Applied Bioremediation Active and Passive Approaches

*Edited by Yogesh B. Patil and Prakash Rao*

**APPLIED**

**BIOREMEDIATION -**

**APPROACHES**

**ACTIVE AND PASSIVE**

Edited by **Yogesh B. Patil** and **Prakash Rao**


# **New Trends and Applications in Femtosecond Laser Micromachining**

Edited by Rebeca Martínez Vázquez, Francesca Bragheri and Petra Paiè Printed Edition of the Special Issue Published in *Micromachines*

www.mdpi.com/journal/micromachines


**2. Materials and Methods**

A near-β phase titanium alloy with a chemical composition of 13.6 Nb, 13.6 Zr, 0.06 Fe, 0.04 C, 0.01–0.02 N, 0.001 H, 0.11 O (in wt.%), provided by Xi'an Saite Metal Materials Development Co., Ltd., Xi'an, China was used for oxygen hardening. The β transformation temperature of this alloy is 735 ◦C [37]. The samples were cut from the bar (27 mm diameter) into discs with a thickness of 1–2 mm. The samples were ground with sandpaper up to 3000-grit until a flat surface was obtained. Next, they were pre-polished with Al2O3 suspension (grain size 1 μm) and finally polished with silica suspension (0.04 μm) until a mirror finish. The prepared alloy had a fine acicular martensitic morphology composed of α (hexagonal close-packed; hcp) and some α" (orthorhombic, Cmcm space group) laths in β (body-centered cubic; bcc) grains (Figure 1). A detailed description of the microstructure of the as-received alloy has been presented in our previous works [24,25]. The grain size, estimated by image analysis, was in the range of 20–80 μm.

**Figure 1.** Microstructure of the as-received alloy.

The samples were placed in a quartz tube furnace. After air evacuation, argon of 99.999% purity was purged into the tube. The pressure was adjusted to 10 Pa by flowing argon (flow rate of 30 standard cm3/min). When the samples reached the desired temperature of oxidation, i.e., 700 ◦C or 850 ◦C or 1000 ◦C, a glow discharge was started. The glow discharge was generated at 1250 V and an electric current of 45 mA. During discharge, high purity oxygen (99.999%) was mixed with argon and pumped to the furnace in 20 cycles of 1 min duration (flow rate of 6 standard cm3/min). During oxygen pumping, the pressure was increased to 12 Pa. After oxidation, cooling to room temperature with working Ar glow discharge was provided.

The microstructure of the oxygen hardened alloy was characterized by light microscopy (LM, ZEISS Axio Imager M1m microscope, Oberkochen, Germany), scanning electron microscopy (SEM, FEI Nova NanoSEM 450 microscope, (Eindhoven, The Nether-

lands) and transmission electron microscopy (TEM, JEOL JEM-2010 ARP microscope, (Tokyo, Japan) techniques. The phase constitution was studied by selected area electron diffraction (SAED). The Java Electron Microscopy Software (JEMS, version 4.4131U2016, Pierre Stadelmann, Switzerland) was used to interpret the SAED patterns. The lamella from the cross-section of the coated alloy was prepared for the TEM investigation. The samples were thinned using FEI Quanta 3D 200i scanning electron microscope equipped with a Ga+ ion gun, Pt precursor gas injection systems (GIS) and OmniProbe micromanipulator for in situ lift-out [38]. Ion beam accelerating voltage of 30 kV and ion currents in the range of 15 nA–30 pA were applied. The lamella was transferred via a micromanipulator to a TEM half ring, where a focused ion beam (FIB) milling to electron transparency was performed (ion currents of 500–30 pA). FIB deposition process from Pt precursor was used to attach the manipulator probe to the lamella and attach it to the grid. The phase identification was supplemented by energy-dispersive X-ray spectroscopy (SEM-EDS, TEM-EDS) microanalysis and STEM-EDS line analysis.

The open-circuit potential (OCP), linear sweep voltamperometry and electrochemical impedance spectroscopy (EIS) were carried out using a potentiostat Autolab PGSTAT302N (Metrohm Autolab B.V., Utrecht, The Nederlands). The reference electrode was a saturated calomel electrode (SCE), and a platinum plate was used as the counter electrode. Ringer's solution was used as the electrolyte for the corrosion study. The chemical composition of the Ringer's solution was: 8.6 g/L NaCl, 0.3 g/L KCl, 0.25 g/L CaCl2. Measurements were performed at pH 7.4 in deaerated solutions at 37 ◦C. The polarization test was performed at a scan rate of 1 mV/s from −1.3 V to +2.2 V vs. SCE. For the EIS measurements, the amplitude was 10 mV; the frequency was 105 Hz to 10−3 Hz. The EIS measurements were performed at the OCP potential. The EIS data were fitted using ZView software.

