Human biomonitoring of trace metals from different altitudinal settings of Pakistan.

Human biomonitoring of toxic trace elements is of critical importance for public health protection. The current study aims to assess the levels of selected trace metals (Cd, Co, Cr, Cu, Mn, Ni, Pb, and Zn) into paired human nail and hair samples (n = 180 each) from different altitudinal setting along the Indus River, and which were measured by using inductively coupled plasma mass spectrometry (ICP-MS). The human samples (hair and nail) were collected from four different ecological zones of Pakistan which include frozen mountain zone (FMZ), wet mountain zone (WMZ), riverine delta zone (RDZ), and low-lying southern areas (LLZ). Our results showed the following occurrence trends into studied hair samples: higher values (ppm) of Zn (281), Co (0.136), and Mn (5.65) at FMZ; Cr (1.37), Mn (7.83), and Ni (1.22) at WMZ; Co (0.15), Mn (11.89), and Ni (0.99) at RDZ; and Mn (8.99) and Ni (0.90) at LLZ. While in the case of nails, the levels (ppm) of Mn (9.91) at FMZ and Mn (9.38, 24.1, and 12.5), Cr (1.84, 3.87, and 2.33), and Ni (10.69, 8.89, and 12.6) at WMZ, RDZ and LLZ, respectively, showed higher concentration. In general, among the studied trace elements, Mn and Ni in hair/nail samples were consistently higher and exceeded the WHO threshold/published reference values in most of the studied samples (> 50-60%) throughout the Indus basin. Similarly, hair/nail Pb values were also higher in few cases (2-10%) at all studied zones and exceeded the WHO threshold/published reference values. Our area-wise comparisons of studied metals exhibited altitudinal trends for Cd, Cr, Zn, and Mn (p < 0.05), and surprisingly, the values were increasing from south to north (at higher altitudes) and indicative of geogenic sources of the studied toxic elements, except Mn, which was higher at lower floodplain areas. Estimated daily intake (EDI) values showed that food and drinking water had the highest contribution towards Zn, Cu, Mn, and Ni and accumulation at all studied zones. Whereas, dust also acts as the main exposure route for Mn, Co, Cr, and Cd followed by the food, and water.

Higher cadmium and zinc accumulation in parsley (Petroselinum crispum) roots activates its antioxidants defense system.

Parsley (Petroselinum crispum) is herb with many biological and medicinal benefits for humans. However, growth on zinc (Zn) and cadmium (Cd) contaminated sites might get severely affected due to over accumulation of heavy metals (HM) in different plant tissues. Antioxidants play a crucial role in minimizing the negative effects of HM. The present study investigates the effects of Zn and Cd stress on P. crispum morphological parameters, enzymatic/non-enzymatic antioxidant profiling and metal accumulation in shoot/root. Plants were exposed to different concentrations of Zn (50, 100, 150 and 200 µM) and Cd (10, 20, 40 and 80 µM) along with control (no stress), in soil-less Hoagland's solution. The results showed that Zn and Cd substantially decrease the growth parameters with increased contents of malondialdehyde (MDA), hydrogen peroxide (H2O2), and electrolyte leakage (EL). Non-enzymatic antioxidant activities, like total phenolic contents (TPC) and ferric reducing antioxidant power (FRAP), were induced high in leaves only upon Cd stress and contrarily decreased upon Zn stress. Total flavonoid contents (TFC) were decreased under Zn and Cd stress. Enzymatic antioxidant activities like superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were also strongly induced upon Cd stress. At the same time, SOD and guaiacol peroxidase (GPX) activity was induced significantly upon Zn stress. Cd uptake and accumulation was notably high in roots as compared to shoots, which suggests P. crispum have a reduced ability to translocate Cd towards aboveground parts (leaves). Additionally, strong induction of antioxidants by P. crispum under Cd stress might indicate the capacity to effectively re-modulate its physiological response. However, further investigations regarding other HMs and experiments at the molecular level are still needed.

