How do novel and conventional agri-food wastes, co-products and by-products improve soil functions and soil quality?

Agriculture is estimated to generate about 700 million tons of waste annually in the EU. Novel valorization technologies are developing continuously to recover and recycle valuable compounds and nutrients from waste materials. To close the nutrient loop, low-value agri-food wastes, co-products and by-products (AFWCBs) produced during the valorization process, need to be returned to the soil. However, knowledge on their reaction in soils that is needed to allow efficient and environmentally sound recycling is largely lacking. To this end, we set up a series of laboratory incubation experiments using 10 AFWCBs including insect frass residues made from three different feedstocks, anaerobic digestates from two feedstocks, potato-pulp, rice bran compost, duckweed and two reference crop residues (wheat straw and sugar beet) and measured net N release, C mineralization, dehydrogenase activity (DHA), microbial biomass C (MBC) and community structure. The suppressing potential of frasses and digestates against Rhizoctonia solani was determined using bean. The digestates released the highest net mineral N (50-70%) followed by rice bran compost (55%) and duckweed (30%), while frass made from general food waste and potato-pulp immobilized N like the reference straw for 91 days after incubation. All AFWCBs except digestates significantly increased MBC compared to the control while frasses, potato-pulp and duckweed increased DHA. Frasses and digestates significantly suppressed the development of Rhizoctonia solani in bean plants. AFWCBs from emerging valorizing technologies have the potential to improve microbial activities, C sequestration and may play a significant role in closing the nutrient loop.

Potential of chopped heath biomass and spent growth media to replace wood chips as bulking agent for composting high N-containing residues.

We investigated the potential of C-rich byproducts to replace wood chips as bulking agent (BA) during composting. The impact of these alternatives on the composting process and on compost stability and characteristics was assessed. Three BA (chopped heath biomass and spent growth media used in strawberry and tomato cultivation) were used for processing leek residues in windrow composting. All BA resulted in stable composts with an organic matter (OM) content suitable for use as soil amendment. Using chopped heath biomass led to high pile temperatures and OM degradation and a nutrient-poor compost with high C/P ratio appropriate for increasing soil organic carbon content in P-rich soils. Spent substrates can replace wood chips, however, due to their dense structure and lower biodegradation potential, adding a more coarse BA is required. Generally, the nutrient content of the composts with growth media was higher than the composts with wood chips and chopped heath biomass.

Simulating the environmental performance of post-harvest management measures to comply with the EU Nitrates Directive.

Nitrate (NO3-) leaching from farmland remains the predominant source of nitrogen (N) loads to European ground- and surface water. As soil mineral N content at harvest is often high and may increase by mineralisation from crop residues and soil organic matter, it is critical to understand which post-harvest management measures can be taken to restrict the average NO3- concentration in ground- and surface waters below the norm of 50 mg l-1. Nitrate leaching was simulated with the EU-rotate_N model on a silty and a sandy soil following the five main arable crops cultivated in Flanders: cut grassland, silage maize, potatoes, sugar beets and winter wheat, in scenarios of optimum fertilisation with and without post-harvest measures. We compared the average NO3- concentration in the leaching water at a depth of 90 cm in these scenarios after dividing it by a factor of 2.1 to include natural attenuation processes occurring during transport towards ground- and surface water. For cut grassland, the average attenuated NO3- concentration remained below the norm on both soils. In order to comply with the Nitrates Directive, post-harvest measures seemed to be necessary on sandy soils for the four other crops and on silty soils for silage maize and for potatoes. Successful measures appeared to be the early sowing of winter crops after harvesting winter wheat, the undersowing of grass in silage maize and the removal of sugar beet leaves. Potatoes remained a problematic crop as N uptake by winter crops was insufficient to prevent excessive NO3- leaching. For each crop, maximum levels of soil mineral N content at harvest were proposed, both with and without additional measures, which could be used in future nutrient legislation. The approach taken here could be upscaled from the field level to the subcatchment level to see how different crops could be arranged within a subcatchment to permit the cultivation of problem crops without adversely affecting the water quality in such a subcatchment.