The hardness and elastic modulus of the as-received and oxygen hardened Ti–13Nb– 13Zr alloys were determined by micro-combitester (CSM Instruments). The indentation tests were performed using a Vickers diamond indenter. The indenter was loaded with a force of 200 mN and kept for 5 s before un-loading. An analysis of the mechanical properties of the alloy was carried out using the loading/unloading curve by Oliver and Pharr method [39]. It allowed for the determination of the elastic modulus (EIT) and hardness (HIT). For each sample, ten consecutive experiments at randomly selected places were performed, and the average of 10 measurements was calculated.

The abrasive wear resistance and the friction coefficient of the as-received and treated alloy were determined by friction tests based on the ISO 20808 standard [40]. The tests were carried out in dry sliding contact with an Al2O3 ball (6 mm diameter) using a ball-on-disc tribotester (ITeE Radom, Radom, Poland). In a typical ball-on-disc arrangement, a counterelement in the form of a ball was pressed against a rotating disc (sample) made of titanium alloy. The tribological tests were repeated three times, with the same parameters: ball load Fn = 5 N, sliding speed v = 0.07 m/s, sliding distance s = 1000 m, room temperature 23 ◦C and relative humidity 55%. The wear rate Wv = V/Fn × s was determined as the ratio of the volume of material removed during friction (V) to the load (Fn) and the sliding distance (s). The volume was calculated based on the size of the cross-sectional area of the wear groove. The groove profile was measured with a stylus profilometer in six places around the wear track.

Friction-wear tests with simultaneous measurement of corrosion potential during friction in Ringer's solution were also performed. To record the changes in the corrosion potential, a 2-electrode system with the working electrode (titanium alloy) and reference electrode (calomel electrode in 3 M KCl) was designed. The schematic of the tribocorrosion device is shown in Figure 2. A specially designed system ensuring an efficient and stable electric contact with the alloy sample was used. In this system, the ball with the holder was moving, whereas the titanium alloy sample was stationary. To fix the ball and sample positions, special polymer holders were used. The same measurement parameters were applied as in the case of dry friction tests. The friction process began after a stable corrosion

potential for the titanium alloy was reached. When the friction was activated, a change in the potential was recorded as a result of the wear of the alloys' surface layer.

**Figure 2.** Schematic of the tribological device with parallel corrosion potential measurement during wear tests of the titanium alloy in sliding contact with a ball.

Before each test, the alumina ball and titanium alloy were ultrasonically cleaned in ethanol and left to dry. Additionally, the ball used in the tribocorrosion test was also washed in Ringer's solution. The holders were subjected to an identical cleaning procedure. The whole measurement took place in a plastic vessel filled with Ringer's solution.


**1. Introduction**

Grass pollen is one of the major causes of allergy, affecting 10–30% of the population around the globe [1,2]. There are over 400 species of grass in Europe, and their pollen is recognized as the leading cause of pollinosis [3]. About 100 species of grass could be found in the European part of Russia [4], flowering periods of which often overlap, and their pollen allergenicity is estimated to be from moderate to very high [5]. Aerobiological monitoring is a necessary element of the complex of anti-allergic measures allowing for tracking and predicting the dynamics of the concentration of major allergens in the air and adjusting the therapy and lifestyle of patients with pollinosis. The standard method

**Citation:** Omelchenko, D.O.; Krinitsina, A.A.; Kasianov, A.S.; Speranskaya, A.S.; Chesnokova, O.V.; Polevova, S.V.; Severova, E.E. Assessment of ITS1, ITS2, 5--ETS, and *trnL-F* DNA Barcodes for Metabarcoding of Poaceae Pollen. *Diversity* **2022**, *14*, 191. https://doi.org/10.3390/d14030191

