Result of the soil chemical analysis on the 11 locations in the Philippines.
\\n\\n
More than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\\n\\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\\n\\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\\n\\nAdditionally, each book published by IntechOpen contains original content and research findings.
\\n\\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\\n\\n\\n\\n
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'
Simba Information has released its Open Access Book Publishing 2020 - 2024 report and has again identified IntechOpen as the world’s largest Open Access book publisher by title count.
\n\nSimba Information is a leading provider for market intelligence and forecasts in the media and publishing industry. The report, published every year, provides an overview and financial outlook for the global professional e-book publishing market.
\n\nIntechOpen, De Gruyter, and Frontiers are the largest OA book publishers by title count, with IntechOpen coming in at first place with 5,101 OA books published, a good 1,782 titles ahead of the nearest competitor.
\n\nSince the first Open Access Book Publishing report published in 2016, IntechOpen has held the top stop each year.
\n\n\n\nMore than half of the publishers listed alongside IntechOpen (18 out of 30) are Social Science and Humanities publishers. IntechOpen is an exception to this as a leader in not only Open Access content but Open Access content across all scientific disciplines, including Physical Sciences, Engineering and Technology, Health Sciences, Life Science, and Social Sciences and Humanities.
\n\nOur breakdown of titles published demonstrates this with 47% PET, 31% HS, 18% LS, and 4% SSH books published.
\n\n“Even though ItechOpen has shown the potential of sci-tech books using an OA approach,” other publishers “have shown little interest in OA books.”
\n\nAdditionally, each book published by IntechOpen contains original content and research findings.
\n\nWe are honored to be among such prestigious publishers and we hope to continue to spearhead that growth in our quest to promote Open Access as a true pioneer in OA book publishing.
\n\n\n\n
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\r\n\tOrganic synthesis has always been one of the central topics of research for the scientific community in the academic laboratories and industrial world. Many striking journal articles and remarkable reviews and books have been published in the past year describing the practicability and applications of the subject demonstrating the importance of organic synthesis. In the present book, we will be putting together the topics in organic synthesis which may include but not limited to, (1) the basic terms and concepts, (2) various organic reactions including reduction, oxidation, addition, elimination, rearrangements, and cycloadditions, (3) Total Synthesis of Natural products, (4) transition metal catalysts, organocatalysts, enzymes and biotransformations, (5) applications in medicinal chemistry and drug design and development, (6) purification methods and characterization techniques, etc. To set a limit and to increase the scope of the book, author(s) are encouraged to send the chapters that include selected examples with practical applications and good yielding reactions reported within the past decade. Older topics with significant findings or their essence to prepare the foundation may be included in the chapter are welcomed as well.
",isbn:null,printIsbn:"979-953-307-X-X",pdfIsbn:null,doi:null,price:0,priceEur:null,priceUsd:null,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"f3bbbd989d0896f142d317ccb8abcc35",bookSignature:"Dr. Prashant S Deore",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8807.jpg",keywords:"Natural Product Synthesis, Organic Reaction Mechanism, Stereoselective synthesis, Chirality, C-H Functionalization, Cross-Coupling Reactions, Heterogeneous Catalysis, Homogeneous Catalysis, Green Synthesis, Green Solvents and Reagents, Bioorganic synthesis, Click Chemistry",numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"December 10th 2018",dateEndSecondStepPublish:"January 14th 2019",dateEndThirdStepPublish:"March 15th 2019",dateEndFourthStepPublish:"May 20th 2019",dateEndFifthStepPublish:"July 19th 2019",remainingDaysToSecondStep:"2 years",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"251769",title:"Dr.",name:"Prashant",middleName:"S",surname:"Deore",slug:"prashant-deore",fullName:"Prashant Deore",profilePictureURL:"https://mts.intechopen.com/storage/users/251769/images/system/251769.png",biography:"Dr. Prashant S. Deore was born in India. He received a Master’s degree in organic chemistry from Pune University in 2007. In the same year, he qualified with the SET and CSIR-NET (JRF) and joined in the group of Prof. Narshinha P. Argade for the doctoral studies in National Chemical Laboratory, India. In 2014, he awarded with a Ph. D. in Chemistry and was a recipient of the 2nd prize in “2014 Eli Lilly and Company Asia Outstanding Thesis Awards”. In July 2014 he moved to Canada and joined as a postdoctoral researcher in the group of Prof. Richard Manderville at the University of Guelph, Canada. Presently, Dr. Deore is working on the collaborative project between the University of Guelph and Aterica health Inc., and providing consulting to the company. His research interest includes organic synthesis, fluorescent probes development, nucleic acid synthesis and modifications, and aptasensor development for proteins and food toxins.",institutionString:"University of Guelph",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:null}],coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"8",title:"Chemistry",slug:"chemistry"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"270935",firstName:"Rozmari",lastName:"Marijan",middleName:null,title:"Ms.",imageUrl:"https://mts.intechopen.com/storage/users/270935/images/7974_n.png",email:"rozmari@intechopen.com",biography:"As an Author Service Manager my responsibilities include monitoring and facilitating all publishing activities for authors and editors. From chapter submission and review, to approval and revision, copyediting and design, until final publication, I work closely with authors and editors to ensure a simple and easy publishing process. I maintain constant and effective communication with authors, editors and reviewers, which allows for a level of personal support that enables contributors to fully commit and concentrate on the chapters they are writing, editing, or reviewing. I assist authors in the preparation of their full chapter submissions and track important deadlines and ensure they are met. I help to coordinate internal processes such as linguistic review, and monitor the technical aspects of the process. As an ASM I am also involved in the acquisition of editors. 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MOFs are crystalline nanoporous materials composed of metal complexes that are linked by organic ligands to create highly porous frameworks [1, 2]. MOFs become strong alternatives to more traditional nanoporous materials such as zeolites due to their fascinating physical and chemical properties. MOFs typically have very large surface areas (500–6000 m2/g), high pore volumes (1–4 cm3/g), wide range of pore sizes from micro- to mesoscale (1–98 Å), and reasonable thermal and mechanical stabilities. The most important characteristic of MOFs is that their physical, chemical, and structural properties can be tuned during synthesis. This controllable synthesis leads to a large diversity of materials having different geometry, pore size, and chemical functionality [3, 4]. As a result, thousands of MOFs have been reported in the Cambridge Crystallographic Database [5]. The family of MOFs can be divided into subgroups such as isoreticular MOFs (IRMOFs), zeolitic imidazolate frameworks (ZIFs), zeolite-like MOFs (ZMOFs), and covalent organic frameworks (COFs).
\nMOFs have been examined for a variety of chemical applications including gas storage [6, 7], gas sensing [8], gas separating membranes [9], mixed matrix membranes [10], catalysis [11], and biomedical applications [12, 13]. Among these applications, gas separation has received a significant interest because the pore sizes of MOFs can be tuned to selectively separate gases at the molecular level. Gas separation using MOFs has been generally studied in two categories: equilibrium-based gas separations and kinetic-based gas separations [14]. In equilibrium-based gas separations, MOFs are used as adsorbents and in kinetic-based separations, MOFs are used as membranes. Adsorption-based gas separation is governed by the thermodynamic equilibrium. Gas components are reversibly adsorbed into the pores of the adsorbent. An ideal adsorbent material must have a good combination of adsorption selectivity and working capacity in addition to high stability, high void volume, and well-defined pore sizes. High porosities, large surface areas, different pore sizes and shapes, and reasonable stabilities of MOFs suggest that these materials can be ideal adsorbents in equilibrium-based gas separation applications. Several experimental studies have been carried out for adsorption-based gas separations using MOFs [15–17].
