Methodological aspects of microcalorimetry used to assess the dynamics of microbial activity during composting

Isothermal microcalorimetry is a sensitive non-invasive analytical tool that can become useful in research on compost and other biosolids. The aim of the present study was to address several methodological aspects that are critical to the use of microcalorimetry to assess the dynamics of microbial activity in such systems. The results show that: (1) The calorimetric baseline is strongly influenced by the run temperature in the range relevant to composting systems (20–60 °C), and is also affected by addition of the water that is required to maintain or optimize microbial activity, presumably because some water evaporates through ampoule gaskets. (2) Amending mature compost with readily available substrates requires additional careful baseline treatment. (3) Sample heterogeneity can be successfully minimized by passing through a 2-mm sieve. Additional size separation can be useful to enable focusing on the more active fractions. (4) Oxygen depletion is a key feature in batch calorimetric analysis; for samples of highly active composts or manure, the total amount of heat released relative to the oxygen available in the ampoule may indicate the co-existence of anaerobic and aerobic metabolic pathways. Finally, practical recommendations for microcalorimetry analyses of pre-mature and mature composts are outlined.     

Soil as the source of trace elements

The nature of the parent rock determines the trace element content of soils. Ultrabasic and basic rocks, which solidified first from the molten magma, incorporated bioessential trace elements such as Co, Ni, Zn and Cr by isomorphous replacement of Fe and Mg in ferromagnesian minerals, while acidic rocks, the last to solidify, tended to be richer in other elements such as Ba and Pb. Cu, Mn and, to a lesser extent, B, Mo and Se are more evenly distributed. The weathering of rocks by pedological and biological processes such as glacial and hydrodynamic comminution, secretion of acids and liganding species by microbes and plants leads to the formation of sands, silts and clays, and finally the incorporation of organic matter causes humification and the formation of soils as we know them. Part of the soil's store of bioessential elements is held in forms that are available to plants. Availability is controlled by the forms of occurrence and the nature of binding of the trace elements in the soil, which in turn is affected by soil acidity, redox balance (drainage) and organic matter content. These and other factors are discussed along with measures for alleviation of deficiency problems. Future progress in this area will depend to a large extent on interdisciplinary research by biologists, chemists, physicists and statisticians.     

Methodological considerations for the use of faecal nitrogen to assess diet quality in ungulates: The Alpine ibex as a case study

Faecal indices are used to describe spatiotemporal patterns of diet quality in wild herbivores, and near infrared spectroscopy methods (NIRS) distinctively reduce costs and labour compared to conventional chemical analyses. In this study we compared the prediction accuracy of faecal nitrogen (N), ash and fibrous fractions of a laboratory instrument (NIRS) with that of a lightweight portable instrument (VisNIRS) using faecal samples of Alpine ibex (Capra ibex). We then compared how different indices based on faecal N and fibrous fractions were able to detect the expected temporal trend and difference between sexes in diet quality, and verified whether the presumably lower accuracy of NIRS and VisNIRS (compared to wet chemistry) influenced the ability of indices to detect such trends. The laboratory NIRS was more accurate than the portable VisNIRS, especially for fibrous fractions. Predicted data from both instruments clearly identified the temporal trend identified by chemically analysed data, but only NIRS identified the small difference between sexes. Expressing faecal N as a proportion of organic matter (OM) or neutral detergent fibre (NDF) improved the power of the index as compared to expressing it as a proportion of dry matter (DM), irrespective of the method used to estimate the data. Faecal NDF contents followed the patterns expected from the variation of faecal N, while faecal acid detergent fibre (ADF) and especially lignin (ADL) followed contradictory patterns, which might be due to a selection of different forage sources rather than to changing quality of a homogenous forage source. We conclude that expressing faecal N as a proportion of OM is advised with wild herbivores faeces, where ash content can be biased by accidental soil contamination or ingestion. Faecal fibrous fractions are useful to complement faecal N and to avoid simplified interpretations of the results. For this purpose, NIRS methods can be extremely useful to predict multiple indices on large numbers of samples. VisNIRS portable instruments might be unable to detect subtle biological patterns in small numbers of samples. However, developing procedures for portable instruments outside laboratory facilities might further improve practicability, cost effectiveness, and, through allowing on-field predictions on large numbers of samples, power of statistical testing of diet quality indices.     