Academic Editors: W. John Kress, Morgan Gostel and Michael Wink

Received: 30 January 2022 Accepted: 2 March 2022 Published: 5 March 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

of pollen identification in the air samples is light microscopy. However, one of the major disadvantages of pollen light microscopy analysis is that similar pollen morphology of Poaceae species makes it challenging to discriminate species in airborne pollen mixes, which impairs the quality of aerobiological monitoring [6,7]. DNA metabarcoding is an alternative approach that has been actively developing recently, allowing qualitative (to the level of species or genus for some taxa) and quantitative (to some extent) composition analysis of complex biological mixes. It employs high-throughput sequencing (HTS) and comparative analysis of specific DNA sequences called "DNA barcodes" to discriminate species present in the mix. DNA barcoding has been widely used in various areas of botanical research; for example, the phylogeny of wild cherry [8], archaeobotany of grapevine [9], authentication and identification of medicinal [10] and poisonous [11] plants, and plant species composition of honey [12,13].

Choosing the correct DNA barcode for the target taxa is one of the main problems of plant barcoding [14,15]. The resolution capacity of each of the primary chloroplast markers (first a combination of *matK* and *rbcL* recommended by the CBOL group [16] and later the nuclear ribosomal internal transcribed spacer (ITS) regions and several intergenic spacers) vary significantly between different taxa (for a review, see [17]). Many studies focused on the DNA barcoding of plants; note, that the identification at the high-rank taxa (order, family) is successful in more than 90% of cases, while insufficient data on reference DNA barcode sequences prevents determination to the level of genus or species [13,18]. Therefore, the right choice of DNA barcode and the primers to amplify them is the key to successful species identification.

The regions of the chloroplast genome *rbcL*, *matK*, *trnL*, *trnH-psbA,* and nuclear ITS2 are most often used as plant DNA barcodes. Some of these barcodes have been used with varying success for metabarcoding pollen (airborne or from food products such as honey). However, only *rbcL*, *matK*, ITS2, and *trnL* barcodes have been studied compared to the palynological analysis for assessing qualitative and quantitative consistency [19]. In particular, a comprehensive study of ITS2 and *rbcL* has shown their usability in metabarcoding of pollen for the construction of pollinator networks and qualitative analysis of pollen mixes. Though, the quantitative relativity of the metabarcoding results and real pollen abundance of mixture components has been low [20,21]. Another study has assessed *trnL* and ITS1 for quantitative pollen analysis using metabarcoding and concluded that *trnL* demonstrates the best sequence-to-pollen prediction [22]. Furthermore, comparative studies have shown a good capability of *trnL* intron and *trnL-trnF* (*trnL-F*) intergenic spacers, ITS region, and their combinations to resolve grass species [23–26]. Indel and SNP patterns of the *trnL-F* intergenic spacer and ITS region have been employed for infrageneric classification and phylogeny study of *Chascolytrum* and *Festuca* genera [27,28].

External transcribed spacer (ETS) is another nuclear DNA barcode closely related to the ITS region in rDNA, but it is less frequently used than ITS. However, ETS is regarded as a promising DNA barcode as the taxon-specific informativity of the ETS sequence has proved to be the highest among nuclear and plastid barcodes in several studies [29–31].

Many published studies report the species identification of different grasses using only some of these barcodes and focusing on a particular plant taxon (e.g., [32,33]). In this study, we have compared the plastome *trnL-F* and nuclear ITS1 and ITS2 barcodes with the 5--ETS barcode and assessed their capability to identify the pollen of a diverse set of 14 grass species of different genera from the Poaceae family. New Poaceae-specific primers were designed to amplify the 5--ETS fragment suitable for the HTS sequencing as its length is less than 600 bp for all species in the study (maximum length for Illumina paired-end sequencing at present). Additionally, we have optimized the protocol for DNA extraction from pollen grains to obtain high-quality DNA for amplification and sequencing. To identify the pollen composition, we have created a local barcode sequence database for the reference Poaceae species using Sanger sequenced *trnL-F*, *ITS1*, *ITS2*, and 5- -*ETS* sequences of the live field samples, herbarium specimens, and available GenBank records. All four barcode sequences were tested by their capacity to resolve the composition of the grass pollen mixes using artificial pollen mixes of various complexity.