\nTwo criteria are widely investigated to assess the potential of MOF adsorbents: adsorption selectivity and working capacity. Adsorption selectivity is determined by the adsorption affinity of the MOF for one gas species relative to another. High adsorption selectivity means a high-purity product and hence lower energy requirements. Working capacity is defined as the difference between the adsorbed amounts of gas at the adsorption and desorption pressures. High working capacity means easy regeneration of the adsorbent material. For an efficient and economic adsorption-based gas separation, both high selectivity and high working capacity are desired. Therefore, experimental studies on MOF adsorbents generally examine selectivity and working capacity of the materials [18].
\nMost of the experimental studies have focused on CO2 separation. Because of the growing environmental concerns, removal of CO2 from natural gas (CO2/CH4), flue gas (CO2/N2), and other gases (CO2/H2) becomes an important issue. Experimentally measured selectivity and gas uptake capacity of several MOFs for separation of CO2 from CH4 and N2 have been summarized in the literature [19]. Currently available adsorbents such as activated carbons, carbon molecular sieves, and zeolites are not highly selective for CO2 separation, especially for separation of CO2 from flue gas [20]. A good comparison of CO2 separation performances of different nanoporous materials such as MOFs, zeolites, and activated carbons is available in a recent review [21]. It is shown that CO2/N2 selectivity changes from low in zeolites to moderate in carbon-based absorbents and becomes high in MOFs. Therefore, research on adsorption-based gas separations has focused on identifying highly selective MOF adsorbents with high CO2 capacities that can replace traditional adsorbents.
\nConsidering the very large number of available MOFs, it is not possible to test thousands of different MOFs as adsorbents using purely experimental methods. Molecular simulations play an increasingly important role in understanding the potential of MOFs in adsorption-based gas separations. Among molecular simulation methods, grand canonical Monte Carlo (GCMC) simulations have been widely used to accurately predict adsorption isotherms of various gases in MOFs [22]. Gas selectivities calculated from simulated adsorption isotherms are generally found to be in good agreement with the experiments [23]. In most studies, single-component gas adsorption isotherms are computed using GCMC simulations; mixture adsorption isotherms are then predicted based on pure gas adsorption data using ideal adsorbed solution theory (IAST). IAST is a well-developed technique to describe adsorption equilibria for gas components in a mixture using only single-component adsorption data at the same temperature and on the same adsorbent [24]. GCMC simulations can be also performed to obtain mixture adsorption data directly. This data is then used to predict adsorption selectivity and working capacity of the MOF. Results of molecular simulations provide molecular-level insights which can be used to design new MOFs with better separation performances. In the early years of these studies, simulations examined only one or a few MOFs at a time. With the development of new computational approaches and with the quick increase in the number of synthesized MOFs, molecular simulations have started to screen a large numbers of materials. The results of large-scale MOF screening studies are highly useful to direct experimental efforts, resources, and time to the most promising MOF materials.
\nThis chapter aims to address the importance of molecular simulations to evaluate the potential of MOFs in adsorption-based CO2 separations. Section 2.1 introduces details of GCMC simulations to study CO2 adsorption in MOFs. Section 2.2 describes evaluation criteria used to assess CO2 separation potential of MOF adsorbents. Studies on large-scale computational screening of MOF adsorbents are discussed in Section 2.3. Structure-separation performance relations obtained from molecular simulations of MOFs are summarized in Section 2.4. Section 3 closes by addressing the opportunities of using molecular simulations for examining the potential of MOF adsorbents in CO2 separations.
\nGCMC is a well-known method to estimate the adsorption equilibria of gases in nanoporous materials. This simulation mainly mimics an adsorption experiment. In an experimental setup, the adsorbed gas is in equilibrium with the gas in the reservoir at fixed temperature, volume, and chemical potential [25]. GCMC simulations are run at an ensemble where the temperature, volume, and chemical potential are kept constant and the number of gas molecules is allowed to fluctuate during the simulation at the imposed temperature and chemical potential. The output of a GCMC simulation is the number of adsorbed gas molecules per unit cell of the MOF structure at predetermined temperature and pressure. These simulations provide single-component adsorption isotherms, binary mixture adsorption isotherms, and isosteric heat of adsorptions which are directly comparable with the output of adsorption experiments. In GCMC simulations where only a single-component gas such as CO2 is studied, four different types of moves are considered including translation, rotation, insertion, and deletion of a molecule. In mixture GCMC simulations where a binary gas mixture is considered such as CO2/H2, CO2/CH4, and CO2/N2, another trial move, exchange of molecules is also performed in order to speed up the equilibrium. The adsorbed amounts of each gas component are calculated by specifying pressure, temperature, and composition of the bulk gas mixture in GCMC simulations.
\nIn a typical GCMC simulation of CO2 adsorption in an MOF, CO2 is the adsorbate and MOF is the adsorbent. Adsorbate molecules interact with the MOF atoms and with other adsorbates through dispersive and electrostatic interactions. These interactions are defined using a force field. A force field is the functional form and parameter sets are used to calculate the potential energy of a system of atoms in molecular simulations [26]. There are several potentials such as Lennard-Jones (LJ) [27] and Morse [28] potentials. In almost all molecular simulations of gases in MOFs, Lennard-Jones (LJ) potential is used. Results of a GCMC simulation may vary depending on the force field choice. General-purpose force fields such as universal force field (UFF) [29] and DREIDING force field [30] have been widely employed for simulations of MOFs. At the early stages of the molecular simulation studies of MOFs, efforts have been made to develop new force fields specific to MOF-gas interactions using quantum-level calculations [31, 32]. Some studies refined the force field parameters to match the predictions of molecular simulations with the available experimental measurements of gas adsorption in MOFs [32–36]. However, considering the large number and variety of MOFs, it is challenging to develop a new force field or refine an existing one for every single MOF. Therefore, generic force fields such as UFF and DREIDING are mostly preferred in molecular simulations of CO2 adsorption in MOFs, especially for large-scale computational screening of MOFs. The reliability of the molecular simulation studies, of course, hinges on the accuracy of the force fields used. Schmidt et al. [37] computed CO2 adsorption isotherms in a very large number of MOFs using ab initio force fields to probe the accuracy of common force fields. They concluded that there are significant quantitative differences between gas uptakes predicted by generic force fields such as UFF and ab initio force fields, but the force fields predict similar ranking of the MOFs, supporting the further use of generic force fields in large-scale material screening studies.
\nThe LJ potential parameters of CO2 are generally taken from the force field developed by Potoff and Siepmann [38]. A rigid linear triatomic molecule with three charged LJ interaction sites located at each atom is used for CO2 molecules. Partial point charges centered at each LJ site approximately represent the first-order electrostatic and second-order induction interactions. Charge-quadrupole interactions between MOF atoms and CO2 molecules significantly contribute to the adsorption of CO2. It was shown that if these interactions are not taken into account, adsorption isotherms of CO2 molecules in MOFs can be significantly underestimated [39]. In order to compute the electrostatic interactions between CO2 molecules and MOF, partial point charges must be assigned to the MOF atoms. Several different methods are available in the literature to assign partial charges such as density-derived electrostatic and chemical charge (DDEC) method [40], connectivity-based atom contribution (CBAC) method [41], extended charge equilibration (EQeq) method [42], and quantum mechanical methods based on the ChelpG [43] density functional theory (DFT) calculations. Charges obtained from different methods generally do not agree well with each other. This is acceptable because atomic charges are not experimentally observable and the methods used to derive them depend on the physical phenomenon the charges are intended to be reproduced [44]. A simulated CO2 adsorption isotherm is generally compared with the experimentally measured one to tune the charges if necessary. After atomic models, force fields and charges are defined for CO2 molecules and MOF atoms, GCMC simulations can be carried out to obtain the adsorbed gas amounts. Results of these simulations are directly used to calculate the adsorption-based gas separation potential of MOFs based on several criteria as discussed below.