Opportunities and barriers to on-farm composting and compost application: A case study from northwestern Europe.

Maintaining and increasing soil quality and fertility in a sustainable way is an important challenge for modern agriculture. The burgeoning bioeconomy is likely to put further pressure on soil resources unless they are managed carefully. Compost has the potential to be an effective soil improver because of its multiple beneficial effects on soil quality. Additionally, it fits within the bioeconomy vision because it can valorize biomass from prior biomass processing or valorize biomass unsuitable for other processes. However, compost is rarely used in intensive agriculture, especially in regions with high manure surpluses. The aim of this research is to identify the barriers to on-farm composting and the application of compost in agriculture, using a mixed method approach for the case of Flanders. The significance of the 28 identified barriers is analyzed and they are categorized as market and financial, policy and institutional, scientific and technological and informational and behavioral barriers. More specifically, the shortage of woody biomass, strict regulation, considerable financial and time investment, and lack of experience and knowledge are hindering on-farm composting. The complex regulation, manure surplus, variable availability and transport of compost, and variable compost quality and composition are barriers to apply compost. In conclusion, five recommendations are suggested that could alleviate certain hindering factors and thus increase attractiveness of compost use in agriculture.

Characterization of aerosols and fibers emitted from composite materials combustion.

This work investigates the aerosols emitted during combustion of aircraft and naval structural composite materials (epoxy resin/carbon fibers and vinyl ester/glass fibers and carbon nanotubes). Combustion tests were performed at lab-scale using a modified cone calorimeter. The aerosols emitted have been characterized using various metrological devices devoted to the analysis of aerosols. The influence of the nature of polymer matrices, the incorporation of fibers and carbon nanotubes as well as glass reinforcements on the number concentration and the size distribution of airborne particles produced, was studied in the 5 nm-10 µm range. Incorporation of carbon fibers into epoxy resin significantly reduced the total particle number concentration. In addition, the interlaced orientation of carbon fibers limited the particles production compared to the composites with unidirectional one. The carbon nanotubes loading in vinyl ester resin composites influenced the total particles production during the flaming combustion with changes during kinetics emission. Predominant populations of airborne particles generated during combustion of all tested composites were characterized by a PN50 following by PN(100-500).

Hypereosinophilic syndrome in an atopic patient.

Hypereosinophilic syndrome (HES) is a rare, heterogeneous group of systemic diseases characterized by sustained overproduction of eosinophils leading to variable end-organ damage. The skin is affected in 45-60% of patients and may be of diagnostic and prognostic value. In 1975, three criteria were suggested for the diagnosis of HES: (i) blood eosinophilia of > 1.5 x 10(9)/L present for > 6 months, (ii) no apparent cause for the hypereosinophilia, and (iii) signs of end-organ dysfunction. We present a patient with hypereosinophilia in whom a delay in diagnosing HES occurred, partly due to his atopic constitution. However, atopy is not associated with such high or longstanding eosinophilia.

Automatic inducer addition and harvesting of recombinant Escherichia coli cultures based on indirect on-line estimation of biomass concentration and specific growth rate.

