Co-pyrolysis of chicken manure with tree bark for reduced biochar toxicity and enhanced plant growth in Arabidopsis thaliana.

Co-pyrolysis of chicken manure with tree bark was investigated to mitigate salinity and potentially toxic element (PTE) concentrations of chicken manure-derived biochar. The effect of tree bark addition (0, 25, 50, 75 and 100 wt%) on the biochar composition, surface functional groups, PTEs and polycyclic aromatic hydrocarbons (PAH) concentration in the biochar was evaluated. Biochar-induced toxicity was assessed using an in-house plant growth assay with Arabidopsis thaliana. This study shows that PTE concentrations can be controlled through co-pyrolysis. More than 50 wt% of tree bark must be added to chicken manure to reduce the concentrations below the European Biochar Certificate-AGRO (EBC-AGRO) threshold. However, the amount of PAH does not show a trend with tree bark addition. Furthermore, co-pyrolysis biochar promotes plant growth at different application concentrations, whereas pure application of 100 wt% tree bark or chicken manure biochar results in decreased growth compared to the reference. In addition, increased plant stress was observed for 100 wt% chicken manure biochar. These data indicate that co-pyrolysis of chicken manure and tree bark produces EBC-AGRO-compliant biochar with the potential to stimulate plant growth. Further studies need to assess the effect of these biochars in long-term growth experiments.

Application of biochar to anaerobic digestion versus digestate: Effects on N emissions and C stability.

Research attention is growing for biochar as amendment for anaerobic digestion (AD), as it may improve both the AD process and digestate properties. In this study, two biochars (from insect frass or the woody fraction of green waste, both pyrolyzed at 450 °C) were added (5 % w/w) during semi-continuous AD of organic kitchen waste and chicken manure. Biochar was mixed either during the AD process or to the digestate post-treatment. The impact of biochar addition on the AD process, NH3 and greenhouse gas emissions, as well as C and N mineralization following soil application, was examined. Biochar did not affect the biogas yield, but did lead to a reduction in NH3 levels in the biogas and NH4+-N sorption onto the biochars, reducing the risk of AD inhibition. N sorbed as NH4+-N onto the biochar was more available for mineralization than N sorbed as NH3. N sorption on biochar-amended digestates did not result in trade-offs when these digestates were applied to soil: the sorbed N was not released as NH3 or N2O after soil application and was not released as mineral N in either the short or medium term. The C-rich biochars increased the OC content of the digestate, which enhanced its value as soil improver. Additionally, mixing biochar with digestate decreased the soil CO2 emissions up to 33 % when it was added after AD; no priming effects were observed. These findings highlight the potential benefits of incorporating biochar into AD systems and the subsequent application of biochar-amended digestate in agricultural practices.

The screening of various biochars for Cd2+ removal at relevant soil pH.

Fourteen biochars from seven biomass sources were investigated on their long-term Cd2+ removal. The experiments consisted of a ten-day batch Cd2+ adsorption in a pH-buffered solution (pH = 6) to minimise pH effects. Insect frass, spent peat and chicken manure-derived biochars are promising Cd2+ adsorbents. Pyrolysis temperature was crucial for optimising Cd2+ removal by insect frass and spent peat-derived biochars. For these biochars, a pyrolysis temperature of 450 °C was optimal. In contrast, the Cd2+ removal by chicken manure biochars was independent of pyrolysis temperature. The Cd2+ removal by insect-frass and spent peat-derived biochars was associated with chemisorption on surface functionalities, while using chicken manure biochars was more associated with Cd2+ precipitation. The kinetics of Cd2+ removal over the course of ten days showed that insect frass biochar (450 °C) showed a gradual increase from 36 to 75 % Cd2+ removal, while chicken manure and spent peat-derived biochar (450 °C) already showed a higher Cd2+ removal (72 - 89 %) after day 1. This evidences that a long-term Cd2+ removal effect can be expected for some biochars. This should certainly be taken into consideration in future soil-based experiments.

Biochar amendment to cattle slurry reduces NH3 emissions during storage without risk of higher NH3 emissions after soil application of the solid fraction.

