration (MSWI) ashes as substitutes for natural materials in landfill cover designs. The leaching of substances from a full-scale landfill cover test area built with different fly and bottom ashes was evaluated based on laboratory tests and field monitoring. The water that drained off above the liner (drainage) and the water that percolated through the liner into the landfill (leachate) were contaminated with Cl(-), nitrogen and several trace elements (e.g., As, Cu, Mo, Ni and Se). The drainage from layers containing ash will probably require pre-treatment before discharge. The leachate quality from the ash cover is expected to have a minor influence on overall landfill leachate quality because the amounts generated from the ash covers were low, <3-30l (m(2)yr)(-1). Geochemical modelling indicated that precipitation of clay minerals and other secondary compounds in the ash liner was possible within 3 years after construction, which could contribute to the retention of trace elements in the liner in the long term. Hence, from an environmental view point, the placement of ashes in layers above the liner is more critical than within the liner.

Evaluation of leachate emissions from crushed rock and municipal solid waste incineration bottom ash used in road construction.

Three years of leachate emissions from municipal solid waste incineration bottom ash and crushed rock in a full-scale test road were evaluated. The impact of time, construction design, and climate on the emissions was studied, and the predicted release from standard leaching tests was compared with the measured release from the road. The main pollutants and their respective concentrations in leachate from the roadside slope were Al (12.8-85.3 mg l(-1)), Cr (2-125 microg l(-1)), and Cu (0.15-1.9 mg l(-1)) in ash leachate and Zn (1-780 microg l(-1)) in crushed rock leachate. From the ash, the initial Cl(-) release was high ( approximately 20 g l(-1)). After three years, the amount of Cu and Cl(-) was in the same range in both leachates, while that of Al and Cr still was more than one order of magnitude higher in ash leachate. Generally, the release was faster from material in the uncovered slopes than below the pavement. Whether the road was asphalted or not, however, had minor impacts on the leachate quality. During rain events, diluted leachates with respect to, e.g., salts were observed. The leaching tests failed to simulate field leaching from the crushed rock, whereas better agreement was observed for the ash. Comparisons of constituent release from bottom ash and conventional materials solely based on such tests should be avoided.

Effect of accelerated carbonation and zero valent iron on metal leaching from bottom ash.

About 85% of the ashes produced in Sweden originated from the incineration of municipal solid waste and biofuel. The rest comes from the thermal treatment of recycled wood, peat, charcoal and others. About 68% of all ashes annually produced in Sweden are used for constructions on landfills, mainly slopes, roads and embankments, and only 3% for construction of roads and working surfaces outside the landfills (SCB, 2013). Since waste bottom ash (BA) often has similar properties to crushed bedrock or gravel, it could be used for road constructions to a larger extent. However, the leaching of e.g. Cr, Cu, Mo, Pb and Zn can cause a threat to the surrounding environment if the material is used as it is. Carbonation is a commonly used pre-treatment method, yet it is not always sufficient. As leaching from aged ash is often controlled by adsorption to iron oxides, increasing the number of Fe oxide sorption sites can be a way to control the leaching of several critical elements. The importance of iron oxides as sorption sites for metals is known from both mineralogical studies of bottom ash and from the remediation of contaminated soil, where iron is used as an amendment. In this study, zero valent iron (Fe(0)) was added prior to accelerated carbonation in order to increase the number of adsorption sites for metals and thereby reduce leaching. Batch, column and pHstat leaching tests were performed and the leaching behaviour was evaluated with multivariate data analysis. It showed that leaching changed distinctly after the tested treatments, in particular after the combined treatment. Especially, the leaching of Cr and Cu clearly decreased as a result of accelerated carbonation. The combination of accelerated carbonation with Fe(0) addition reduced the leaching of Cr and Cu even further and reduced also the leaching of Mo, Zn, Pb and Cd compared to untreated BA. Compared with only accelerated carbonation, the Fe(0) addition significantly reduced the leaching of Cr, Cu and Mo. The effects of Fe(0) addition can be related to binding of the studied elements to newly formed iron oxides. The effects of Fe(0) addition were often more distinct at pH values between 7 and 9, which indicates that a single treatment with only Fe addition would be less effective and a combined treatment is recommended. The pHstat results showed that accelerated carbonation in combination with Fe(0)(0) addition widens the pH range for low solubility of about one unit for several of the studied elements. This indicates that pre-treating the bottom ash with a combination of accelerated carbonation and Fe(0) addition makes the leaching properties of the ash less sensitive to pH changes that may occur during reuse. All in all, the addition of Fe(0) in combination with carbonation could be an effective pre-treatment method for decreasing the mobility of potentially harmful components in bottom ash.