\nIn adsorption-based gas separation processes such as pressure swing adsorption (PSA), selectivity and working capacity of the adsorbent are the two important parameters that define the efficiency of the process [45, 46]. Adsorption selectivity is described as the ratio of mole fractions of gases in the adsorbed phase normalized by the bulk composition of the gas mixture:\n
Here, x stands for the molar fraction of the adsorbed phase obtained from the GCMC simulations, while y represents the molar fraction of the bulk gas phase. Eq. (1) defines the adsorption selectivity of an MOF adsorbent with respect to component 1, meaning that if selectivity is greater than 1, then the adsorbent is selective for component 1 over 2. Selectivities for CO2/CH4, CO2/H2, and CO2/N2 mixtures are calculated using the results of binary mixture GCMC simulations where 1 is CO2 and 2 is the other gas component in Eq. (1). The bulk gas compositions of CO2/CH4, CO2/H2, and CO2/N2 mixtures are generally set to 50/50, 15/85, and 15/85, respectively, in molecular simulations to represent industrial operating conditions.
\nWorking capacity (ΔN) is described as the difference between the loading amounts of the strongly adsorbed gas at the corresponding adsorption (Nads) and desorption (Ndes) pressures [46]. It is defined in the unit of mol gas per kg of MOF adsorbent. GCMC simulations are performed at specific adsorption and desorption pressures to calculate the working capacity as shown in Eq. (2).\n
Bae and Snurr [45] recently suggested some other adsorbent evaluation criteria in addition to selectivity and working capacity. The CO2 uptake of an MOF under adsorption conditions (Ndes), sorbent selection parameter (Ssp), and regenerability (R%) are also considered as adsorbent evaluation criteria to assess the potentials of MOFs in CO2 separation processes such as natural gas purification, landfill gas separation, and CO2 capture from power-plant flue gas. The CO2 uptake of an MOF under adsorption conditions (Nads) is the direct output of GCMC simulations. Sorbent selection parameter (Ssp) is defined as the combination of adsorption selectivity and working capacity. It is used to compare performances of different nanoporous materials in adsorption-based separation processes and is defined as shown in Eq. (3). Here, subscripts 1 and 2 indicate the strongly adsorbed component and the weakly adsorbed component, respectively. In CO2-related mixtures, CO2 is generally the strongly adsorbed component (1), whereas other gases such as CH4 and N2 are weakly adsorbed (2).\n
Regenerability (R%) is defined as the ratio of working capacity to the amount of the adsorbed gas at the adsorption pressure and it is an important parameter to evaluate the practical usage of an adsorbent for cyclic PSA and vacuum swing adsorption (VSA) processes:\n
At that point, it is important to mention that none of these criteria are perfect but they are complementary with each other to assess adsorbent potential of MOFs under practical conditions. Bae and Snurr [45] calculated adsorption selectivity, working capacity, gas uptake capacity, sorbent selection parameter, and regenerability values of several MOFs for adsorption-based separation of CO2/CH4:10/90, CO2/CH4:50/50, and CO2/N2:10/90 mixtures. Although mixtures are considered, they used the experimental single-component adsorption isotherm data from the literature and obtained mixture amounts under adsorption and desorption conditions at the partial pressure of the specific component. In order to investigate the effect of mixture data, Ozturk and Keskin [47] calculated the same separation properties of MOFs using both single-component and mixture GCMC data. They showed that selectivity calculated from single-component GCMC simulations can be enormously different than the selectivity calculated from mixture GCMC simulations due to strong competition effects between different gas species. Therefore, it is better to characterize adsorbent materials based on their performance for mixed-gas feeds to reflect the real operation conditions [48].
\nLlewellyn et al. [49] recently suggested a new criterion named as adsorbent performance indicator (API) as shown in Eq. (5) to initially highlight porous materials of potential interest for PSA processes. This indicator takes into account working capacity (represented as WC in Eq. (5)), adsorption energy (ΔHads,1) of the most adsorbed species, and selectivity (Sads). It additionally uses weighting factors to reflect the specific requirements of a given process.\n
They calculated APIs of seven MOFs for two different CO2/CH4 separation scenarios using the experimental gas adsorption data [49]. Results showed that API can be more versatile than previously discussed comparison criteria for an initial indication of potential adsorbent performance.
\nIt is beyond the scope of this chapter to give a complete account of all GCMC studies of MOFs for CO2 separation. Several molecular simulation studies examined a single MOF or a few MOFs at a time [50–59]. Most of these studies focused on adsorption-based separation of CO2 using the two most widely studied MOFs, MOF-5 and CuBTC [60–64]. This section will focus on molecular simulation studies that examine a family of MOFs for adsorption-based CO2 separations and large-scale computational screening studies to provide a relation between structure and separation performance of MOFs.
\nIRMOFs are isoreticular MOFs. The “IR” stands for isoreticular, which essentially means that the molecular system is “stitched together” into a netlike structure through strong chemical bonds. Molecular simulations were used to compare the separation of CO2/N2 mixtures in two different classes of nanoporous materials, traditional zeolites (MFI, LTA, and DDR) and MOFs (IRMOF-1, -11, -12, -13, -14, CuBTC, and MIL-47 (V)) [65]. Results showed that although MOFs perform much better for gas storage than zeolites, their CO2 separation performance is comparable to zeolites with adsorption selectivities in the range of 5–35. Krishna and van Baten [46] used configurational-bias Monte Carlo (CBMC) simulations to examine zeolites and IRMOF-1, MOF-177, rho-ZMOF, CuBTC, ZnMOF-74, and MgMOF-74 for separation of CO2/CH4, CO2/N2, and CO2/H2 mixtures using PSA units. The best CO2 capture performance was obtained with MgMOF-74 that offers strong electrostatic interactions of CO2 molecules with the exposed metal cation sites. Selectivity of MgMOF-74 was reported as ∼300, 50, and 10 for CO2/H2, CO2/N2, and CO2/CH4 separations, respectively. Traditional zeolite adsorbents such as NaX and NaY show higher adsorption selectivities for these gas separations but these two zeolites suffer from relatively low working capacities that are important in PSA units. For CO2/N2 and CO2/H2 separation in PSA units, MgMOF-74 was found to offer the best combination of high adsorption selectivity and high working capacity. Han et al. [66] used GCMC simulations with first-principles-based force fields to report the effects of interpenetration on the CO2/H2 separation of MOFs. Non-interpenetrating IRMOF-1, -7, -8, -10, -14, -16, MOF-177, and MOF-200 and interpenetrating IRMOF-9, -13, -62, and SUMOF-4 were considered for comparison. For example, IRMOF-9 and IRMOF-13 are interpenetrating versions of IRMOF-10 and -14, respectively. The interpenetration of MOFs at low pressure remarkably enhanced the selectivity of CO2 over H2 by creating new adsorption sites for CO2. However, selectivity of the interpenetrating and non-interpenetrating MOFs was reversed at higher pressures. Since interpenetration lowers the pore volume of MOFs, it significantly reduced CO2 uptake at high pressures. The decrease in the H2 uptake resulting from interpenetration was found to be marginal. Therefore, selectivity of the interpenetrating MOFs was reported to be lower than that of non-interpenetrating MOFs at high pressures.