This article describes a novel bioreactor configuration for production optimization of recombinant proteins in Escherichia coli. Inducer addition and harvesting are controlled on-line based on indirect estimation of biomass concentration and specific growth rate from addition of NaOH to maintain constant pH. When either a predetermined biomass concentration is reached or the cultures have obtained, a constant specific growth rate inducer is introduced automatically. The induction period is ended by automatic harvesting of the cultures either at a predetermined biomass concentration or when substrate (in this study glucose) is depleted, detected as an increase of pH, or dissolved oxygen tension. During harvesting, metabolic activities are quenched within 3 min by cooling of the cell suspension. The system has been used to optimize expression of glutathione S-transferase (GST) fusion protein of the ligand binding domain of mouse peroxisome proliferator-activated receptor, GST-PPARalpha LBD. Total yield of GST-PPARalpha LBD was independent of the time of inducer addition as long as the length of induction period corresponded to at least 0.25 cell divisions while the yield of soluble GST-PPARalpha LBD, the only active form, increased with the length of induction period. Highest yields were obtained when the inducer was added at low cell concentration as soon as constant specific growth rate was detected, resulting in induction periods corresponding to 3.4 +/- 0.4 cell divisions. The specific growth rate remained almost constant for one cell division after inducer addition, whereafter it decreased. No decrease of specific growth rate was observed when inducer was added in the lag-phase, and no soluble protein was produced. These results suggest that solely soluble GST-PPARalpha LBD acts as a growth inhibitor and that GST-PPARalpha LBD is expressed predominantly as inclusion bodies immediately after inducer addition whereas the proportion expressed as soluble protein is increased after 1 h of induction. Compared to the procedures, which are generally used for protein expression in the laboratory, this system is less labor intensive, it automatically provides recording of biomass concentration and specific growth rate, and it allows direct comparisons between expression of different proteins and performance of different constructs since the induction period is linked to growth.

Long-chain acyl-CoA esters and acyl-CoA binding protein are present in the nucleus of rat liver cells.

A detailed analysis of the subcellular distribution of acyl-CoA esters in rat liver revealed that significant amounts of long-chain acyl-CoA esters are present in highly purified nuclei. No contamination of microsomal or mitochondrial marker enzymes was detectable in the nuclear fraction. C16:1 and C18:3-CoA esters were the most abundant species, and thus, the composition of acyl-CoA esters in the nuclear fraction deviates notably from the overall composition of acyl-CoA esters in the cell. After intravenous administration of the non-beta-oxidizable [(14)C]tetradecylthioacetic acid (TTA), the TTA-CoA ester could be recovered from the nuclear fraction. Acyl-CoA esters bind with high affinity to the ubiquitously expressed acyl-CoA binding protein (ACBP), and several lines of evidence suggest that ACBP functions as a pool former and transporter of acyl-CoA esters in the cytoplasm. By using immunohistochemistry, immunofluorescence microscopy, and immunoelectron microscopy we demonstrate that ACBP localizes to the nucleus as well as the cytoplasm of rat liver cell and rat hepatoma cells, suggesting that ACBP may also be involved in regulation of acyl-CoA-dependent processes in the nucleus.

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum.

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.

Human health implications of environmental contaminants in Arctic Canada: a review.

This paper assesses the impact on human health of exposure to current levels of environmental contaminants in the Canadian Arctic, and identifies the data gaps that need to be filled by future human health research and monitoring. The concept of health in indigenous groups of the Arctic includes social, cultural, and spiritual dimensions. The harvesting, sharing and consumption of traditional foods are an integral component to good health among Aboriginal people influencing both physical health and social well-being. Traditional foods are also an economic necessity in many communities. Consequently, the contamination of country food raises problems which go far beyond the usual confines of public health and cannot be resolved by health advisories or food substitutions alone. The primary exposure pathway for the contaminants considered in this paper is through the traditional northern diet. For the Inuit, the OCs of primary concern at this time from the point of view of exposure are chlordane, toxaphene, and PCBs. Exposures are higher in the eastern than in the western region of the North. For Dene/Metis, exposure to OCs is in general below a level of concern. However, estimated intake of chlordane and toxaphene has been found to be elevated for certain groups and is a cause for concern if exposures are elevated on a regular basis. The developing foetus and breast-fed infant are likely to be more sensitive to the effects of OCs and metals than individual adults and are the age groups at greatest risk in the Arctic. Extensive sampling of human tissues in the Canadian north indicate that a significant proportion of Dene, Cree and Inuit had mean maternal hair mercury levels within the 5% risk-range proposed by the WHO for neonatal neurological damage. Based on current levels, lead does not appear to pose a health threat while cadmium is likely only a major risk factor for heavy smokers or consumers of large amounts of organ meats. Consumers of traditional foods are exposed to an approximately seven-fold higher radiation dose than non-consumers of traditional foods due predominantly to the bioaccumulation of natural radionuclides in the food chain. Risk determination for contaminants in country food involves a consideration of the type and amounts of food consumed and the sociocultural, nutritional, economic, and spiritual benefits associated with country foods. Risk management options that minimize the extent to which nutritional and sociocultural aspects of Aboriginal societies are compromised must always be considered.

