An Investigation of the Soil Property Changes and Heavy Metal Accumulation in Relation to Long-term Wastewater Irrigation in the Semi-arid Region of Iran

Concentrations and spatial distribution of Zn, Cu, Cd, and Pb along two landscapes including a wastewater-irrigated area and a control area were determined to assess the impact of long-term wastewater irrigation and landscape properties on heavy metal contamination. Some disturbed and undisturbed soil samples were taken from soil trenches and soil cores, located on three main landscape positions (upper slope, midslope, and lower slope) in northwestern Iran. The investigation showed that the mean concentration of the heavy metals followed the order Zn > Cu > Pb > Cd in the wastewater-irrigated soil and Pb > Zn > Cu > Cd in the control soils. On average, compared to similar positions in the control region, the wastewater-irrigated regions contained 3.0 (midslope) to 4.9 (lower slope), 2.7 (midslope) to 4.6 (lower slope), 3.3 (upper slope) to 4.1 (lower slope), and 1.7 (upper slope) to 2.6 (lower slope) times higher amounts of Zn, Cu, Cd, and Pb, respectively. Significant positive relationships (P < 0.05) were recorded between the heavy metals concentration with <0.002 mm particle-size fraction and organic matter content, the fractions linked to runoff and soil erosion. It is believed that the two soil fractions play a crucial role in the distribution of the metals along the wastewater-irrigated landscape. Despite the significant increase of heavy metals (P < 0.05) in the wastewater-irrigated soils compared with control soils, the concentration of all evaluated metals was below the maximum accepted limits (Zn < 300 mg/kg, Cu < 100 mg/kg, Cd < 5 mg/kg, and Pb < 100 mg/kg), and grouped as “not-enriched” to “moderately-enriched” categories regarding the topsoil enrichment index. Overall, the lower slope was shown to be more contaminated with the heavy metals compared to the other positions.     

The Influence of Soil Properties on the Mobility of Metals Following a Single Application of Polluted Sewage Sludge to Seventy Agricultural Topsoils: A Laboratory Column Study

The main aim of this study was to examine the influence of soil properties on the leaching of Cd, Cr, Cu, Ni, Pb, and Zn following the application of polluted sewage sludge to contrasting topsoils. Seventy agricultural soil samples from different parts of Spain were amended with a single dose of sewage sludge (equivalent to 50 t dry weight ha^?1) and a column study was performed under controlled conditions. After two, four, and six months of incubation, 283 ml of distilled water (equivalent to a rainfall event of 25 l m^?2) was applied. The leachates were then collected and analyzed for metals. For all of the soils considered, the pH was the most important parameter for the control of mobility metals (except for Cu, determined by the sand and soil organic carbon and only to a lesser extent by the soil pH r² = 0.604, p < 0.001) and was negatively related to all of the studied metals. For Pb and Zn, soil pH was the single soil property explaining their mobility (r² = 0.411, p < 0.001 for Pb; r² = 0.713, p < 0.001 for Zn) while for Cd, Cr and Ni, EC, sand and silt also appeared in the statistical models (r² = 0.753, p < 0.001 for Cd; r² = 0.366, p < 0.001 for Cr; r² = 0.784, p < 0.001 for Ni). In the basic soils, soil texture was the most important soil property controlling the mobility of metals (except for that of Pb, which it only weakly predicted). For the acidic-neutral soils, the soil pH was the most important soil property controlling metal mobility (except for that of Cr, which was mainly determined by the pseudo-total Cr content).     

Detection of adeno- and lentiviral (HIV1) contaminations on laboratory surfaces as a tool for the surveillance of biosafety standards

Aims: As a biosafety laboratory, we survey the handling of adenovirus type 5 (Ad5) and HIV1‐derived lentivirus in contained‐use facilities in Switzerland to identify insufficiencies of the safety precautions taken by the laboratories. Methods and Results: In the past 9 years, we took 430 swab samples from various types of surfaces in research laboratories. Samples were examined for Ad5 contaminations by real‐time PCR and infectivity assay or for the presence of lentivirus (HIV1) nucleic acids by real‐time (RT) PCR. Samples collected from centrifuges did not only contain Ad5 DNA more frequently but also exhibited higher numbers of Ad5 and lentiviral (HIV1) DNA copies than swabs from any other area of sampling. Five of ten samples containing infectious Ad5 particles or lentivirus (HIV1) RNA were found in samples taken from centrifuges. Ad5 contamination rates were higher in the tube holder and lower on the inner wall of the rotor chamber in centrifuges that were fitted with aerosol tight covers compared to centrifuges without covers. Conclusions: Our results allowed the comparison of hygiene standards of different laboratories and lead to the identification of centrifuges as hotspots for contaminations. Significance and Impact of the Study: Based on our results, we propose to use the collected data as a tool for rating future swab results. Furthermore, the amount of Ad5 and HIV1‐derived lentivirus DNA could serve as an indicator of the level of good laboratory practice in contained‐use laboratories handling these viral vectors.     