Characterization of alkaline metalloprotease isolated from halophilic bacterium Bacillus cereus and its applications in various industrial processes.

Microbial proteases are one of the most demanding enzymes for various industries with diverse applications in food, pharmaceutics, and textile industries to name the few. An extracellular alkaline metalloprotease was produced and purified from moderate halophilic bacterial strain, Bacillus cereus TS2, with some unique characteristics required for various industrial applications. The protease was produced in basal medium supplemented with casein and was partially purified by ion exchange chromatography followed by ammonium sulphate precipitation. The alkaline metalloprotease has molecular weight of 35 kDa with specific activity of 535.4 µM/min/mg. It can work at wide range of pH from 3 to 12, while showing optimum activity at pH 10. Similarly, the alkaline metalloprotease is stable till the temperature of 80 °C and works at wide range of temperature from 20 to 90 °C with optimum activity at 60 °C. The turnover rate increases in the presence of NaCl and Co+2 with k cat/KM of 1.42 × 103 and 1.27 × 103 s-1.M-1 respectively, while without NaCl and Co+2 it has a value of 7.58× 102. The alkaline metalloprotease was relatively resistant to thermal and solvent mediated denaturation. Applications revealed that the metalloprotease was efficient to remove hair from goat skin, remove blood stains and degrade milk, thus can be a potential candidate for leather, detergent, and food industry.

Amides as modifiable directing groups in electrophilic borylation.

Amide directed C-H borylation using ≥two equiv. of BBr3 forms borenium cations containing a R2N(R')C[double bond, length as m-dash]O→B(Ar)Br unit which has significant Lewis acidity at the carbonyl carbon. This enables reduction of the amide unit to an amine using hydrosilanes. This approach can be applied sequentially in a one-pot electrophilic borylation-reduction process, which for phenyl-acetylamides generates ortho borylated compounds that can be directly oxidised to the 2-(2-aminoethyl)-phenol. Other substrates amenable to the C-H borylation-reduction sequence include mono and diamino-arenes and carbazoles. This represents a simple method to make borylated molecules that would be convoluted to access otherwise (e.g. N-octyl-1-BPin-carbazole). Substituent variation is tolerated at boron as well as in the amide unit, with diarylborenium cations also amenable to reduction. This enables a double C-H borylation-reduction-hydrolysis sequence to access B,N-polycyclic aromatic hydrocarbons (PAHs), including an example where both the boron and nitrogen centres contain functionalisable handles (N-H and B-OH). This method is therefore a useful addition to the metal-free borylation toolbox for accessing useful intermediates (ArylBPin) and novel B,N-PAHs.

Effect of low levels of oxytetracycline on anaerobic digestion of cattle manure.

Cattle manure is rich in organic matter and nutrients, but it may also contain harmful substances such as residual antibiotics and other toxic compounds. Oxytetracycline (OTC) is a widely used veterinary antibiotic and its presence in manure can affect the subsequent anaerobic digestion process. This study evaluated the effect of OTC concentrations viz. 0.12, 0.3, 0.6, 1.2, 3, 6 and 12 mg L-1 on batch mesophilic anaerobic digestion of cattle manure. The results showed that cumulative biogas yield decreased by 25, 29 and 55% at 3, 6 and 12 mg OTC L-1 in contrast to control. Volatile solids removal was 39% for control which reduced to 13% in 12 mg L-1 OTC spiked reactor. Effect on stability parameters was significant at OTC concentrations from 1.2 to 12 mg L-1. Two different kinetic models were used for biogas data validation and the modified Gompertz model best fitted to the experimental data.

Lead tolerant endophyte Trametes hirsuta improved the growth and lead accumulation in the vegetative parts of Triticum aestivum L.

