Utilization of Particle Induced X-ray Emission (PIXE) for Plant Elemental Profiling

Abstract

Proton Induced X-ray Emission (PIXE) is a non-destructive, versatile, feasible and reliable technique for trace element profiling. PIXE utilizations extremely depends on the X-ray production, the fluorescence yield, particular energy of the proton beam, choice of sample, sample preparation, high resolution X-ray detector and the multi-target vacuum chambers. The application of this technique to analyze trace elements of aerosol, archaeological, geological, medical samples is well known due to ability to analyze low molecular weight samples without any pre-treatment (cause of any potential cross contamination) which makes it relatively useful for several biological samples. Here, we highlighted the precision of PIXE technique to carry out rapid and accurate investigations of several elements from diverse group of plant samples including, micro algae, crops and fruits to answer different biological questions. For the ease of reader, we divided this dissertation into three different parts as given below, Part-I aims to profile elemental composition with special focus on detection of heavy metals in artificially ripened fruits including mango (Magnefera indica) and Banana (Musa balbisiana). Ethylene exposure for artificial fruit ripening is non-toxic and human safe method but relatively cost effective due to the requirements of environmentally controlled chambers and transport technologies. Therefore, a very inexpensive and banned chemical known as calcium carbide (CaC2) is still very popular for fruit ripening purpose in many developing countries, including Pakistan. CaC2 produces acetylene gas (acts as ethylene) in presence of moisture on fruit surface to accelerate fruit ripening. CaC2 isusually contaminated even industrial grade CaC2 contains traces of heavy metals including arsenic and phosphines and their application directly or indirectly to fruits can lead to severe human health hazards. We therefore utilized PIXE to analyze the elemental composition of mango and banana with and without CaC2treatment. Fruits were categorized into five groups on the basis of CaC2 treatments (T2- T4), control without any treatment (T1), and fully ripened fruits from market (T5). All the fruits were carefully analysed for the organoleptic characteristics during whole experiments and compared with control fruit samples. Fruits were cut into pieces and were dried to remove the moisture. Dried samples were grinded to fine powder. Pellets of 2 mm thickness and 13 mm diameter were prepared using Laboratory Hydraulic Press. Pulverized mango samples were irradiated by 3MeV collimated protons from 5MV tandem accelerator and emitted X-ray spectrum was analyzed using GUPIXWIN to detect significant amount of As traces with differential presence of several other elements. PIXE elemental profiling of the fruit samples was further validated by Graphite Furnace Atomic Absorption Spectroscopy (GFAAS) and Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). Both state of the art technologies confirmed the presence of As traces in different groups (T2-T4) including market mangoes (T5). Estimated daily intake of arsenic in mango (0.019 mg/kg) and banana (0.024 mg/kg) was more than the WHO recommended guidelines (0.0003 mg/kg) and higher rate of such fruits consumption during season can accumulate dangerous levels of As in our bodies so, this chemical should be totally ban. Furthermore, this study provides an easy and reliable method to do the fruit elemental profiling and provides a platform to develop mass awareness programs among different stakeholders and government agencies, farmers, vendors, scientists and mainly consumers to address different aspects of artificial fruit ripening issues and the implementations of the existing rules for food quality in Pakistan. Part II, sheds light on utilization of PIXE for elemental profiling of our major crops wheat and maize treated with Plant Growth Promoting Rhizobacteria (PGPR) to analyze compare the growth and nutrient up take capacity under drought stress. Drought stress affect crops by decreasing turgor pressure which results in decrease in photosynthesis and reduced nutrient uptake by roots. Therefore , we inoculated wheat and maize seeds with Pseudomonas fluoresces and were grown with and without drought stress in growth chambers at their optimal growth temperatures followed by comparisons with control plants initially for physiological parameters related to growth plants such as, plant height, root length, chlorophyll content, relative water contents, shoots and roots fresh and dry weight. PGPR inoculation resulted in enhanced plant height, root length, chlorophyll content, plant biomass and relative water content as compared to non-inoculated plants. Leaf samples of PGPR treated and control wheat and maize plants were oven dried and ground to fine powder. Pellets of 2 mm thickness and 13 mm diameter were prepared using Laboratory Hydraulic Press and samples were irradiated with 3 MeV collimated protons from the 5 MV tandem accelerator. The X-rays emitted during irradiation from the samples were measured by Silicon Drift Detector (SDD) and the acquired X-ray spectrum was analysed by using the software GUPIXWIN. Enhanced nutrients amount was observed in PGPR treated plants in comparisons to control plants. In PGPR