Efficient proteolysis using a regenerable metal-ion chelate immobilized enzyme reactor supported on organic-inorganic hybrid silica monolith

A metal‐ion chelate immobilized enzyme reactor (IMER) supported on organic–inorganic hybrid silica monolith was developed for rapid digestion of proteins. The monolithic support was in situ prepared in a fused silica capillary via the polycondensation between tetraethoxysilane hydrolytic sol and iminodiacetic acid conjugated glycidoxypropyltrimethoxysilane. After activated by Cu²⁺, trypsin was immobilized onto the monolithic support via metal chelation. Proteolytic capability of such an IMER was evaluated by the digestion of myoglobin and BSA, and the digests were further analyzed by microflow reversed‐phase liquid chromatography with ESI‐MS/MS. Similar sequence coverages of myoglobin and BSA were obtained by IMER, in comparison to those obtained by in‐solution digestion (91 versus 92% for 200 ng myoglobin, and 26 versus 26% for 200 ng BSA). However, the digestion time was shortened from 12 h to 50 s. When the enzymatic activity was decreased after seven runs, the IMER could be easily regenerated by removing Cu²⁺ via EDTA followed by trypsin immobilization with fresh Cu²⁺ introduced, yielding the equal sequence coverage (26% for 200 ng BSA). For ～5 μg rat liver extract, even more proteins were identified with the immobilized trypsin digestion within 150 s in comparison to the in‐solution digestion for 24 h (541 versus 483), demonstrating that the IMER could be a promising tool for efficient and high‐throughput proteome profiling.     

Putative copper- and zinc-binding motifs in Streptococcus pneumoniae identified by immobilized metal affinity chromatography and mass spectrometry

The aim of metalloproteomics is to identify and characterize putative metal-binding proteins and metal-binding motifs. In this study, we performed a systematical metalloproteomic analysis on Streptococcus pneumoniae through the combined use of efficient immobilized metal affinity chromatography enrichment and high-accuracy linear ion trap-Orbitrap MS to identify metal-binding proteins and metal-binding peptides. In total, 232 and 166 putative metal-binding proteins were respectively isolated by Cu- and Zn-immobilized metal affinity chromatography columns, in which 133 proteins were present in both preparations. The putative metalloproteins are mainly involved in protein, nucleotide and carbon metabolisms, oxidation and cell cycle regulation. Based on the sequence of the putative Cu- and Zn-binding peptides, putative Cu-binding motifs were identified: H(X)mH (m=0-11), C(X)(2) C, C(X)nH (n=2-4, 6, 9), H(X)iM (i=0-10) and M(X)tM (t=8 or 12), while putative Zn-binding motifs were identified as follows: H(X)mH (m=1-12), H(X)iM (i=0-12), M(X)tM (t=0, 3 and 4), C(X)nH (n=1, 2, 7, 10 and 11). Equilibrium dialysis and inductively coupled plasma-MS experiments confirmed that the artificially synthesized peptides harboring differential identified metal-binding motifs interacted directly with the metal ions. The metalloproteomic study presented here suggests that the comparably large size and diverse functions of the S. pneumoniae metalloproteome may play important roles in various biological processes and thus contribute to the bacterial pathologies.     

Zinc-induced Cell Death in Rice (<Emphasis Type="Italic">Oryza Sativa</Emphasis> L.) Roots

Cell death in rice roots due to zinc (Zn) toxicity was investigated using inhibitors of signal molecules known to regulate programmed cell death in plants. Zn (5.0– 25.0 mM) induced cell death in a dose- and time-dependent manner. Sodium benzoate, a scavenger of reactive oxygen species (ROS), increased the cell viability under toxic Zn level (25.0 mM), suggesting a role of ROS in Zn-induced cell death. The protective role of rotenone in cell death indicated the involvement of mitochondrial electron transport chain in this Zn-induced ROS generation. Cantharidin and endothall, two serine/threonine phosphatase inhibitors, and sodium orthovanadate (Na₃VO₄) and phenylarsine oxide (PAO), two protein tyrosine phosphatase inhibitors, blocked Zn-induced root cell death. Conversely, K252-a, a serine/threonine kinase inhibitor, increased Zn-induced cell death. Furthermore, the phosphatidylinositol 3-Kinase (PI-3K) inhibitors, LY 294002 and wortmannin inhibited Zn-induced root cell death. These results suggest that the ROS, protein phosphatase and PI-3K may function in the Zn-induced cellular toxicity in rice roots.     