A human homologue of Escherichia coli ClpP caseinolytic protease: recombinant expression, intracellular processing and subcellular localization.

We have recently cloned a human cDNA (hClpP) with significant sequence similarity to the ATP-dependent Escherichia coli ClpP protease [Bross, Andresen, Knudsen, Kruse and Gregersen (1995) FEBS Lett. 377, 249-252]. In the present study, synthesis, intracellular processing and subcellular localization of hClpP have been analysed in intact cells and in a cell-free system. Using pulse-labelling/immunoprecipitation of Chang cells transfected with the hClpP cDNA, we observed two major bands with apparent molecular masses of approx. 39 and 37 kDa. A pulse-chase experiment showed that these bands were converted into one mature-enzyme band with a molecular mass of approx. 32 kDa that was stable for at least 24 h. The 37 kDa band co-migrated with a band produced upon expression of full-length hClpP in E. coli, and the 32 kDa band co-migrated with the product of E. coli-expressed hClpP in which the 56 N-terminal residues had been deleted, indicating that the 37 kDa moiety represents the precursor and that approx. 56 residues are cleaved off during maturation. The processing of hClpP in intact cells was dependent on mitochondrial membrane potential. These results were confirmed in an import assay system using in vitro transcription and translation directed by the hClpP cDNA and isolated rat liver mitochondria. No protease activity towards a series of fluorogenic peptides could be observed in extracts of Chang cells overexpressing hClpP, indicating that the protease may not be active without co-factors. Immunofluorescence studies using confocal-laser-scanning microscopy showed co-localization of the hClpP and the mitochondrially located Hsp60 (heat-shock protein 60). Taken together, the results reported here show that hClpP is localized inside mitochondria and that the trafficking and processing of hClpP resembles the typical biogenesis pathway for nuclear-encoded mitochondrial proteins.

A common W556S mutation in the LDL receptor gene of Danish patients with familial hypercholesterolemia encodes a transport-defective protein.

In a group of unrelated Danish patients with familial hypercholesterolemia (FH) we recently reported two common low-density lipoprotein (LDL) receptor mutations, W23X and W66G, accounting for 30% of the cases. In this study, we describe another common LDL receptor mutation, a G to C transition at cDNA position 1730 in exon 12, causing a tryptophan to serine substitution in amino acid position 556 (W556S). In the Danish patients, the W556S mutation was present in 12% of 65 possible mutant alleles. The pathogenicity of the W556S mutation, which is located in one of the five conserved motifs Tyr-Trp-Thr-Asp in the epidermal growth factor homology region, was studied in transfected COS-7 cells expressing normal and mutant LDL receptor cDNAs. Results obtained by immunofluorescence flow cytometry and confocal microscopy, as well as by immunoprecipitation, were compatible with complete retention of the mutant protein in the endoplasmic reticulum. The transport-defective W556S mutation and the W23X and W66G mutations seem to account for about 40% of the LDL receptor defects in Danish families with FH.

Ethylmalonic aciduria is associated with an amino acid variant of short chain acyl-coenzyme A dehydrogenase.