\nAdsorption-based separation performance of ZIFs for (a) CH4/H2, (b) CO2/CH4, and (c) CO2/H2 mixtures. The compositions of the bulk gas mixtures are (a) 10/90, (b) 10/90, and (c) 1/99 for ZIFs at 298 K and (a) 50/50, (b) 50/50, and (c) 15/85 for zeolites at 300 K. Reprinted with permission from Ref. [68]. Copyright (2012) American Chemical Society.
Zeolitic imidazolate frameworks (ZIFs) are composed of tetrahedral networks that resemble those of zeolites with transition metals connected by imidazolate ligands. Zeolites are known with the Al(Si)O2 unit formula, whereas ZIFs are recognized by M(Im)2, where M is the transition metal and Im is the imidazolate-type linker. Battisti et al. [67] calculated CO2/CH4, CO2/H2, and CO2/N2 adsorption selectivity in the zero-pressure limit for nine ZIFs, ZIF-2, -3, -4, -5, -6, -7, -8, -9, and -10 using GCMC simulations. These ZIFs were characterized by pores of medium to small size compared to other MOFs. Therefore, they were able to store at most half of the amount of gases than other MOFs. ZIF-7 and ZIF-9 were shown to be promising due to their high adsorption selectivities of 280 and 15 for CO2/H2 and CO2/N2 separations, respectively. Atci and Keskin [68] used GCMC simulations to predict the performance of 15 different ZIFs (ZIF-1, -2, -3, -6, -8, -10, -60, -65, -67, -68, -69, -70, -79, -81, and -90) in both adsorption-based and membrane-based separations of CH4/H2, CO2/CH4, and CO2/H2 mixtures. Adsorption selectivity, working capacity, membrane selectivity, and gas permeability of ZIFs were predicted using GCMC and molecular dynamics simulations. Figure 1 compares adsorption selectivities and working capacities (shown as delta loadings) of ZIFs calculated at an adsorption pressure of 10 bar and desorption pressure of 1 bar with the data for zeolites and MOFs [68]. Several ZIFs were identified to outperform traditional zeolites and widely studied MOFs in CO2/CH4 and CO2/H2 separations. Keskin’s group [69] also calculated adsorption of both single-component gases (CH4, CO2, H2, and N2) and binary gas mixtures (CO2/CH4, CO2/N2, and CO2/H2) using GCMC simulations and predicted the ideal and mixture adsorption selectivities of ZIFs. They showed that the adsorption selectivity calculated from mixture GCMC simulations can be significantly higher than the ideal adsorption selectivity calculated from single-component adsorption isotherms. This result highlighted the importance of using mixture selectivity to assess the performance of MOF adsorbents.
\nThe calculated adsorption selectivity of ZIFs may vary significantly depending on the force field parameters used in molecular simulations [70]. Generic force fields generally tend to overestimate gas adsorption capacities; hence, adsorption selectivities of ZIF-68 and ZIF-69 compared to the tailored-force fields. However, the difference between predicted adsorption selectivities of different force fields is not high enough to change the assessment about the separation performance of the material. Both generic and tailored-force fields were able to identify the promising adsorbent materials that exhibit high adsorption selectivities. In other words, generic force fields can be safely used to screen large number of MOFs to differentiate between the promising and non-promising materials.
\nMOFs are also referred as porous coordination networks (PCNs) in the literature and several synthesized materials have been named as PCNs [71]. Ozturk and Keskin [72] studied 20 different PCNs (PCN-6, 6’, 9-Co, 9-Mn, 9-Fe, 10, 11, 13, 14, 16, 16’, 18, 19, 20, 26, 39, 46, 80, 131’, and 224-Ni) using molecular simulations to identify the most promising adsorbent and membrane candidates for CO2/H2, CO2/CH4, and CO2/N2 mixtures. PCN-9-Co, -9-Mn, -14, and -16 were found to be strong adsorbents for CO2 capture, especially for CO2/CH4 separations because of their high working capacities. They also developed a simple model that can predict adsorption selectivities of PCNs for CO2/H2 mixtures without performing extensive molecular simulations. The model was based on the structural and chemical properties of the materials that can be simply measured or computed, such as pore volume, surface area, and the inverse of difference of heat of adsorption of components in the mixture. Predictions of the model for adsorbent selectivities were found to be in good agreement with the direct results of detailed molecular simulations.
\nCovalent organic frameworks (COFs) are not strictly MOFs but similar materials. They are formed by building units linked into a periodic framework but in contrast to MOFs, all components are organic in COFs. Zhong’s group [73] examined a diverse set of 46 COFs to predict their separation performance for industrial gas mixtures, CO2/H2 and CO2/CH4, using PSA process. Results show that COFs outperform most commonly used zeolites and widely studied MOFs in the separation of CH4/H2 while they have a comparable performance in separating CO2/H2 and CO2/CH4. The same group then studied the performance of 151 MOFs with large chemical and topological diversity for CO2/CH4 separation for temperature swing adsorption (TSA) process [74]. The thermal regeneration energy was used as an evaluation criterion in addition to adsorption selectivity, working capacity, and regenerability. Cu-TDPAT and IRMOF-1-2Li MOFs were reported as the most promising candidates for CO2/CH4 separation in TSA processes based on the ranking of the materials according to the four evaluation criteria. Cu-TDPAT was identified as the best adsorbent candidate because of its high thermal stability and water-stable property.
\nIn 2012, Sholl’s group [75] used GCMC simulations to calculate adsorption of CO2 and N2 in 500 different MOFs. This was the largest set of structures for which this information has been reported until 2012. Adsorption selectivities of MOFs were calculated using Henry’s constant at infinite dilute loading. More detailed calculations such as quantum chemistry methods and binary mixture GCMC simulations were then carried out to assess adsorption selectivities of highly promising MOFs. Watanabe and Sholl [76] later on screened a larger number of MOFs for CO2/N2 separation. They first analyzed pore characteristics of 1163 MOFs using a simple steric model developed by Haldoupis et al. [77]. Adsorption selectivity of the selected 201 MOFs was calculated using single-component GCMC simulations at infinite dilute loading. Selectivities were plotted as a function of largest cavity diameter (LCD) of MOFs as shown in Figure 2. This figure demonstrates that MOFs are promising materials for CO2/N2 separations. There is a significant number of MOFs with high selectivities of 100–1000. A small number of materials have extremely high adsorption selectivities, greater than 1000. Selectivities of the top 10 promising MOFs were also computed considering CO2/N2:15/85 mixture and results showed that CO2/N2 selectivities of MOFs remained high even for binary gas mixtures with the composition of dry flue gas.
\nLin et al. [78] screened hundreds of thousands of hypothetical zeolite and ZIF structures for CO2 capture from flue gas. They determined the optimal process conditions of each material by minimizing the electric load imposed on a power plant by a temperature-pressure swing capture process using that material followed by compression. This minimum load was called as parasitic energy and it was introduced as a metric to compare different materials. Results of that study showed that parasitic energy for ZIFs is higher than for zeolites. Wilmer et al. [79] drastically expanded the scope of previous MOF screening studies by examining over 130,000 hypothetical MOFs. They used molecular simulation to calculate adsorption selectivity, working capacity, regenerability, and sorbent selection parameter of MOFs for CO2/CH4 and CO2/N2 separations. Single-component GCMC simulations to obtain the pure component CO2, CH4, and N2 adsorption data are required to calculate the five adsorbent evaluation criteria. The resulting simulation data exhibited sharply defined structure-property relationships as we will discuss in Section 2.4. These type of relationships were not apparent when smaller collections of MOFs were studied in the previous works, indicating that screening large number of MOFs is important to understand the effect of structure on the gas separation performance of MOFs.