Rapid industrialization and increasing population are continuously adding contaminants to our environment. Among those, heavy metals are considered to be one of the serious threats to the ecosystem due to their persistent nature. Microbe assisted phytoremediation is an effective tool for metal remediation as microbes enhance the metal availability and uptake to the host plants or reduce it by binding them intracellularly or extracellularly. An endophytic fungus, Trametes hirsuta, was isolated from Chenopodium album L. plant growing in the lead (Pb) contaminated soil of an industrial area. This is the first study citing Trametes hirsuta, as a root endophyte of Chenopodium album L. This endophytic fungus was found to be tolerant to high concentration of Pb i.e., 1500 mg L-1, when tested in-vitro. Wheat (Triticum aestivum L.) seedlings were infected by Trametes hirsuta and Pb tolerance was observed. With the fungal inoculation plants cumulative growth and total chlorophyll content increased by 24% and 18%, respectively as compared to their respective non-inoculated controls at 1000 mg kg-1 Pb. Similary, 50% more Pb accumulation was measured in the shoots of fungal inoculated plants at 1500 mg kg-1 Pb as compared to control. Thus, the results of the present study suggest that mutualism with endophytic fungi can improve the survival of host plants in metal contaminated soils, additionally it can also assist the phytoextraction of heavy metals from polluted sites by increasing their uptake by the host plants.

Enhanced uptake of Cd, Cr, and Cu in Catharanthus roseus (L.) G.Don by Bacillus cereus: application of moss and compost to reduce metal availability.

Heavy metals (HMs) being the notorious and toxic are being introduced into the environment credited to natural and anthropogenic activities. The use of ornamental plants is being ignored as potential candidates for HMs phytoremediation. In this study, pot experiments were conducted on Catharanthus roseus (L.) G.Don to evaluate selected heavy metals tolerance and accumulation potential with reference to the bacterial endophyte (Bacillus cereus) and organic amendments (moss and compost at 5% v/v). Results indicated improvement in uptake of Cd (230 mg kg-1), Cu (229 mg kg-1), and Cr (458 mg kg-1) by C. roseus with B. cereus. The concentration of Ni and Pb was found highest in controls (without strain) that were 420 and 904 mg kg-1, respectively. Conversely, the addition of organic amendments enhanced biomass production, as compared to controls, 441, 471, and 763% by peat moss (T3), compost (T4), and peat moss + compost + inoculum treatments (T6), respectively, while reduction of plant HMs content was observed. Microbial-aided phytoremediation/phytoextraction could be a potential method for removal of Cd, Cr, and Cu, while organic amendments can significantly improve plant growth in the presence of heavy metals.

Reduced growth response of ornamental plant Nicotiana alata L. upon selected heavy metals uptake, with co-application of ethylenediaminetetraacetic acid.

The use of ornamental plant will increase with the improvement in living standards in green and blue-green infrastructure of urban settings. Nicotiana alata is an ornamental plant, frequently grown as a model plant for horticulture, medicine, and scientific research studies throughout the world. Despite its popularity, little is known about the response of N. alata against heavy metals (HMs). This work is based on the hydroponic study to identify the impacts of selected HMs (Cd, Cr, Cu, Ni and Pb) on N. alata, at 0, 50 and 100 µM concentration, with the co-application of EDTA, at 0 and 2.5 mM in hydroponics system. The HMs uptake was found to be dose dependent, with significant higher uptake at 100 µM of respective HM. Highest cumulative uptake (mg kg-1 of HMs in root, shoot, and leaf dried weight) noted were 767.50 ±â€¯50.83, 862.30 ±â€¯23.83, 271.29 ±â€¯18.68, 1117.49 ±â€¯46.10 and 2166.81 ±â€¯102.09, for Cd, Cr, Cu, Ni, and Pb at 100 µM, respectively. It was identified that EDTA co-application with HMs resulted in boosted HMs uptake, with cumulative uptake percentage increment of 41.62, 54.67, 53.98, 34.48 and 19.92% for 100 µM of Cd, Cr, Cu, Ni, and Pb, respectively. Higher uptake led to negative impact on plant physiology, photosynthetic pigments, and higher lipid peroxidation, H2O2 contents, and electrolyte leakage that increased the stress. Higher HMs uptake induced higher antioxidant enzymatic response. It is recommended to incorporate appropriate soil modification to grow N. alata in sustainable infrastructures.