Variations in plant metallothioneins: the heavy metal hyperaccumulator <Emphasis Type="Italic">Thlaspi caerulescens</Emphasis> as a study case

Plant metallothioneins (MTs) are extremely diverse and are thought to be involved in metal homeostasis or detoxification. Thlaspi caerulescens is a model Zn/Cd hyperaccumulator and thus constitutes an ideal system to study the variability of these MTs. Two T. caerulescens cDNAs (accession: 665511; accession: 665515), that are highly homologous to type 1 and type 2 Arabidopsis thaliana MTs, have been isolated using a functional screen for plant cDNAs that confer Cd tolerance to yeast. However, TcMT1 has a much shorter N-terminal domain than that of A. thaliana and so lacks Cys motifs conserved through all the plant MTs classified as type 1. A systematic search in plant databases allowed the detection of MT-related sequences. Sixty-four percent fulfil the criteria for MT classification described in Cobbett and Goldsbrough (2002) and further extend our knowledge about other conserved residues that might play an important role in plant MT structure. In addition, 34% of the total MT-related sequences cannot be classified strictly as they display modifications in the conserved residues according to the current plant MTs’ classification. The significance of this variability in plant MT sequences is discussed. Functional complementation in yeast was used to assess whether these variations may alter the MTs’ function in T. caerulescens. Regulation of the expression of MTs in T. caerulescens was also investigated. TcMT1 and TcMT2 display higher expression in T. caerulescens than in A. thaliana. Moreover, their differential expression patterns in organs and in response to metal exposure, suggest that the two types of MTs may have diverse roles and functions in T. caerulescens.     

Metal-ion-mediated oxidative stress in the gill homogenate of rainbow trout (Oncorhynchus mykiss)

Human activities play a major role in toxic and carcinogenic metal pollution of the environment. This study was undertaken to evaluate the effects of copper and mercury at the 400-to 1000-μM concentration range on some biochemical markers of oxidative stress, such as lipid peroxidation (LPO), glutathione-S-transferase (GST) activity, and reduced glutathione (GSH) content in the rainbow trout gill homogenates with or without supplementation of manganese, selenium, and bovine serum albumin (BSA). The integrity of DNA was also measured to assess metal ion toxicity. The results showed that the LPO and specific activity of GST were elevated. This indicated that cell-protecting antioxidant mechanisms were overtaxed and could not prevent membrane peroxidation. Following the addition of metals, the GSH content was also significantly reduced in a concentration-dependent manner. Mercury was found to be more effective than copper. The application of antioxidants proved beneficial in inhibiting LPO, reducing GST activity, and elevating the GSH levels in the gill samples. Manganese was more effective than selenium and BSA. Surprisingly, when BSA (1.0%) was added to the gill homogenates treated with a 1000-μM concentration of metal ions, instead of alleviating malondialdehyde (MDA) generation, a drastic elevation in the MDA levels, alleviation in GST activity, and a further decrease in glutathione (GSH) levels were observed, which were most likely the result of pro-oxidant activity of BSA. The results also indicated that mercury and copper functioned as genotoxic pollutants, which altered the DNA integrity by inducing the single and double-stranded DNA breaks in the gill cell nuclei. Collectively, toxicity of metal ions is related to the depletion of GSH content and inhibition of antioxidant enzyme GST, resulting in the propagation of LPO and DNA damage.     

Ethanol production in multistage continuous, single stage continuous, Lactobacillus-contaminated continuous, and batch fermentations

Saccharomyces cerevisiae-based ethanol fermentations were conducted in batch culture, in a single stage continuous stirred tank reactor (CSTR), a multistage CSTR, and in a fermentor contaminated with Lactobacillus that corresponded to the first fermentor of the multistage CSTR system. Using a glucose concentration of 260 g l^–1 in the medium, the highest ethanol concentration reached was in batch (116gl^–1), followed by the multistage CSTR (106gl^–1), and the single stage CSTR continuous production system (60gl^–1). The highest ethanol productivity at this sugar concentration was achieved in the multistage CSTR system where a productivity of 12.7gl^–1h^–1 was seen. The other fermentation systems in comparison did not exceed an ethanol productivity of 3gl^–1h^–1. By performing a continuous ethanol fermentation in multiple stages (having a total equivalent working volume of the tested single stage), a 4-fold higher ethanol productivity was achieved as compared to either the single stage CSTR, or the batch fermentation.     

Pb hyperaccumulation and tolerance in common buckwheat (Fagopyrum esculentum Moench)

Common buckwheat grown in Pb-contaminated soil was found to accumulate a large amount of Pb in its leaves (8,000 mg/kg DW), stem (2,000 mg/kg DW), and roots (3,300 mg/kg DW), without significant damage. This indicates that buckwheat is a newly recognized Pb hyperaccumulator, which is defined as a plant containing over 1,000 mg/kg of Pb in its shoots on a dry-weight basis. Moreover, it was shown that application of the biodegradable chelator methylglycinediacetic acid trisodium salt at concentrations of up to 20 mmol/kg resulted in a more than five times higher concentration of Pb in the shoot without notable growth inhibitation at up to 10 mmol/kg. These results indicate that buckwheat is a potential phytoremediator of Pb-contaminated soils.