Synthesis of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and its use as a substrate to assess feruloyl esterase activity

A synthetic scheme was developed for the production of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside (FA-Ara) in gram quantities. This molecule accurately models the chemical attachment of ferulic acid to polysaccharides found in cell walls of plants in the Gramineae family. It is therefore a realistic substrate that can be used to monitor feruloyl esterase activity. Ultraviolet spectral analysis indicated that FA-Ara has an absorption maximum distinct from the hydrolytic product, ferulic acid (FA), over a wide range of solution pH values. The log molar extinction coefficient ranges from 4.16 to 4.36 for FA-Ara and 4.16 to 4.33 for FA depending upon the pH of the buffered solution. Consequently a convenient spectrophotometric assay can be utilized to monitor esterase activity. Three different methods were developed for using this model substrate to assess esterase activity, including thin-layer chromatography, a spectrophotometric assay, and the use of high-performance liquid chromatography.     

Dopamine as trace amine in the dorsal root ganglia

It has been shown that in the chick dorsal root ganglion (DRG) about 8% of neurons, belonging to both the A and B classes of sensory neurons exhibit a clear dopamine immunoreactivity. In the present study are reported the results of measurements, by mean of HPLC-electrochemical detection (HPLC-ED), of DA and of the DA metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the rat DRG and their central nerves. Very low levels of DA, about 10 folds lower than the levels found in the dorsal horn of the spinal cord, were found in the DRG. However the levels of DOPAC and HVA were approximately equivalent to the levels found in the cord. The immunocytochemical study performed in parallel has shown that some dopaminergic-immunoreactive fibers in the DRG are located around the blood vessels. Few dopamine-immunoreactive sensory neurons were identified in the DRG and immunoreactive fibers, not linked to blood vessels, were identified in the dorsal root nerves. The present work indicates that there is a dopaminergic innervation of the blood vessels in the rat DRG but that dopamine may also be, as in the chick, a transmitter of primary afferent fibers.     

Experimental tests to assess emotionality in horses

Different tests were used to assess different aspects of the emotionality of 1–3 year-old horses: arena test; a `novel object' test; and a handling test. In reaction to the test situations no important differences were observed according to age or sex in the behaviour patterns, but clear individual differences were observed within these classes. The arena test seemed to reveal the degree of gregariousness of the animals whereas the results in the two other tests were correlated and seemed to reflect an inherent degree of fearfulness in the horse. Indices were developed that enabled to rank the animals, by taking into account all behaviour patterns shown. Such individual characteristics might have some genetic basis: half-siblings tended to behave the same way in most cases.     

The uptake of trace elements by spinach and bean varieties of different root parameters

The uptake by plants of trace elements from the soil depends to a large extent on root characteristics and activities. Differences between plant species and varieties in the uptake of trace elements are well known. Less understood, however, are the mechanisms governing these differences and the relative significance of various root parameters.Spinach and bean varieties were, therefore, compared with respect to their root lengths and number of root apices, and to the uptake of Mn, Fe and Zn from soil-sand mixtures. The results showed significant differences among the varieties tested, both in root characteristics and in the uptake of trace elements. However, no relationships were evident between the trace element uptake by spinach varieties and their root characteristics. Contrary to this the Fe-uptake by bean varieies exhibited a clear dependence on the total number of root apices. The uptake of Mn and Zn showed a similar relationship with some exceptions. Whether the apical regions of individual roots are the most active sites of uptake or rather affect the solubility of trace elements will be the subject of further investigations.     