Influence of Pseudomonas japonica and organic amendments on the growth and metal tolerance of Celosia argentea L.

In this study, a pot experiment was piloted in a greenhouse to evaluate the potential of Celosia argentea var. cristata L. for tolerating/accumulating heavy metals in synthetic wastewater in the presence of Pseudomonas japonica and organic amendment, i.e., moss and compost. Two-week-old seedlings were transferred to pots, and after 4 weeks, the bacterial strain was inoculated, then watered with synthetic wastewater for 5 weeks and harvested after 9 weeks. After harvesting, physiological and biochemical parameters, as well as metal contents of plants, were quantified. The results indicated highest growth and biomass production in moss- and compost-associated plants while highest metal uptake has been found in the presence of P. japonica and synthetic wastewater-irrigated plants. Synthetic wastewater-irrigated plants have shown highest Pb uptake of 2899 mg kg-1 DW, while with P. japonica in soil those plants have shown highest Cd, Cu, Ni, and Cr uptake of 962, 1479, 1042, and 956 mg kg-1 DW, respectively. The production of antioxidant enzymes, i.e., catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), and glutathione-s-transferase (GST), was high in P. japonica-amended plants because of increased uptake of metals. It is concluded that moss and compost have improved growth while P. japonica improved metal accumulation and translocation to aerial parts with little involvement in plant growth.

Immobilized cells of a novel bacterium increased the degradation of N-methylated carbamates under low temperature conditions.

Carbamates are synthetic pesticides, extensively used throughout the world due to their broad specificity against various insect pests. However, their enormous and inadequate use have made them a potential threat to the environment. At low temperature, degradation of carbamates becomes difficult mainly because of low biological activity. In the present study, we isolated a bacterial strain from a low temperature climate where the N-methylated carbamates are used for crop protection. The bacterium, was identified as Pseudomonas plecoglossicida strain (TA3) by 16S rRNA analysis. Degradation experiments with both free and immobilized cells in minimal salt medium indicated that the strain TA3 utilized carbaryl, carbofuran and aldicarb as both carbon and nitrogen source. TA3 can grow well at 4 °C and demonstrated the ability to degrade three carbamates (50 µgml-1) at low temperature. The immobilized cells were found more efficient than their free cells counter parts. Immobilized cells has ability to degrade 100% of carbamates at 30 °C while 80% at 4 °C but incase of their free cells counter parts the efficiency to degrade carbamates was less which was 60% at 4 °C and 80% at 30 °C. TA3 free cellsextract also depicted high activity against all the three carbamates even at 4 °C indicating a possible enzymatic mechanism of degradation.

Soil amendments enhanced the growth of Nicotiana alata L. and Petunia hydrida L. by stabilizing heavy metals from wastewater.

Due to the non-degradable nature of heavy metals (HMs), the industrial effluent, whether treated or untreated, carrying HMs, eventually end up into the water bodies, soil, and sediments. Numerous countermeasures were applied, but the use of ornamental plants for the stress mitigation associated with HMs on the environment is a neglected research domain. The composition of wastewater influences bioremediation strategies. As the wastewater is contaminated with multiple HMs, many lab studies, with the plants, failed in the industrial field. This work focuses on the potential of Nicotiana alata L. and Petunia hydrida L. against multiple HMs contaminated synthetic wastewater. To improve plant tolerance, soil amendments (biochar, compost, and moss, each at 5% v/v in soil) were used, individually and in combination. After 6 weeks of the exposure, plant physiological, biochemical and enzymatic parameters, as well as the distribution of HMs, (Cd, Cr, Cu, Pb, Mn, Ni, and Zn) in the plant (flower, leaves, root, and shoot) and soil, were measured. The HMs uptake positivity influenced the malondialdehyde content, hydrogen peroxide content and electrolyte leakage, while negatively to photosynthetic pigments, and resulted in increased catalase, guaiacol peroxidase, glutathione s-transferase, ascorbate peroxidase, while reduced superoxide dismutase activity. It was found that all amendments improved the plant growth by metal stabilization, and best results were obtained with the combined application of biochar + compost + moss. So, HMs stabilization can be achieved by growing ornamental plants, like Nicotiana alata L. and Petunia hydrida L. along with soil amendments.