\nCO2/N2 sorption selectivity of the MOFs at 303 K. The data include only the materials with CO2 diffusivity greater than 10−8 cm2/s. Reprinted with permission from Ref. [76]. Copyright (2012) American Chemical Society.
We so far discussed the molecular simulation studies in the literature that mimic a classical adsorption experiment. Other than isotherm experiments, breakthrough experiments are also carried out on nanoporous adsorbents in order to investigate the materials’ kinetics. However, these experiments are labor-intensive and can present a range of technical challenges to achieve accurate results. Krishna and Long [80] suggested a new metric, breakthrough time (τbreak), that is based on the analysis of the transient response of an adsorber to a step input of a gaseous mixture. This metric determines the frequency of required regeneration of an adsorbent. High value of τbreak is desirable in practice because it reduces the frequency of required regeneration. Breakthrough calculations were done for separation of CO2/H2, CO2/CH4, and CO2/CH4/H2 mixtures using five MOFs, MgMOF-74, CuBTTri, MOF-177, BeBTB, and Co(BDP) and results were compared with traditional zeolites. MgMOF-74 emerged as the best material from the viewpoints of both frequency of regeneration and productivity. The advantage of MgMOF-74 over traditionally used NaX zeolite was found to be evident at pressures exceeding 10 bar. Krishna and van Baten [81] later studied breakthrough characteristics of an adsorber packed with a number of zeolites (MFI, JBW, AFX, and NaX) and MOFs (MgMOF-74, MOF-177, and CuBTTri-mmen) for CO2 capture from a CO2/N2 mixture. These calculations demonstrated that high capacities could have a dominant influence on the overall performance of PSA units. MgMOF-74 was again identified as a promising adsorbent with a CO2 capture capacity more than twice that of other materials investigated. For separation of CO2/H2, CO2/CH4, and CH4/H2 mixtures, Jiang’s group [82] recently studied seven different rht-MOFs namely Cu-TDPAT, PCN-61,-66,-68, NOTT-112, NU-111, and NU-110. These MOFs have the same rht topology with different ligands. The breakthrough profiles for CO2-containing mixtures were predicted from the simulation results. Due to the presence of small ligands, unsaturated metals, and amine groups, Cu-TDPAT was found to exhibit the highest adsorption capacity and separation performance among the seven rht-MOFs. Upon substituting the phenyl rings in Cu-TDPAT by pyridine rings, Cu-TDPAT-N was designed and the breakthrough time for CO2 in Cu-TDPAT-N was found to be extended by twofold. This result shows the importance of understanding structure-separation performance relations for MOFs as we discuss below.
\nHigh-throughput computational screening is a very useful approach to identify promising MOF materials for gas separation applications and to uncover structure-property relations [83]. With the development of new computational methodologies, it is now easier to perform large-scale computational screening studies where the properties of thousands of MOF candidates can be evaluated and compared. When this type of large-scale MOF screening is performed, a large amount of data is produced and used to investigate correlations between MOFs’ structural properties and their gas separation performances.
\nThe interplay map of ϕ and ΔQst0 on their impact on the selectivity at 1 bar for CO2/N2 mixture in MOFs, where the design strategy based on UiO-66(Zr) is also given. Reprinted with permission from Ref. [84]. Copyright (2012) American Chemical Society.
Maurin’s group [84] used molecular simulations to examine separation performance of 105 MOFs with a large chemical and topological diversity for CO2 capture from flue gas under industrial operating conditions. They developed a quantitative structure-property relationship (QSPR) model from this extended series of MOFs to rationalize the resulting CO2/N2 selectivity. The difference of isosteric heats of adsorption between CO2 and N2 at infinite dilution (ΔQst0) and porosity (ϕ) were found to be the main features of the MOFs that strongly impact the CO2/N2 adsorption selectivity at 1 bar. Figure 3 shows the interplay map of these two factors on the calculated CO2/N2 adsorption selectivity. Results of QSPR analysis suggested that increasing ΔQst0 and simultaneously decreasing ϕ seems to be an appropriate route to enhance the CO2/N2 selectivity of MOFs. Motivated from this structure-performance relation, a new functionalized MOF, UiO-66(Zr)-(SO3H)2, was computationally designed and predicted to exhibit a high CO2/N2 selectivity as shown in Figure 3.
\nThe CO2 separation potential of a new class of porous aromatic frameworks (PAFs) with diamond-like structure was studied by molecular simulations [85]. It was discussed that selectivity might be only determined by the difference of the gas-material interactions of the mixtures at a pressure close to zero. The CO2/H2, CO2/N2, and CO2/CH4 selectivities and the difference of isosteric heats (ΔQst0) were calculated at the pressure close to zero. The ΔQst0 was found to be linear with the logarithm of the selectivity no matter what the gas mixtures and materials were. This result suggested that at zero pressure the selectivity is only dependent on the values of ΔQst0 and is independent of the type of the gases and the materials. With the increase of ΔQst0, the selectivity increases correspondingly, which means that the ΔQst0 can be used instead of the selectivity to screen out the promising nanoporous materials for gas separation. Finding a correlation between adsorption selectivity and ΔQst0 is useful. However, it is difficult to design new MOF materials that have a priori chosen Qst value. It is easier to design materials based on measurable structural properties such as porosity, pore size, or surface area. Wilmer et al. [79] studied a very large number of hypothetical MOFs and showed clear correlations between purely structural characteristics such as pore size, surface area, and pore volume as well as chemical characteristics such as functional groups with five adsorbent evaluation criteria listed in Section 2.2. For example, it was shown that adsorption selectivity correlates well with the maximum pore diameter for flue gas separation. Adsorption selectivity also correlates with the heat of adsorption of CO2 for flue gas and natural gas separation. Certain chemical functional groups, particularly those with fluorine and chlorine atoms, were frequently found among the best performing MOF adsorbents. These type of structure-property relationships can be used as a guide for experimental MOF synthesis studies.
\nIn a recent study, in silico screening of 4764 MOFs was performed for adsorption-based CO2/CH4 and CO2/N2 separations [86]. Quantitative relations between the metal type and adsorbent properties such as selectivity, working capacity, and regenerability were investigated for the first time in the literature. A wide variety of metals exists in MOFs such as alkalis, alkalines, lanthanides (Ln), and transition metals. Figure 4 shows the probabilities of different metals in the selected MOFs based on their selectivity and working capacity for CO2/CH4 and CO2/N2 separations. For instance, the probability of K is about 0.8 for CO2/N2 separation, meaning that 80% of K-based MOFs show high selectivity and working capacity. Combining selectivity, working capacity, and regenerability, however, alkali- and alkaline-MOFs possess the lowest performance for CO2 separation. Among 4764 MOFs, about 1000 were found to contain Ln metals, 50% contain Ln as open metal sites. These open metal sites have high adsorption affinity for CO2; therefore, MOFs with Ln metals have the highest CO2 separation performance. The 30 best candidates identified for CO2/CH4 and CO2/N2 separations have Ln metals. These results can be used to synthesize MOFs having predetermined metal atoms to enhance the CO2 separation performance of materials.
\nProbabilities of different metals in the selected MOFs (based on S and NCO2). The red lines indicate the percentages of the selected MOFs from the total. Reprinted with permission from Ref. [84]. Copyright (2015) The Royal Society of Chemistry.