The use of transgenic plants in the bioremediation of soils contaminated with trace elements

The use of plants to clean-up soils contaminated with trace elements could provide a cheap and sustainable technology for bioremediation. Field trials suggested that the rate of contaminant removal using conventional plants and growth conditions is insufficient. The introduction of novel traits into high biomass plants in a transgenic approach is a promising strategy for the development of effective phytoremediation technologies. This has been exemplified by generating plants able to convert organic and ionic forms of mercury into the less toxic, volatile, elemental mercury, a trait that occurs naturally only in some bacteria and not at all in plants. The engineering of a phytoremediator plant requires the optimization of a number of processes, including trace element mobilization in the soil, uptake into the root, detoxification and allocation within the plant. A number of transgenic plants have been generated in an attempt to modify the tolerance, uptake or homeostasis of trace elements. The phenotypes of these plants provide important insights for the improvement of engineering strategies. A better understanding, both of micronutrient acquisition and homeostasis, and of the genetic, biochemical and physiological basis of metal hyperaccumulation in plants, will be of key importance for the success of phytoremediation.     

The use of the in vitro micronucleus assay to detect and assess the aneugenic activity of chemicals

The successful validation of the in vitro micronucleus assay by the SFTG now provides the opportunity for this highly cost effective assay to be used to screen chemicals for their ability to induce both structural (clastogenic) and numerical (aneugenic) chromosome changes using interphase cells. The use of interphase cells and a relatively simple experimental protocol provides the opportunity to greatly increase the statistical power of cytogenetic studies on chemical interactions. The application of molecular probes capable of detecting kinetochores and centromeres provides the opportunity to classify mechanisms of micronucleus induction into those which are primarily due to chromosome loss or breakage. When a predominant mechanism of micronucleus induction has been shown to be based upon chromosome loss then further investigation can involve the determination of the role of non-disjunction in the induction of aneuploidy. The binucleate cell modification of the in vitro micronucleus assay can be combined with the use of chromosome specific centromere probes to determine the segregation of individual chromosomes into daughter nuclei. The combination of these methods provides us with powerful tools for the investigation of mechanisms of genotoxicity particularly in the low dose regions.     

Fish early life stage tests as a tool to assess embryotoxic potentials in small streams

Early life stage (ELS) studies with brown trout(Salmo trutta f. fario L.) andstone loach (Barbatula barbatula L.)were performed between 1995 and 2000 toevaluate embryotoxic potentials in twodifferently polluted streams in southwestGermany. With both species, semistatic exposureexperiments with water samples and sedimenteluates were conducted in the laboratory.Additionally, brown trout ELS tests wereperformed in flow-through systems in thesemi-field and in the field. Thus, differentlevels of complexity of environmentalconditions were addressed which allowed thestudy of effects of xenobiotic contamination,temperature, and sediments on the success ofembryonic development. Additionally, effects ofwater from the polluted stream on fertilizationof brown trout eggs were determined. In themore polluted stream, xenobiotics caused anembryotoxic potential for both brown trout andstone loach, and viability of exposed browntrout eggs was drastically reduced by suspendedsolids in the water which covered the eggs.Additionally, fertilization rates of browntrout eggs were significantly decreased inwater of the more polluted stream. In the lesspolluted stream, low water temperature andinfestations by protozoic ectoparasites causedmortality of embryos. In this stream, pollutionand sediment effects were not observed. Resultsmade evident that in the more polluted streamrecruitment of brown trout was drasticallyimpaired.     

Motor activity as an unbiased variable to assess anaphylaxis in allergic rats

The release of mediators by mast cells triggers allergic symptoms involving various physiological systems and, in the most severe cases, the development of anaphylactic shock compromising mainly the nervous and cardiovascular systems. We aimed to establish variables to objectively study the anaphylactic response (AR) after an oral challenge in an allergy model. Brown Norway rats were immunized by intraperitoneal injection of ovalbumin with alum and toxin from Bordetella pertussis. Specific immunoglobulin (Ig) E antibodies were developed in immunized animals. Forty days after immunization, the rats were orally challenged with the allergen, and motor activity, body temperature and serum mast cell protease concentration were determined. The anaphylaxis induced a reduction in body temperature and a decrease in the number of animal movements, which was inversely correlated with serum mast cell protease release. In summary, motor activity is a reliable tool for assessing AR and also an unbiased method for screening new anti-allergic drugs.     