Cattle slurry storage is a major source of gaseous N emissions. The aim of this study was to evaluate the effects of biochar, clinoptilolite and elemental sulfur (S°) on (1) NH3 and greenhouse gas emissions during storage of cattle slurry and (2) after soil application of the enriched solid fractions; and (3) on the agronomic quality of the solid and liquid fractions. In the first phase, biochar was added to the slurry (10 g L-1); subsequently in the second phase, clinoptilolite (50 g L-1), S° (1 g L-1) and 40 g L-1 extra biochar were added. Gaseous emissions were monitored by a semi-continuous multi-gas analyzer and the agronomic quality of solid and liquid fractions was assessed after separation. The enriched solid fractions were applied to soil to study the effects on gaseous emissions, N and C mineralization. Amendment of biochar reduced NH3 emissions during cattle slurry storage by 12% during the first 7 days. Extra amendment of biochar, clinoptilolite and S° in combination with biochar resulted in a decrease of NH3 emissions of approximately 20%. The N sorbed from the slurry by the biochar was not released as NH3 during soil application of the solid fractions and was not released as mineral N in the short term (within 28 days). A short-term positive priming effect of biochar on the C mineralization of manure and biochar-manure mixture applied to soil was observed. The biochar-enriched solid fractions contained more C, total and organic N and water-available P with a slow release.

Screening tests for N sorption allow to select and engineer biochars for N mitigation during biomass processing.

Biochar amendment during biomass processing can improve those processes and products, and reduce the emissions of greenhouse gases and NH3, resulting in ecologic and economic benefits. The potential positive effects of biochar are related to NH4+-N and NH3 sorption, which in turn are depending on different biochar characteristics. By knowing the relationship between biochar characteristics and NH4+-N and NH3 sorption, biochar production can be steered towards a higher N sorption or existing biochars can be selected for targeted applications for high N sorption. Therefore, this study aims to develop fast screening tests to estimate the potential for both NH4+-N and NH3 sorption of biochar, before application in biomass processing. Further, the effects of feedstock, pyrolysis temperature, biochar characteristics and biochar treatments on N sorption are studied. The results show that NH4+-N sorption varied between 0 and 1.54 mg NH4+-N/g fresh biochar and was highest for manure-based biochars with a high nutrient content and cation exchange capacity, produced at lower temperatures (300-450 °C). For some biochars, the feedstock itself had a higher NH4+-N sorption than the biochar. Grinding and washing increased the NH4+-N sorption. In addition, a general linear model was proposed to predict the NH4+-N sorption based on three chemical characteristics· NH3 sorption varied between 0 and 100 % of the negative control and showed a linear positive relationship with the NH4+-N sorption, moisture retention factor and cation exchange capacity. Pyrolysis temperature and feedstock type did not significantly affect NH3 sorption. NH4+-N and NH3 desorption varied amongst biochar type.

A comparative techno-economic assessment of biochar production from different residue streams using conventional and microwave pyrolysis.

A comparative techno-economic assessment and Monte Carlo risk analysis is performed on large scale (3 tonne/h) biochar production plants for conventional (CPS) and microwave (MWP) pyrolysis using six different residue streams. Both plants are viable with minimum selling prices between € 436/tonne and € 863/tonne for CPS, and between € 564/tonne and € 979/tonne for MWP. The CPS is therefore more viable than MWP as it is a simpler and more established technology. However, a 20% biochar price increase due to higher biochar quality makes the MWP technology more viable. Nevertheless, the discounted payback period remains higher than this of CPS due to the increased CAPEX. Biochar price is the most important determinant of a biochar production plant's feasibility, motivating the need for economic and market research on biochar prices in function of biochar characteristics to reduce fluctuations in widely varying biochar prices.

Long-term cadmium exposure influences the abundance of proteins that impact the cell wall structure in Medicago sativa stems.