Analysis of Arabidopsis thaliana HKT1 and Eutrema salsugineum/botschantzevii HKT1;2 Promoters in Response to Salt Stress in Athkt1:1 Mutant.

Soil salinity imposes a serious threat to the productivity of agricultural crops. Among several other transporters, high-affinity K+ transporter (HKT)'s play an important role in reducing the phytotoxicity of Na+. Expression of Eutrema salsugineum (a halophyte) HKT1;2 is induced upon salt exposure. To elucidate the role of its promoter, we compared the sequences of HKT1;2 promoters from E. salsugineum (1822 bp) and E. botschantzevii (1811 bp) with Arabidopsis thaliana HKT1;1 (846 bp) promoter. In silico analysis predicted several cis-acting regulatory elements (GT-1 elements, core motifs of DRE/CRT, MYC/MYB-recognition sites and ACGT elements). Activities of the three promoters were analyzed by measuring HKT1;1 and/or HKT1;2 transcript level in the Athkt1;1 mutant plants. NaCl tolerance of the transgenics was also assessed. Our results depicted that expressing either AtHKT1;1 or EsHKT1;2 coding regions under the control of AtHKT1;1 promoter, almost reversed the hypersensitivity of the mutant for salt, on contrarily, when AtHKT1;1 coding sequence expressed under either Es or EbHKT1;2 promoters did not. Changes in shoot Na+/K+ concentrations under salt exposure is significantly consistent with the complementation ability of the mutant. The transcript concentration for genes under the control of either of Eutrema promoters, at control level was very less. This may suggest that either an important upstream response motif is missed or that A. thaliana misses a transcriptional regulator that is essential for salt-inducible HKT1 expression in Eutrema.

Combined application of selected heavy metals and EDTA reduced the growth of Petunia hybrida L.

Up till now, despite of well-developed ornamental market, very little information is available on Petunia hybrida L. tolerance against heavy metals (HMs), which can contribute in both beautification of urban dwellings, as well as potential in phytoremediation. Therefore, hydroponic study was conducted to check the effects of Cd, Cr, Cu, Ni and Pb individually (50 and 100 µM) and with co-application of EDTA (2.5 mM) in Hoagland's nutrient solution. Results indicated higher uptake of Cd, Cr, Ni and Pb in above ground parts, and Cu in roots, further the co-application of EDTA enhanced HMs uptake in P. hybrida L. This uptake accompanied changes in biochemical stress indicators, included significantly higher MDA, H2O2 contents and electrolyte leakage with reduced chlorophyll a, chlorophyll b, total chlorophyll and carotenoid content. Upon exposure to HMs increased antioxidant enzyme activities (CAT, POX, GST, APX, and SOD) were noted. Though selected HMs can be removed by using P. hybrida L., the findings of current study indicated that the direct exposure of P. hybrida L. to Cd, Cr, Cu, Ni and Pb damaged the plant's aesthetics, and to use P. hybrida L. for beautification of urban landscape or phytoremediation, appropriate soil modification should be included.

Synergistic effect of alkaline pretreatment and magnetite nanoparticle application on biogas production from rice straw.