The use of bacteriophage as tracers of aerosols liberated by sludge suction appliances

Three bacteriophage tracers were added to 600-1 containers of water and simulated latrine sludge to provide high titres of tracer in aqueous and semi-aqueous media. After a period of mixing and stabilization, media were removed from the containers with suction hoses coupled to the vacuum pump of one of two sludge suction tankers. Exhaust air from the appliances was sampled by cyclone sampler and assayed for the presence of tracer organisms carried over during the emptying process. In the experiments the appliances were operated at different vacuum pump speeds, drawing both aqueous and semi-aqueous (simulated sludge) media. Air around one tanker was also sampled during the emptying, under pressure, of the vacuum vessel. The degree of aerosolization and expulsion of tracer bacteriophage by the vacuum appliances was consistently low, regardless of medium and pump air flow. In contrast, the proportion of tracer retained within the appliances was very high, exceeding the proportion expelled by a minimum of 8 log10 orders of magnitude and a maximum of greater than 11 log10 orders. The highest total of tracer bacteriophage was recovered during the pressure emptying of the vacuum vessel of one tanker. The results may be used for assessing and comparing potential public health hazards associated with the handling of wastewater sludge by vacuum appliances.     

The use of bacteriophage as tracers of aerosols liberated by sludge suction appliances

Three bacteriophage tracers were added to 600–1 containers of water and simulated latrine sludge to provide high titres of tracer in aqueous and semi-aqueous media. After a period of mixing and stabilization, media were removed from the containers with suction hoses coupled to the vacuum pump of one of two sludge suction tankers. Exhaust air from the appliances was sampled by cyclone sampler and assayed for the presence of tracer organisms carried over during the emptying process. In the experiments the appliances were operated at different vacuum pump speeds, drawing both aqueous and semi-aqueous (simulated sludge) media. Air around one tanker was also sampled during the emptying, under pressure, of the vacuum vessel. The degree of aerosolization and expulsion of tracer bacteriophage by the vacuum appliances was consistently low, regardless of medium and pump air flow. In contrast, the proportion of tracer retained within the appliances was very high, exceeding the proportion expelled by a minimum of 8log₁₀ orders of magnitude and a maximum of greater than 11log₁₀ orders. The highest total of tracer bacteriophage was recovered during the pressure emptying of the vacuum vessel of one tanker. The results may be used for assessing and comparing potential public health hazards associated with the handling of wastewater sludge by vacuum appliances.     

Methodological proposal to assess the water footprint accounting of direct water use at an urban level: A case study of the Municipality of Vicenza

Climate change, world population growth and industrialization have placed considerable stress on the local availability of water resources. Considering that most of the world's population currently resides in cities, this issue has particular relevance in an urban environment. One of the methods that is recognized to support better water management is water footprint accounting. However, due to the requirement of large amounts of data and their limited availability, applications of this method at an urban scale use national or regional water use data. The goal of this study is to develop a methodological proposal to assess the water footprint accounting of direct water use at an urban level to support the local management of water resources by using local data, by adopting a bottom-up approach and by verifying its applicability in a real case study. The water footprint accounting approach was modified by adopting a modular approach in the definition of boundaries and by adapting the blue, green and gray water footprint accounting formulations. The proposed method was successfully tested in the municipality of Vicenza (north east Italy) and provided interesting responses in terms of water management. Herein, we also discuss the latest developments in impact assessments related to water based on a life cycle assessment, which should be used as a framework for the future development of the model that is presented.