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen.

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage-antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.

Single, antigen-specific B cells used to generate Fab fragments using CD40-mediated amplification or direct PCR cloning.

A simple procedure for the generation of human antibody fragments directly from single B cells or B-cell clones is described here. The procedure is based on antigen-specific selection of single human B cells, using antigen-coated magnetic beads and a cellular amplification step based on a culture system involving both EL-4 thymoma cells and anti-CD40 antibodies, presented by CD32-expressing fibroblasts. Nested PCR was applied to rescue V-regions from both single B cells and B-cell clones obtained using the cellular amplification step. This amplification step both increased the cell number as well as activated the cells that amplified mRNA levels, thereby facilitating immortalization by cloning. The V-regions were cloned and expressed as Fab fragments and characterized by biosensor analysis. This approach allowed us to bypass cumbersome hybridoma technology and to obtain human antibody fragments that retained the original VH/VL pairing, a feature of importance when studying, e.g., the V-gene usage in various human diseases and in normal B-cell repertoires.

A comparison of small-scale, pilot-scale and large-scale tests for predicting leaching behaviour of landfilled wastes.

Landfills generate emissions over long periods, often longer than a lifetime. The longest lasting emission is leachate. In order to estimate the future requirements for leachate treatment, different kinds of leaching tests may be applied. In this paper, shaking leaching tests (SLT), landfill-simulator leaching tests and a field-cell leaching test performed with ash, municipal solid waste (MSW) and MSW+ash are evaluated. The tests are compared and the factors influencing leaching are identified and discussed. The factors are: liquid to solid (L/S) ratio, water withdrawal, recirculation rate, presence or absence of biological processes, size of particles, duration of experiment, temperature and pre-treatment of the waste. The presence of biological processes has the greatest impact on leaching and is the main reason why SLT is less useful for long-term predictions. The landfill simulator tests were found to be useful for several different kinds of predictions. However, they are not reliable for predicting the L/S required for reaching a certain concentration. The possibilities for reliable long-term predictions would be facilitated by a better knowledge of the influence of various factors on leaching. Such an increased knowledge would make it possible to enhance waste stabilisation in leaching tests as well as in full-scale landfills.

Chaperonin assisted phage display of antibody fragments on filamentous bacteriophages.

We have used the GroE chaperonins to assist in the packing of a new phage display vector, pEXmide3. Titers of the packed phagemid increased almost 200-fold from approximately 4 x 10(11) cfu/ml, without coexpression of the GroE proteins, to approximately 7 x 10(13) cfu/ml with their coexpression. Equal titers of non-assisted and assisted phagestocks exhibited the same antigen specificity and ELISA reactivity, indicating the same frequency of displayed Fab-fragments. While the diversity of antibody libraries depends on the bacterial transformation efficiency, the copy number of each antibody is determined by subsequent amplification of the phage, thus chaperonin assisted phagemid packing in bacteriophage M13 can be used as a general and simple tool to increase the amplification level of expressed Fab fragments. pEXmide3 was developed for display of Fab and single chain Fv-fragments (scFv), using restriction enzymes that do not cut, or cut with low frequencies, in genes encoding immunoglobulin variable domains. The vector allows cloning of genes for the variable domains linking these to predetermined human constant domains or cloning of the entire light and heavy Fab chains. A modification of the pelB leader sequence, with a glutamine to alanine substitution at residue 18, was used for export of the light chain.

Steel slags in a landfill top cover--experiences from a full-scale experiment.