As can be seen from this literature review, current studies have generally focused on establishing relations between adsorption selectivity and a single chemical or structural property such as difference of isosteric heat of adsorption of gases or metal type. However, separation performances of materials are determined by the interplay of various factors and cannot be easily correlated to only one or two properties. All physical and chemical properties of MOFs including pore size, shape, porosity, surface area, topology, metal and organic linker type must be considered to better understand structure-performance relations. Deriving structure-performance relations for MOFs is a new and developing research area and more studies in this area will be valuable to synthesize new MOFs with useful physical and chemical properties to achieve targeted gas separations.
\nMolecular simulations are very useful to quickly evaluate the potential of new MOF materials in adsorption-based gas separation processes. The outcome of molecular simulations can be used as a guide to design and develop new materials with enhanced separation properties. There is a continuous growth in the number of molecular simulation studies of MOFs for adsorption-based CO2 separations. However, there are still several open areas in which future studies will be valuable. Opportunities and challenges related with these open research areas are discussed below:
\nStrategies to improve the ability of MOFs to selectively adsorb CO2 are reviewed in detail in the literature [87]. Some of these strategies are control of pore size, using materials with open metal sites, introduction of alkali-metal cations into MOFs, interpenetration, and using materials with polar functional groups [45, 88]. Among these, rational design of functionalized materials is a feasible way to improve the CO2 separation efficiency of MOFs. GCMC simulations were recently used to study the effect of amine functionalization on the CO2/CH4 separation performance of MIL-53 [89]. Results showed that CO2/CH4 separation factor of −(NH2)4 amine-functionalized MIL-53 is the best and predicted separation performance of −NH2 and −NHCO functionalized MIL-53 surpasses that of the original one. Future molecular simulation studies examining the effects of functionalization on the separation performance of MOFs will be very useful to establish guidelines for the experimental design and development of new materials.
\nAs discussed in Section 2.3., most molecular simulation studies of MOFs focus on the separation of CO2 from its binary mixtures such as CO2/CH4 and CO2/N2. However, in reality, these gas mixtures include some impurities. Water and the other minor components mostly H2O, O2, SO2, and NOx cannot be ignored in assessing the performance of MOFs especially for post-combustion CO2 capture. However, the number of molecular simulation studies examining the effects of trace gases on the CO2/N2 and CO2/CH4 separation performance of MOFs is limited. Bahamon and Vega [90] recently used GCMC simulations to study 11 materials including zeolites and MOFs for separation of CO2 from N2, including water as an impurity. Sun et al. [91] studied 12 materials including MOFs, ZIFs, and zeolites for removal of SO2 and NOx from flue gas using GCMC simulations. The influences of water and SO2 on CO2 adsorption and separation in UiO-66(Zr) MOFs with different functional groups were evaluated using a combination of GCMC and DFT simulations [92]. Babarao et al. [93] considered small amounts of O2, H2O, and SO2 impurities typically found in flue gas and evaluated the CO2/N2 selectivity of four PCNs in the presence of these impurities. Zhong’s group [94] used molecular simulations to investigate the effect of trace amount of water on CO2 capture in natural gas upgrading process in a diverse collection of 25 MOFs. These studies concluded that the effect of H2O impurities on the CO2 selectivity is highly specific to the chemistry of the framework and needs to be evaluated on an individual case-by-case basis. The CO2 selectivity of MOFs was generally reported to decrease in the presence of water. Future studies on GCMC simulations of MOFs considering impurities in CO2-related mixtures must be conducted to evaluate the potential use of MOFs in industrial CO2 capture processes.
\nWhile CO2 separation using MOF adsorbents has been extensively investigated in different MOFs, their performance under practical process conditions is scarcely examined. A multi-scale modeling study was recently carried out to examine CO2 capture from flue gas by vacuum swing adsorption (VSA) process using rho-ZMOFs as adsorbents [95]. The full adsorption process was simulated and optimized and results showed that the operating spaces of rho-ZMOFs are similar to that of traditional 13X zeolite. Further studies that employ multi-scale modeling approaches will be useful to design and develop MOF-based industrial CO2 separation processes. A related point is to test the long-term stability of MOF adsorbents under industrial operating conditions. An ideal MOF adsorbent must have good thermal and mechanical stability. Several MOFs are sensitive to atmospheric moisture and lose their crystal structures when exposed to water. This may be a significant problem when MOFs are used as adsorbents in flue gas separations since flue gas contains water. Molecular simulation studies that can provide information about the long-term stability of MOF adsorbents will be useful to evaluate the real performance of MOFs.
\nSoybean (Glycine max [L.]) is a leguminous plant that can form a symbiotic relationship with the nitrogen-fixing group of bacteria living in the rhizosphere, which are generally termed as rhizobia. In the Philippines, soybean production has been limited by the poor grain yield which leads to the importation of more than 90% of the country’s demand. Thus, it is essential to look for an alternative way to increase the volume of production per unit area.
The research about tropical bradyrhizobia indicated a high diversity of species and their distribution has been reported to be due to several abiotic and biotic factors such as soil acidity [1, 2, 3], alkalinity [3, 4], temperature [1, 5, 6, 7, 8, 9, 10, 11], climate [12, 13], soil water status [14, 15], soil type [2, 14, 16, 17, 18], and soil management or cultural practices [2, 14, 19, 20, 21, 22]. In case of the Philippines, the pioneer research that was able to identify the most dominant species of bradyrhizobia in the country reported that B. elkanii species was the most abundant, followed by the B. diazoefficiens, B. japonicum, and some yet unclassified Bradyrhizobium sp. [14]. In this later study, it was identified that the distribution of these indigenous species of bradyrhizobia were influenced mainly by the water status of the soil, followed by soil pH, nutrient content, and soil type.
Previous studies have reported that aside from the various agro-environmental factors, the competition with the native rhizobia is a hindrance for a successful inoculation [23, 24]. The utilization of inoculants for legumes had shown promising results for the increase in grain yield as evidenced by recent reports [25, 26]. The role of the biological nitrogen fixation (BNF) in providing the N requirement of the plant in a natural way has been deemed necessary especially these times that the soil has become more degraded due to over-fertilization. The indiscriminate use of NPK fertilizer could cause soil pollution and less crop production [27]. Therefore, it is essential to select and evaluate the symbiotic competitiveness of the indigenous strains which are native and existing in high density in the country. The use of different genetic markers to accurately identify the rhizobia for taxonomic purposes has been proposed [28] and so we have used three genetic markers such as the 16S rRNA gene, 16S-23S rRNA gene internal transcribed spacer (ITS) region, and the rpoB housekeeping gene.
Thus, this study was formulated with the aim to utilize the recently identified indigenous bradyrhizobia in the Philippines and characterize their symbiotic performance with the local soybean cultivars.
The soil samples were collected from 11 locations in the Philippines, where some basic information on the sites are listed in Table 1. The collection of soil was conducted by first removing the surface litters then, obtaining a bar of soil with a dimension of approximately 20 cm in depth and 3 cm in thickness that weighs about 1 kg. A total of 10 subsamples per location were obtained and were mixed thoroughly until a 1 kg of composite soil sample was taken. A 0.5 kg soil was air-dried for the chemical analyses while the remaining 0.5 kg of the fresh soil was used for the soybean cultivation.
Result of the soil chemical analysis on the 11 locations in the Philippines.
Mason et al., 2018
This study
The cultivation of soybean was performed using a 1-L capacity culture pots (n = 3). Each pot was filled with vermiculite and a N-free solution [29] was added at 40% (vol/vol) water content. The culture pots were sterilized by autoclaving for 20 min at 121°C. Meanwhile, the soybean seeds were surface-sterilized by soaking into a 70% EtOh for 30 s, then by a diluted sodium hypochlorite solution (0.25% available chlorine) for 3 min and followed by washing with sterile distilled water for about 6–8 times. Then, a 2–3 g of soil sample was placed on the vermiculite at a depth of about 2–3 cm, the seeds were sown on the soil and the pot was weighed and recorded. The plants were grown inside a growth chamber for 28 days at 28°C (8 h, night) and 33°C (16 h, day) then were supplied weekly with sterile distilled water until the initial weight of the pot was reached.