Cadmium (Cd) is a non-essential, toxic heavy metal that poses serious threats to both ecosystems and human health. Plants employ various cellular and molecular mechanisms to minimise the impact of Cd toxicity and cell walls function as a defensive barrier during Cd exposure. In this study, we adopted a quantitative gel-based proteomic approach (two-dimensional difference gel electrophoresis) to investigate changes in the abundance of cell wall and soluble proteins in stems of Medicago sativa L. upon long-term exposure to Cd (10 mg·Cd·kg-1 soil as CdSO4 ). Obtained protein data were complemented with targeted gene expression analyses. Plants were affected by Cd exposure at an early growth stage but seemed to recover at a more mature stage as no difference in biomass was observed. The accumulation of Cd was highest in roots followed by stems and leaves. Quantitative proteomics revealed a changed abundance for 179 cell wall proteins and 30 proteins in the soluble fraction upon long-term Cd exposure. These proteins are involved in cell wall remodelling, defence response, carbohydrate metabolism and promotion of the lignification process. The data indicate that Cd exposure alters the cell wall proteome and underline the role of cell wall proteins in defence against Cd stress. The identified proteins are linked to alterations in cell wall structure and lignification process in stems of M. sativa, underpinning the function of the cell wall as an effective barrier against Cd stress.

Mycorrhization protects Betula pubescens Ehr. from metal-induced oxidative stress increasing its tolerance to grow in an industrial polluted soil.

In recent years, the use of woody plants in phytoremediation has gained popularity due to their high biomass production and their association with mycorrhizal fungi, which can improve their survival and development rates under stress conditions. In this study, mycorrhized and non-mycorrhized white birch plants (Betula pubescens Ehr.) were grown in control and a metal-polluted industrial soil. After 60days of culture, plant growth and metal accumulation, the content of photosynthetic pigments and oxidative-stress markers, as well as the enzymatic activities and gene expressions of antioxidant enzymes were measured. According to our results, mycorrhized birch plants grown in control soil showed an increased activity and gene expression of catalase and ascorbate peroxidase, along with hydrogen peroxide overproduction, which could support the importance of the reactive oxygen species as signaling molecules in the regulation of plant-fungus interactions. Additionally, in polluted soil mycorrhized plants had higher biomass but lower metal accumulation, probably because the symbiotic fungus acted as a barrier to the entrance of metals into the host plants. This behavior led to mitigation in the oxidative challenge, reduced hydrogen peroxide content and diminished activities of the antioxidant enzymes in comparison to non-mycorrhized plants.

The functional role of the photosynthetic apparatus in the recovery of Brassica napus plants from pre-emergent metazachlor exposure.

Metazachlor is a chloroacetamide herbicide, frequently used in Brassica napus cultivations around the world. Its primary target is the inhibition of very long chain fatty acid biosynthesis. This study included a morphological and physiological screening of hydroponically grown B. napus, exposed to a concentration range of 0, 0.25, 0.50, 0.75 and 1.0kg metazachlor per hectare. The results indicate that within a month after application, growth and development of B. napus are severely affected by low metazachlor doses. At intermediate metazachlor concentrations, loss of phosphorous and potassium from the plant tissues suggests destabilisation of cellular membranes, which may be a direct consequence of metazachlor application. This membrane instability could be indirectly linked with alterations of electron transport and a reduction of carbon assimilation. At increased metazachlor doses of 0.75kga.i.ha(-1), pigment concentrations are strongly reduced. However, chlorophyll fluorescence parameters seem to remain unaffected at metazachlor doses up to 0.75kga.i.ha(-1). At a metazachlor concentration of 1.0kga.i.ha(-1), negative effects are observed on all tested parameters, resulting in limited survival. The results indicate photosynthesis is assured at intermediate metazachlor concentrations for the cost of growth and development. It is clear that photosynthesis plays a key role in the survival strategy of young plants to overcome initially induced chemical stress.

Cadmium-induced and trans-generational changes in the cultivable and total seed endophytic community of Arabidopsis thaliana.