A full scale field study has been carried out in order to test and evaluate the use of slags from high-alloy steel production as the construction materials for a final cover of an old municipal landfill. Five test areas were built using different slag mixtures within the barrier layer (liner). The cover consisted of a foundation layer, a liner with a thickness of 0.7 m, a drainage layer of 0.3 m, a protection layer of 1.5 m and a vegetation layer of 0.25 m. The infiltration varied depending on the cover design used, mainly the liner recipe but also over time and was related to seasons and precipitation intensity. The test areas with liners composed of 50% electric arc furnace (EAF) slag and 50% cementitious ladle slag (LS) on a weight basis and with a proper consistence of the protection layer were found to meet the Swedish infiltration criteria of ⩽50 l (m(2)a)(-1) for final covers for landfills for non-hazardous waste: the cumulative infiltration rates to date were 44, 19 and 0.4 l (m(2)a)(-1) for A1, A4 and A5, respectively. Compared to the precipitation, the portion of leachate was always lower after the summer despite high precipitation from June to August. The main reason for this is evapotranspiration but also the fact that the time delay in the leachate formation following a precipitation event has a stronger effect during the shorter summer sampling periods than the long winter periods. Conventional techniques and equipment can be used but close cooperation between all involved partners is crucial in order to achieve the required performance of the cover. This includes planning, method and equipment testing and quality assurance.

Accelerated carbonation of steel slags in a landfill cover construction.

Steel slags from high-alloyed tool steel production were used in a full scale cover construction of a municipal solid waste (MSW) landfill. In order to study the long-term stability of the steel slags within the final cover, a laboratory experiment was performed. The effect on the ageing process, due to i.e. carbonation, exerted by five different factors resembling both the material characteristics and the environmental conditions is investigated. Leaching behaviour, acid neutralization capacity and mineralogy (evaluated by means of X-ray diffraction, XRD, and thermogravimetry/differential thermal analysis, TG/DTA) are tested after different periods of ageing under different conditions. Samples aged for 3 and 10 months were evaluated in this paper. Multivariate data analysis was used for data evaluation. The results indicate that among the investigated factors, ageing time and carbon dioxide content of the atmosphere were able to exert the most relevant effect. However, further investigations are required in order to clarify the role of the temperature.

Manifold sequencing: efficient processing of large sets of sequencing reactions.

Automated instruments for DNA sequencing greatly simplify data collection in the Sanger sequencing procedure. By contrast, the so-called front-end problems of preparing sequencing templates, performing sequencing reactions, and loading these on the instruments remain major obstacles to extensive sequencing projects. We describe here the use of a manifold support to prepare and perform sequencing reactions on large sets of templates in parallel, as well as to load the reaction products on a sequencing instrument. In this manner, all reaction steps are performed without pipetting the samples. The strategy is applied to sequencing PCR-amplified clones of the human mitochondrial D-loop and for detection of heterozygous positions in the human major histocompatibility complex class II gene HLA-DQB, amplified from genomic DNA samples. This technique will promote sequencing in a clinical context and could form the basis of more efficient genomic sequencing strategies.

Stimulation of human peripheral lymphocytes via CD3 and soluble antigen abrogates specific antibody production by reducing memory B cell numbers.

Human B cells are polyclonally activated in vitro by T cells stimulated with immobilized anti-CD3 monoclonal antibodies. We have analysed the effect of CD3 ligation on the production of antigen-specific antibodies, using peripheral blood lymphocytes from tetanus toxoid vaccinated blood donors. High levels of antigen-specific antibodies were obtained after stimulation with anti-CD3 antibodies for 7 days. Addition of soluble recall antigen did not affect the total amount of Ig produced, but dramatically decreased the antigen-specific response. The addition of IL-2, IL-4, and anti-CD40 or anti-CD28 antibodies or the removal of antigen did not restore the B cell response. Analysis using limiting dilution of B cells showed that the frequency of antigen-specific memory B cells decreased significantly in cultures stimulated with antigen. The antigen-specific B cell response could be completely restored only if the soluble antigen was cross-linked on the surface of the B cells. These results suggested that peripheral memory B cells were eliminated or anergized in the presence of soluble antigen.

Efficient detection of mutations in Wilson disease by manifold sequencing.