After 28 days, approximately 20 random nodules were collected from the roots of each soybean plants and were sterilized with 70% EtOh and sodium hypochlorite solution as previously described [29]. Each nodule was homogenized with sterile distilled water in a microtube and streaked on to a yeast-extract mannitol agar (YMA) plate [30]. The YMA plate was incubated in the dark at 28°C for about 1 week until a single colony was formed. After then, the single colony was streaked on to a YMA plate containing a 0.002% (wt/wt) bromothymol blue (BTB) [31] and was incubated as above. Repeated streaking was done until a pure single colony was obtained which was cultured for about 3–4 days in a HEPES-MES (HM) broth culture [32, 33] at 28°C in a shaker for 120 rpm. After then, the bacteria cells were collected by centrifugation at 9000×g and washed with sterile distilled water. The DNA was extracted by using BL buffer as described [34] from the method reported by Hiraishi et al. [35].
For the amplification of the 16S rRNA gene, the primer set: 16S-F: 5′ AGAG TTTGATCCTGGCTCAG-3′ and 16S-R2: 5′- CGGCTACCTTGTTACGACTT-3′ [36]. The PCR tubes were then placed in the PCR Thermal Cycler (TaKaRa Co. Ltd.) with the following conditions: pre-run at 94°C for 5 min; followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min. Final extension was set at 72°C for 10 min and indefinite preservation at 4°C.
On the other hand, the PCR amplification of the ITS region was conducted using the following primer set: Bra-ITS-F: 5-GACTGGGGTGAAGTCGTAAC-3′ and Bra-ITS-R1: 5′-ACGTCCTTCATCGCC TC-3′ [6]. The PCR cycle for the ITS region was almost the same with the 16S rRNA gene except for a shorter denaturation and annealing periods which were conducted at 30 s for each step.
For the rpoB gene, simplification was done using the following primer sets: rpoB83F: 5′-CCTSATCGAGGTTCAC AGAAGGC-3′ and rpoB1540R: 5′-AGCTGCGAGGAACCGAAG-3′ [37]. The PCR cycle conditions were as follows: pre-run at 94°C for 5 min; followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 1 min, and extension at 72°C for 1 min. Final extension was set at 72°C for 5 min and indefinite preservation at 4°C.
The successfully amplified products were subjected to the RFLP treatment using four restriction enzymes which were HhaI, HaeIII, MspI, and XspI. For the rpoB gene, the enzymes that were used for RFLP are HaeIII, MspI, and AluI. The reference strains that were used in this study are the Bradyrhizobium USDA strains (B. japonicum USDA 4, 6T, 38, 62, 115, 122, 123, 124, 125, 127, 129, 135, B. diazoefficiens USDA 110T, B. elkanii 31, 46, 61, 76T, 94, 130, and B. liaoningense 3622T) which were previously described [38]. This was done in a 10-μL reaction mixture containing a 2.5-μL amplified PCR product and was incubated in a 37°C for 16 h. Afterward, a 3–4% agarose gel was used in a submerged gel electrophoresis for about 60 min, stained with ethidium bromide and the patterns were visualized using a Luminiscent Image Analyzer LAS-4000 (FUJIFILM Tokyo, Japan).
After the amplification and the RFLP treatment of the 16S rRNA gene, a single-strain inoculation test was conducted for all the amplified isolates that shared the same restriction enzymes’ fragment patterns with the USDA Bradyrhizobium reference strains. This was done to confirm the strain’s capability to nodulate soybean and was tested on two local varieties which are the PSB-SY2 and Collection 1 which are both commercially available across the country.
The cultivation of soybean was conducted as described above, but without soil. Each isolate was cultured in a YM broth (YMB) [30] at 28°C for about 1 week on a shaker. After then, the cultures were diluted with sterile distilled water at about 106 cells mL−1 and were inoculated on the cultivated soybean at a rate of 1.0 mL per seed. This was done with three replications. After inoculation, the weight of the pot was recorded and it was placed inside a growth chamber with a condition set to mimic the average temperature in the Philippines at 26°C (8 h, night) and 33°C (16 h, day). The same condition was used for the cultivation of an uninoculated control and a positive control pot that was inoculated with B. diazoefficiens USDA110. The pots were kept inside the growth chamber for 28 days and were supplied weekly with sterile distilled water until the initial weight of each pot was reached.
According to the similarities of the band patterns through the RFLP treatment, a representative of the most abundant isolates was chosen for each location. In total, there were 11 isolates that were selected to confirm the nucleotide sequence of the 16S rRNA gene and the ITS region. The sequence primers that were used were reported previously [22]. From the PCR amplified product, the samples were purified according to the protocol of the manufacturer (Nucleospin® Gel and PCR Clean-up; Macherey-Nagel, Germany). Then, the samples were sent to the company for the sequence analysis (Eurofins Genomics, Tokyo, Japan).
Then, the Basic Local Alignment Search Tool (BLAST) program in DNA Databank of Japan (DDBJ) was used to determine the nucleotide homology of the isolates. Only the sequences with a similarity of at least 99% for the 16S rRNA and 96% for the ITS region with our isolates were retrieved from the BLAST database. The alignment was performed using the ClustalW and Neighbor-Joining [21] method was used to construct the phylogenetic trees. The genetic distances were computed using the Kimura 2-parameter model [39] in the Molecular Evolutionary Genetic Analysis (MEGA v7) software [40]. Subsequently, the phylogenetic trees were bootstrapped with 1000 replications. All the nucleotide sequences determined in this study were deposited in DDBJ at
The soil samples that were used in this study were all slightly to moderately acidic (5.22–6.64) with non-saline condition (0.05–0.20 dS/m), low nutrient status as evidenced by low amounts of NPK and CEC (Table 1). These values are generally typical of agricultural soils that are used for crop production all throughout the year. These results showed that the soils used in this study have low fertility status that indicated the need for soil restoration strategies.
The growth morphologies of the pure single colony for each strain of bradyrhizobia were characterized and listed in Table 2. All the isolates were slow growers which were able to form single colonies measuring about 2 mm between 5 and 7 days upon streaking on YMA plates and incubation in a dark room. Based on the morphology, the isolates were grouped into three. Group I include the isolates IS-2, NE1–6, NR-2, and BO-4 which were translucent and the colonies are circular in shape with slightly convex elevation and an entire margin. When they were manipulated with a needle, the colony was liquid. Group II include the isolates BA-24, SO-1, LT-3, and SK-5 were translucent with circular colonies, convex elevation with entire margin. When manipulated with a needle, the colonies have mucoid viscosity. On the other hand, last group (III) are the isolates GI-4 and NE2-37 which have similar growth morphology with Group II except that their viscosity was intermediate between liquid and mucoid. All the isolates produced alkaline substances when grown on YMA plate with BTB which is an indication of the Bradyrhizobium genus.
Characterization of the morphology of the indigenous bradyrhizobia isolated from Philippines’ soil according to their growth on Yeast-Extract Mannitol Agar plate medium [30].