Trans-generational adaptation is important to respond rapidly to environmental challenges and increase overall plant fitness. Besides well-known mechanisms such as epigenetic modifications, vertically transmitted endophytic bacteria might contribute to this process. The cultivable and total endophytic communities of several generations of Arabidopsis thaliana seeds harvested from plants exposed to cadmium (Cd) or not exposed were investigated. The diversity and richness of the seed endophytic community decreased with an increasing number of generations. Aeromicrobium and Pseudonocardia were identified as indicator species in seeds from Cd-exposed plants, while Rhizobium was abundantly present in both seed types. Remarkably, Rhizobium was the only genus that was consistently detected in seeds of all generations, which suggests that the phenotypic characteristics were more important as selection criteria for which bacteria are transferred to the next plant generation than the actual genera. Production of IAA was an important trait for endophytes from both seed types, while ACC deaminase activity and Cd tolerance were mainly associated with seed endophytes from Cd-exposed plants. Understanding how different factors influence the seed endophytic community can help us to improve seed quality and plant growth through different biotechnological applications.

Renal cells exposed to cadmium in vitro and in vivo: normalizing gene expression data.

Cadmium (Cd) is a toxic metal with a long half-life in biological systems. This half-life is partly as a result of metallothioneins (MTs), metal-binding proteins with a high affinity for Cd. The high retention properties of the kidneys reside in proximal tubular cells that possess transport mechanisms for Cd-MT uptake, ultimately leading to more Cd accumulation. Researchers have studied MT-metal interactions using various techniques including quantitative real-time PCR (qPCR), an efficient tool for quantifying gene expression. Often a poor choice of reference genes, which is represented by their instability and condition dependency, leads to inefficient normalization of gene expression data and misinterpretations. This study demonstrates the importance of an efficient normalization strategy in toxicological research. A selection of stable reference genes was proposed in order to acquire reliable and reproducible gene quantification under metal stress using MT expression as an example. Moreover, in vitro and in vivo setups were compared to identify the influence of toxicological compounds in function of the experimental design. This study shows that glyceraldehyde-3-phosphate dehydrogenase (Gapdh), tyrosine monooxygenase/tryptophan5-monooxygenase activation-protein, zeta polypeptide (Ywhaz) and beta-actin (Actb) are the most stable reference genes in a kidney proximal tubular cell line exposed to moderate and high Cd concentrations, applied as CdCl2 . A slightly different sequence in reference gene stability was found in renal cells isolated from rats in vivo exposed to Cd. It was further shown that three reference genes are required for efficient normalization in this experimental setup. This study demonstrates the importance of an efficient normalization strategy in toxicological research.

The effect of long-term Cd and Ni exposure on seed endophytes of Agrostis capillaris and their potential application in phytoremediation of metal-contaminated soils.

We examined whether long-term Cd exposure leads to beneficial changes in the cultivable endophytic bacteria present in the seeds of Agrostis capillaris. Therefore the cultivable seed endophytes of Agrostis capillaris growing on a long-term Cd/Ni-contaminated plot (Cd/Ni seeds) were compared with those originating from a non-contaminated plot (control seeds). We observed plant- and contaminant-dependent effects on the population composition between control and Cd/Ni seeds. Also differences in phenotypic characteristics were found: endophytes from Cd/Ni seeds exhibited more ACC deaminase activity and production of siderophores and IAA, while endophytes from control seeds, very surprisingly, showed more metal tolerance. Finally, the 3 most promising seed endophytes were selected based on their metal tolerance and plant growth promoting potential, and inoculated in Agrostis capillaris seedlings. In case of non-exposed plants, inoculation resulted in a significantly improved plant growth; after inoculation of Cd-exposed plants an increased Cd uptake was achieved without affecting plant growth. This indicates that inoculation of Agrostis with its seed endophytes might be beneficial for its establishment during phytoextraction and phytostabilisation of Cd-contaminated soils.

Changes in the population of seed bacteria of transgenerationally Cd-exposed Arabidopsis thaliana.