We have applied a solid support for parallel handling and direct loading of sequencing reactions--manifold sequencing--to analyze the coding sequence for the deficient copper transporting P-type ATPase in 24 families with Wilson disease. At least 100 different amplification reactions could be handled in parallel, with a minimal turnaround time of 12 h from isolated genomic DNA to identification of the mutations. Sixteen different mutations were found, accounting for 92% of the mutant genes. Ten of these mutations have not been previously described. Eleven were observed only in single families. Mutation His1069Gln, previously identified as the most prevalent mutation in Northern Europe, was found in one-third of the Northern European chromosomes in our material. Four patients were homozygous for this mutation, and three were homozygous for Thr977Met. The method allowed us to establish the diagnosis of Wilson disease in 24 h in a patient with acute hepatic failure.

Effect of electric arc vitrification of bottom ash on the mobility and fate of metals.

Increasing amounts of municipal solid waste incineration (MSWI) residues are treated prior to landfilling or reuse. In Japan, electric arc melting is used for bottom ash vitrification that generates a glasslike slag. The objective of this paper was to assess this pretreatment technique with respect to its effect on metal mobility and metal content. Both bottom ash and slag were sampled and analyzed on total solids (TS), fixed solids (FS), particle density (pp), specific BET surface area, particle size distribution, and total element content. A six-step wet sequential extraction procedure was used for assessing metal mobility. The results were qualitatively verified by scanning electron microscopy. The major conclusion was that the availability of various metals was affected differently by electric arc vitrification. Metals were solidified, stabilized, and/or separated from the slag. The mobility of Cr, Cu, Zn, Pb, and Ca was reduced. In slag, majorfractions of these elements were found in moderately reducible phases or in the residual slag lattice. The approximately three-fourths of Pb [174 +/- 7 mg (kg of FS)-1] and half of Zn content [676 +/- 352 mg (kg of FS)-1] were most likely removed from bottom ash through evaporation. The total content increases of Al, Cr, Ni, and Cd (51 +/- 3, 621 +/- 27, 138 +/- 19, and 99 +/- 32%, respectively) were probably caused by the wear of furnace refractories.

Antigen-specific activation of B cells in vitro after recruitment of T cell help with superantigen.

BACKGROUND: Human B cells can proliferate in vitro after stimulation with anti-Ig and via the CD40 molecule. Superantigens like SEA which bind to MHC class II antigens on, e.g. B cells can polyclonally activate T cells via interaction with their TcR. The activated T cell subsequently activates the B cells to proliferation and Ig-production. OBJECTIVES: To investigate whether superantigen could be used to direct polyclonal T cell help to human B cells stimulated by antigen in a restricted manner resulting in production of antigen-specific antibodies in vitro. STUDY DESIGN: Purified B cells were preincubated with the antigen in manners allowing crosslinking of surface-Ig. The antigen exposed B cells were then cultured together with autologous CD4+ helper T cells and in the presence of various concentrations of SEA. Antibody production was measured by ELISA after 7-12 days of culture. RESULTS: Antigen-specific activation of B cells could be obtained after stimulating the B cells with antigen or anti-surface-Ig antibodies in the presence of T helper cells and SEA. The degree of B cell activation (proliferation as well as antibody production) depended on the dose of antigen as well as on the dose of SEA used. Increased crosslinking of surface-Ig on antigen-specific B cells enhanced Ig production. Specific antibody production to a secondary recall antigen (tetanus toxoid) and to primary antigens (DNP and GM2) were obtained. The specific B cell response was dependent on contact between T and B cells. CONCLUSION: the results obtained demonstrate that the superantigen SEA can recruit T cell help to human B cells specifically stimulated by antigens, resulting in production of antigen reactive antibodies in vitro.

A manifold support for molecular genetic reactions.

Large numbers of samples can be efficiently processed through sequential reaction steps using a 96-pronged support that projects into individual microtiter wells. The support was constructed by creating a porous surface on a disposable polystyrene manifold, with avidin coupled to this greatly expanded surface. The shape and high binding capacity of the device allow the parallel transfer of large sets of reaction intermediates between different binding or enzymatic processing steps. We have applied the support to increase the efficiency of preparative and analytical molecular genetic reactions. The support also reduce the risks of sample mix-up and contamination in applications such as PCR and DNA sequencing.