Agricultural residues have high potential for biogas production, complex lignocellulosic structure is however the main hindrance in their bioconversion. This research focuses on combined effect of alkaline pretreatment of rice straw and magnetite (Fe3O4) nanoparticle application. Four doses of magnetite nanoparticles viz. 60, 80, 100 and 120 ppm were used in the anaerobic digestion of untreated and 2% NaOH pretreated rice straw. Compared to control, 2% NaOH pretreatment alone increased biogas and methane yield by 57 and 60% respectively. Magnetite nanoparticle (MNP) application alone gave maximum yield at 100 ppm which consisted of 37 and 33% more biogas and methane yield respectively. Combining the effect of 2% NaOH pretreatment and 120 ppm MNPs synergistically increased biogas and methane yield by 100 and 129% as compared to control. In addition, an energy assessment indicated a positive net gain of 3765 kJ for 2% NaOH pretreated rice straw with 120 ppm MNPs.

Salt and heavy metal tolerance and expression levels of candidate tolerance genes among four extremophile Cochlearia species with contrasting habitat preferences.

To test the concept of a general "mineral stress tolerance", we compared four extremophile Cochlearia species for salt (NaCl), zinc (Zn) and cadmium (Cd) tolerance and accumulation, and for expression of candidate tolerance genes for salt and Zn tolerance. Salt tolerance decreased in the order C. anglica>C. x hollandica>C. danica>C. pyrenaica, corresponding with the average salinity levels in the species' natural environments. The glycophytic metallophyte, C. pyrenaica, showed a relatively high level of salt tolerance, compared to other glycophytic Brassicaceae. Salt tolerance was positively correlated with HKT1 expression and the K+ concentration in roots under salt exposure, but uncorrelated with the Na+ concentrations in roots and shoots. All the species accumulated Na+ primarily in their leaves, and exhibited a high NHX1 expression in leaves, in comparison with other glycophytic Brassicaceae, suggesting that salt tolerance in Cochlearia is based on an efficient vacuolar sequestration of Na+ in leaves. The metallicolous C. pyrenaica population was hypertolerant to Zn, but not to Cd, in comparison with the other Cochlearia species. All the Cochlearia species accumulated Zn and Cd primarily in roots, and showed high levels of Cd and Zn tolerance, with unusually low rates of metal accumulation, in comparison with non-metallophytes, or non-metallicolous metallophyte populations, of species belonging to other genera or families. Although Cochlearia, as a genus, shows relatively high levels of tolerance to both salt and heavy metals, this is most probably not due to a common 'mineral stress tolerance' mechanism.

Expression of HMA4 cDNAs of the zinc hyperaccumulator Noccaea caerulescens from endogenous NcHMA4 promoters does not complement the zinc-deficiency phenotype of the Arabidopsis thaliana hma2hma4 double mutant.

Noccaea caerulescens (Nc) exhibits a very high constitutive expression of the heavy metal transporting ATPase, HMA4, as compared to the non-hyperaccumulator Arabidopsis thaliana (At), due to copy number expansion and altered cis-regulation. We screened a BAC library for HMA4 and found that HMA4 is triplicated in the genome of a N. caerulescens accession from a former Zn mine near La Calamine (LC), Belgium. We amplified multiple HMA4 promoter sequences from three calamine N. caerulescens accessions, and expressed AtHMA4 and different NcHMA4 cDNAs under At and Nc HMA4 promoters in the A. thaliana (Col) hma2hma4 double mutant. Transgenic lines expressing HMA4 under the At promoter were always fully complemented for root-to-shoot Zn translocation and developed normally at a 2-µM Zn supply, whereas the lines expressing HMA4 under Nc promoters usually showed only slightly enhanced root to shoot Zn translocation rates in comparison with the double mutant, probably owing to ectopic expression in the roots, respectively. When expression of the Zn deficiency responsive marker gene ZIP4 was tested, the transgenic lines expressing AtHMA4 under an NcHMA4-1-LC promoter showed on average a 7-fold higher expression in the leaves, in comparison with the double hma2hma4 mutant, showing that this construct aggravated, rather than alleviated the severity of foliar Zn deficiency in the mutant, possible owing to expression in the leaf mesophyll.