inoculated wheat plants maximum increase was observed in Cu (111%) followed by Mn (85.5%) while minimum increase was recorded in K which as 7.1%. Other element showed increase as P (51%), S (73%), Cl (67%), Ca (46.76%), Fe (35%) and Zn (17.5%). Similar pattern was noticed in PGPR inoculated maize plants where maximum increase was recorded in Fe (221%) followed by Cu (146%) while lowest increase was noticed for Ca (6.8%) and Cl (9.2%). Other element showed increase as P (57%), S (35%), K (31%), Mn (55%), and Zn (32.5%). Our data revealed that PGPR inoculation not only resulted in enhanced plant growth but it also helped plants to mitigate the effects of drought stress. Since the abiotic stress negatively affects the nutrient uptake ability of plants. This study showed that PIXE technique can be utilized to investigate the effect of stress on plant elemental composition. Particle induced X-ray emission (PIXE), is uniquely suited for the determination of elemental composition in a wide range of concentrations from minor to ultra-trace levels due to many favourable features of the technique, and hence PIXE may be used to verify the accuracy of other analytical techniques. Part III addresses the utilization PIXE to evaluate the elemental profiling of different mutant strains of Chlamydomonas reinhardtii to study the variations in lipid production and nutrient uptake abilities of these microalgae under high temperature conditions for their possible utilization as novel raw materials for biodiesel productions. Microalgae can be used as an excellent source of biodiesel production but temperature is one of the major limiting factors in large scale cultivation of microalgae. Therefore, it is important to evaluate the effects of temperature on the growth and physiology of different strains of microalgae. We initially analyzed and compared the effects of high temperature on the growth and lipid contents of selected mutant strains of C. Reinhardtii (CC-4033, CC 1171 and CC1173) by using various biochemical parameters like photosynthetic pigments, protein, carbohydrate, elemental profiling and different enzymatic activities to examine the immediate response of C. reinhardtii to the given temperature stress followed by PIXE analysis for in-depth comparisons. All the algal strains were grown on medium and well grown cells were then grown at 25°C followed by heat stress at 33°C for 8 days. Algal biomass was harvested and grinded to fine powder using pre cleaned mortar and pestle. Hydraulic press of 24000 psi was used to make pellets of 2 mm thickness and 13 mm diameter.3MeV proton beam collimated to a diameter of 2 mm was used to irradiate the target and the characteristic X-rays emitted were measured by Silicon Drift Detector (SDD).GUPIXWIN software package was used to measure the concentration of different elements. Our results revealed a strong negative correlation between biomass accumulation and lipid contents of the three selected mutant strains. Interestingly, differential preference of temperature among these algal strains was observed e.g., CC-4033 had increased growth rate at 25˚C but CC-1171 showed maximum growth at 33˚C, while, CC-1173 strain performed equally well at both selected temperatures. Similarly lipid contents were also significantly changed at given temperatures in the order CC-4033 >CC-1171 >CC-1173 at 33˚C. In all three mutant strains fatty acid methyl esters (FAMEs) were identified which are considered as the suitable biodiesel components. Our data showed the reduction of antioxidant enzymes (catalase and ascorbate peroxidases) production at 33˚C in all the selected strains. PIXE analysis revealed that elemental profile of C-4033 was badly affected by high temperature stress. Usually increased nutrient contents indicate growth and adoptability but interestingly this mutant strain has potential to overcome the cellular damage caused by high temperature stress. Therefore, based on high lipid production, presence of FAME signatures and high temperature tolerance, we suggest that CC-4033 strain can be considered as a potential mutant among all three mutants strains studied for the exploration of new renewable energy. Conclusively, we successfully utilized PIXE for elemental evaluation of very diverse kind of plant samples with its multi-elemental capability and non-destructive nature. Considering the advantages of PIXE technique for micro analytical investigation involving soluble elements, PIXE can be routinely applied for several interdisciplinary problem such as environmental studies, analysis of materials, biomedicines and plant samples. Publication  Safdar Abbas, Sidra Saeed, Muhammad Ammar, Sitwat Aman and Samina N. Shakeel (2018). Effect of high temperature on activities and lipid production in mutants of Chlamydomonas reinhardtii. Int. J. Agric. Biol., 20: 1331‒1338  Safdar Abbas, Beenish Jehan Azhar, Muhammad Irfan, Shakeel Ahmad, Ishaq Ahmed, Javed Hussain, Samina N Shakeel (2019). Analysis of Organoleptic Parameters and Heavy Metals in Artificially Ripened Mango Fruits in Pakistan (Submitted in Journal of Animal and Plant Sciences).  Beenish J Azhar, Safdar Abbas, Muhammad Irfan, Shakeel Ahmed, Ishaq Ahmed, Javed Hussain, Samina N Shakeel (2019). Heavy Metals in Artificially Ripened Banana Fruits in Pakistan: An Exigency of Alternative Technology (Submitted inInternational Journal of Agriculture & Biology).