Ethylmalonic aciduria is a common biochemical finding in patients with inborn errors of short chain fatty acid beta-oxidation. The urinary excretion of ethylmalonic acid (EMA) may stem from decreased oxidation by short chain acyl-CoA dehydrogenase (SCAD) of butyryl-CoA, which is alternatively metabolized by propionyl-CoA carboxylase to EMA. We have recently detected a guanine to adenine polymorphism in the SCAD gene at position 625 in the SCAD cDNA, which changes glycine 209 to serine (G209S). The variant allele (A625) is present in homozygous and in heterozygous form in 7 and 34.8% of the general population, respectively. One hundred and thirty-five patients from Germany, Denmark, the Czech Republic, Spain, and the United States were selected for this study on the basis of abnormal EMA excretion ranging from 18 to 1185 mmol/mol of creatinine (controls < 18 mmol/mol of creatinine). Among them, we found a significant overrepresentation of the variant allele. Eighty-one patients (60%) were homozygous for the A625 allele, 40 (30%) were heterozygous, and only 14 (10%) harbored the wild-type allele (G625) in homozygous form. By overexpressing the wild-type and variant protein (G209S) in Escherichia coli and COS cells, we showed that the folding of the variant protein was slightly compromised in comparison to the wild-type and that the temperature stability of the tetrameric variant enzyme was lower than that of the wild type. Taken together, the over-representation and the biochemical studies indicate that the A625 allele confers susceptibility to the development of ethylmalonic aciduria.

Cloning and characterization of human very-long-chain acyl-CoA dehydrogenase cDNA, chromosomal assignment of the gene and identification in four patients of nine different mutations within the VLCAD gene.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is one of four straight-chain acyl-CoA dehydrogenase (ACD) enzymes, which are all nuclear encoded mitochondrial flavoproteins catalyzing the initial step in fatty acid beta-oxidation. We have used the very fast, Rapid Amplification of cDNA Ends (RACE) based strategy to obtain the sequence of cDNAs encoding human VLCAD from placenta and fibroblasts. Alignment of the predicted amino acid sequence of human VLCAD with those of the other human ACD enzymes revealed extensive sequence homology. Moreover, human VLCAD and human acyl-CoA oxidase showed extensive sequence homology corroborating the notion that these genes are evolutionarily related. Southern blot analysis of genomic DNA from hybrid cell lines was used to localize the VLCAD gene to human chromosome 17p11.2-p11.13105. Using Northern and Western blot analysis to investigate the tissue specific distribution of VLCAD mRNA and protein in several human tissues we showed that VLCAD is most abundant in heart and skeletal muscle. This agrees well with the fact that cardiac and muscle symptoms are characteristic for patients with VLCAD deficiency. Northern blot analysis and sequencing of cloned PCR amplified VLCAD cDNA from four unrelated patients with VLCAD deficiency showed that VLCAD mRNA was undetectable in one patient and that the other three have mutations in both VLCAD alleles. Western blot analysis of patient fibroblasts showed that the identified mutations result in severely reduced amounts of VLCAD protein. None of the patients harbored identical mutations suggesting that the mutational heterogeneity in VLCAD deficiency is large.

Tetrahymena gene encodes a protein that is homologous with the liver-specific F-antigen and associated with membranes of the Golgi apparatus and transport vesicles.

The F-antigen is a prominent liver protein which has been extensively used in studies on natural and induced immunological tolerance. However, its intracellular localization and biological function have remained elusive. It has generally been assumed that the F-antigen is confined phylogenetically to vertebrates. Now we have cloned and characterized a gene from the ciliated protozoan Tetrahymena thermophila encoding a protein which clearly is homologous with the rat F-antigen. The coding region of the Tetrahymena F-antigen (TF-ag) gene specifies a 46,051 M(r) protein and is interrupted by three introns. In accordance with the predicted molecular mass of the TF-ag protein, antibodies raised against a cro-lacZ'-TF-ag fusion protein specifically recognized a 45,000 M(r) protein in Western blots of total T. thermophila protein. Immunoelectron microscopy demonstrated that the TF-ag is associated with membranes of the Golgi apparatus and transport vesicles pointing to a role of TF-ag in membrane trafficking. Transcription of the TF-ag gene, as determined by run-on analyses, was only detectable in growing cells, and following transfer to starvation condition pre-existing TF-ag mRNA was rapidly degraded. The abundance of the TF-ag protein, however, declined only moderately during prolonged periods of starvation demonstrating that extensive release of the TF-ag did not take place. In combination these results suggest that the TF-ag protein is a recycled constituent of the intracellular membrane network in T. thermophila.