**5. Conclusion**

Dry anaerobic digestion is appropriate for treatment of agricultural waste with high TS content. Optimization of C/N ratio, F/I ratio (or organic loading rate), and TS content is key to avoid failure of digestion. Several batch and continuous dry digestion technologies have been already applied in practice, while new techniques have been also proposed. Solid digestate is beneficial to supply nutrient into the soil and improve soil properties. On the other hand, nitrate leaching is one of the concerns of the digestate application. Digestate C/N ratio adjustment by mixing with crop residue can mitigate nitrate leaching. In addition, it can also mitigate root-knot nematode. More study is needed to optimize dry anaerobic digestion and digestate use for sustainable agricultural waste management.


**5. Conclusions**

It was expected that a coating presenting a well-dispersed second phase and a high amount of micrometric γ-Al2O3 particles presented the best anticorrosive performance. However, these effects had no direct influence on the corrosion performance of these coatings in 0.5 mol L-1 NaCl, and no significant differences could be noted among their Ecorr, as shown in Table 5 and Figure 9, whereas the jcorr values increased when the applied potential became more negative. Moreover, the anticorrosive performance of these coatings, in terms of jcorr, was worse than the values obtained for the pure copper coating (5.27 μA cm-2). The thin layers produced under the deposition conditions could have probably masked the effects of the deposition parameters on the anticorrosive performance of these coatings. In addition, the micrometric size of the γ-Al2O3 particles could have contributed to create defects and voids in the coatings, enhancing the corrosion process, confirming the results earlier shown for Cu-μSiO2 coatings [91], and showing that the size of the particle is an important parameter to produce composite coatings

> 1 -1.20 10.3 -0.411 2 -1.50 62.2 -0.423

**Table 6.** and Ecorr values of composite coatings Cu/γ-Al2O3 in 0.5 mol L-1 NaCl. The coatings were produced at -1.20

**Figure 9.** Polarization curves of Cu/γ-Al2O3 coatings/steel substrate systems in 0.5 mol L-1 NaCl.

Cu/boehmite nanocomposite coatings [AlO(OH) Disperal P2, 25 nm size; Sasol®] was produced onto steel substrate (AISI 1020) by DC electrodeposition using j = 7.0 and 21.0 A

**(VSSE) jcorr (μA cm-2) Ecorr (VSCE)**

with high anticorrosive characteristics.

176 Electrodeposition of Composite Materials

**Experiments Applied cathodic potencial**

and -1.50 VSSE after previous stirring for 5 h of at 800 rpm [61].

It is possible to conclude that, although composite coatings have been under investigation for many years, their application as anticorrosive coatings still needs more research. The effects of the electrical nature of the particle and the bath composition (e.g., complexant, additives, and surfactants) on the anticorrosive characteristics of the MMC coatings must be studied. Other parameters, such as stirring speed, previous stirring time, and solution temperature, must also be optimized to enhance the incorporation of the nanoparticles to the coatings and relate this effect to their anticorrosive properties. In addition, the corrosion processes in different electrolytic media must be evaluated and be related with the deposition parameters. Therefore, there is a great research opportunity involving the production of MMC coatings by electrodeposition and their characterization as anticorrosive coatings.


*Article Ginkgo biloba* **Seeds—An Environmental Pollutant or a Functional Food**

**Teodora Tomova 1,\*, Iva Slavova 1, Desislav Tomov 2, Gergana Kirova 1 and Mariana D. Argirova 1**

**Abstract:** *Ginkgo biloba* has been cultivated in Bulgaria since the end of the 19th century. Ividual specimens can be seen in almost every park. Females of the tree are considered contaminants of the landscape because their ripe seeds have a strong odor and are not utilized. We undertook this study to clarify whether ginkgo seeds of local origin can be converted from an unwanted and unused environmental pollutant into a source of beneficial compounds. Various analytical and chromatographic methods were used to quantify the major constituents and ten biologically active compounds in methanol seed extract. The results showed that the seeds are low in proteins (5%) and fats (1%); the seeds were also rich in unsaturated fatty acids and tocopherols. About 44% of nut starch was resistant to in vitro enzymatic hydrolysis. The amount of terpene trilactones in an aqueous-methanol seed extract was significantly higher than the number of flavonoids. Ginkgotoxin and ginkgolic acid were also found. The extract demonstrated weak antimicrobial activity against thirteen microorganisms. This study revealed that seeds of locally grown Ginkgo trees can be used as a source of biologically active substances. The chemical composition show similarity to those of seeds from other geographical areas.

**Keywords:** *Ginkgo biloba*; trace elements; starch; flavonoids; terpene trilactones; ginkgotoxin; ginkgolik acid; antimicrobial