Plant-associated bacteria can have beneficial effects on the growth and health of their host. Nevertheless, the role of endophytic bacteria present in seeds has not been investigated in depth. In this study, the cultivable endophytic population of seeds from Arabidopsis thaliana exposed to 2 µm cadmium for several generations (Cd seeds) was compared with a population isolated from seeds of plants that were never exposed to Cd (control seeds). We observed obvious differences between the two types of seed concerning genera present and phenotypic characteristics of the different isolates. Sinorhizobium sp. and Micrococcus sp. were only found in control seeds, while Pseudomonas sp., Bosea sp. and Paenibacillus sp. were only found in Cd seeds. Sphingomonas sp., Rhizobium sp., Acidovorax sp., Variovorax sp., Methylobacterium sp., Bacillus sp. and Staphylococcus sp. occurred in varying numbers in both types of seed. Metal tolerance and 1-aminocyclopropane-1-carboxylate deaminase activity were predominantly found in strains isolated from Cd seeds, while the production of siderophores, indole-3-acetic acid and organic acids was more prevalent in endophytes isolated from control seeds. These data support the hypothesis that certain endophytes are selected for transfer to the next generation and that their presence might be important for subsequent germination and early seedling development.

Phytoextraction of toxic metals: a central role for glutathione.

Phytoextraction has a promising potential as an environmentally friendly clean-up method for soils contaminated with toxic metals. To improve the development of efficient phytoextraction strategies, better knowledge regarding metal uptake, translocation and detoxification in planta is a prerequisite. This review highlights our current understanding on these mechanisms, and their impact on plant growth and health. Special attention is paid to the central role of glutathione (GSH) in this process. Because of the high affinity of metals to thiols and as a precursor for phytochelatins (PCs), GSH is an essential metal chelator. Being an important antioxidant, a direct link between metal detoxification and the oxidative challenge in plants growing on contaminated soils is observed, where GSH could be a key player. In addition, as redox couple, oxidized and reduced GSH transmits specific information, in this way tuning cellular signalling pathways under environmental stress conditions. Possible improvements of phytoextraction could be achieved by using transgenic plants or plant-associated microorganisms. Joined efforts should be made to cope with the challenges faced with phytoextraction in order to successfully implement this technique in the field.

Oxidative stress reactions induced in beans (Phaseolus vulgaris) following exposure to uranium.

The present study aimed to analyze the biological effects induced by bioaccumulation of uranium in Phaseolus vulgaris. Ten-day-old seedlings were exposed to 0, 0.1, 1, 10, 100 and 1000 microM U in diluted Hoagland solution. Following 1, 2, 4 and 7 days' exposure, plants were monitored for uranium uptake, biometric parameters, capacities of enzymes involved in the anti-oxidative defense mechanisms (GPOD, SPOD, GLUR, SOD, ICDH, G-6P-DH), glutathione (GSH) pool and DNA integrity. Uranium contents were up to 900-fold higher in roots (31-14,916 mg kg(-1) FW following 7 days' exposure to 0.1 and 1000 microM U, respectively) as compared to primary leaves (1-16 mg kg(-1) FW following 7 days' exposure to 0.1 and 1000 microM U, respectively). Uranium exposure did not significantly affect plant growth compared to the control. For all enzymes studied, except SOD, enzyme capacities in roots were slightly stimulated with increasing contaminant concentrations (though not significantly). For roots exposed to 1000 microM U, enzyme capacities were significantly reduced. Enzyme capacities in leaves were not affected by uranium treatment. Total and reduced GSH levels were higher in primary leaves of uranium (

The chemical behaviour of heavy metals plays a prominent role in the induction of oxidative stress.

It is often described that different environmental stress factors stimulate the production of reactive oxygen species and increase the activity of several enzymes quenching these radicals. The ascorbate-glutathione pathway is also involved in plant defence against oxidative stress. Therefore the effects of 2 metals (Cu, Zn) with different chemical behaviour were investigated on the enzymes of this pathway in the primary leaves of bean seedlings grown on hydroponics and supplied with a 50 microM concentration of both metals. The results obtained demonstrate that the capacities of the enzymes involved in the ascorbate-glutathione pathway increase after metal application, indicating that they induce oxidative stress indeed. However striking differences in the relative induction time of these enzymes suggest that the chemical behaviour of the metals applied, plays an important role in the induction of oxidative stress as well as in the defence mechanism against it.