As seen in Figure 1, it is evident that the 11 most abundant indigenous soybean rhizobia in the Philippines are classified under the genus Bradyrhizobium, and are separated into its two species, B. japonicum and B. elkanii, according to the phylogenetic tree from the sequence analysis of the 16S rRNA gene. To further confirm the classification of the indigenous bradyrhizobia, the phylogenetic trees constructed from the ITS region and the rpoB gene are presented in Figures 2 and 3, respectively. For the ITS region and the rpoB gene, the isolates were distinctly grouped into three species, B. elkanii, B. japonicum, and B. diazoefficiens. Additionally, an independent cluster composed of the representative isolates GI-4 and NE2–37 that are seen in the ITS region and rpoB phylogenetic trees were treated as Bradyrhizobium sp. due to their nucleotide divergence with the known species from the BLAST engine.
Phylogenetic tree based on the sequence analysis of the 16S rRNA gene. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The accession numbers are indicated only for sequences obtained from BLAST. The isolates in this study are indicated with letters and number combinations, for example: BO-4–isolate no. 4 collected from Bohol.
Phylogenetic tree based on the sequence analysis of the 16S-23S rRNA internal transcribed spacer (ITS) region. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The accession numbers are indicated only for sequences obtained from BLAST. The isolates in this study are indicated with letters and number combinations, for example: BO-4–isolate no. 4 collected from Bohol.
Phylogenetic tree based on the sequence analysis of the rpoB housekeeping gene. The tree was constructed using the Neighbor-Joining method with the Kimura 2-parameter (K2P) distance correlation model and 1000 bootstrap replications in MEGA v.7 software. The accession numbers are indicated only for sequences obtained from BLAST. The isolates in this study are indicated with letters and number combinations, for example: BO-4–isolate no.4 collected from Bohol.
Meanwhile, the distribution of the most abundant soybean bradyrhizobia in the country is shown in Table 3, which was classified according to the results of the sequence analysis of the three genetic markers used in this study. From here, it can be seen that 4 of the 11 locations were dominated with B. elkanii species (37.74%), 3 locations were dominated by the isolates under the B. diazoefficiens (28.54%), whereas 2 locations each were dominated by the species of B. japonicum (16.98% and Bradyrhizobium sp. (16.74%). This indicated that in the Philippines, the species of B. elkanii is the most prevalent in terms of population and the most widespread in terms of location as its presence was detected even in minor populations on all the locations except for one, which was Sorsogon.
Percentage distribution of the dominant Bradyrhizobium species in the Philippines as identified from the sequence analysis of the 16S rRNA gene, 16S-23S internal transcribed spacer (ITS) region, and rpoB housekeeping gene.
Upon classification, it is important to determine the capability of the indigenous bradyrhizobia for their symbiotic performance and N-fixation ability. As can be seen in Figure 4A, although USDA110 strain has the highest N-fixation ability, it should be noted that the amount of N that was fixed by B. elkanii IS-2 is the highest among all the indigenous bradyrhizobia isolated from the Philippines’ soil on Rj4 plants. However, the N-fixation ability of IS-2 was comparably similar with other strains (GI-4, NE2–37, and SK-5) with the non-Rj plants. The lowest N-fixation ability was observed from the strain LT-3 which was classified under the B. diazoefficiens species. This suggested that the process of biological N-fixation is a mutual relationship that is influenced by both the plant and the rhizobia and that the plant-rhizobia compatibility should be taken into consideration for inoculation strategies.
Characterization of the dominant indigenous Bradyrhizobium strains isolated from the 11 locations in the Philippines based on the (A) amount of Nitrogen fixed (B) nodulation ability and (C) symbiotic efficiency as influenced by the single-strain inoculation test against the reference strain B. diazoefficiens USDA110 for the two soybean cultivars from the Philippines. Different letters indicate a significant difference by Tukey’s test at p > 0.05, n=3, bar=SE.
Presented in Figure 4B is the nodulation test performed on the strains and it can be seen for Rj4 plants, there was not much significant difference in the nodulation ability of the strains, except for the low nodulation ability that was observed for the BO-4. In contrast, a significant difference in the nodulation ability was detected on the strains upon inoculation on the non-Rj plants. Although all the strains were able to form nodules on both soybean cultivars, the strains GI-4, NR-2, and SK-5 obtained the highest number of nodules for the non-Rj plants.
On the other hand, the symbiotic efficiency of the strains used in this study is presented in Figure 4C. Similar with the N-fixation ability, the USDA110 still possesses the highest symbiotic efficiency. But among all the indigenous bradyrhizobia, the strain IS-2 obtained the highest efficiency regardless of the Rj genotype of the soybean plants. As with the N-fixation, LT-3 obtained the least efficiency for symbiosis. This result indicated that the symbiotic efficiency of the rhizobia might not be directly influenced by the Rj genotype of the plant.
The distribution of the most dominant and abundant species of soybean bradyrhizobia in the Philippines are reported in this study along with the characterization of their growth morphology. According to our earlier reports, we have elucidated that the Philippines was dominated by the soybean-nodulating bradyrhizobia that were classified under the B. elkanii species and the most important agro-environmental factors that affected their diversity and prevalence in the country was the similarity of soil pH, salinity, and temperature in the study locations [5, 14]. Our observation that there are abundant and high diversity of indigenous bradyrhizobia in the Philippines is similar with previous reports in other sub-tropical and tropical regions [12, 25, 41, 42, 43, 44]. The temperate regions of Japan and USA were studied in the past and were reported to be dominated by species of B. japonicum and B. diazoefficiens [6, 9, 10, 11, 13, 45]. Our report showed that the distribution of bradyrhizobia in a tropical region like the Philippines seemed to be different from those of temperate regions.
Meanwhile, it was included in a recent report that the distribution and abundance of B. diazoefficiens and B. japonicum at specific locations were due to the longer period of flooding conditions [14]. The effect of nutrient content and soil type were also correlated with the abundance of these two species. In a report by Shiina et al. [17], it was stated that the predominance of B. diazoefficiens was observed on more anaerobic condition; whereas, B. japonicum was predominant on aerobic soils which was supported by another study [18]. Additionally, it was reported that B. diazoefficiens becomes predominant with enhanced flooding condition [15]. These results confirmed that our observations for the abundant of B. diazoefficiens, followed by B. japonicum and Bradyrhizobium sp. on flooded areas in the country which were usually used for planting rice.
In this report, the symbiotic performance, N-fixation and nodulation ability of the indigenous soybean bradyrhizobia form the Philippines were evaluated against that of the B. diazoefficiens USDA110 strain. The USDA110 has been extensively used in the world as a model strain for soybean inoculation due to its high ability for N-fixation and symbiotic efficiency [25, 46, 47]. Additionally, its possession of a complete set of denitrification genes that allows the release of N2 back into the atmosphere makes it an ideal strain also for climate change mitigation studies [17, 48, 49, 50].
Therefore, we hypothesized that the indigenous isolates SO-1, LT-3, and SK-5, which were phylogenetically clustered under the USDA110 would also prove to be as effective N-fixer and efficient microsymbiont of soybean cultivars from the Philippines. However, our results indicated that the N-fixation ability and symbiotic efficiency of LT-3 and SO-1 were very low in comparison to the other indigenous isolates. For the low performance of these two isolates, it is hypothesized that the inherent ability of these strains to fix N and establish a symbiotic relationship with soybean is low. This could be explained by the fact that their nodulation ability was comparably similar with the other strains which possess higher N-fixation ability and symbiotic efficiency. In contrast, the isolate IS-2, which was clustered under the B. elkanii species, showed the highest symbiotic efficiency for both Rj-genotypes of the soybean cultivar used and the highest N-fixation ability for Rj4 plants. In a previous report, the Rj genes that could restrict the nodulation of soybean by some strains of bradyrhizobia was summarized [51] but in case of our present report, all the strains in this study were not restricted by the two Rj-plants that were used. This led us to consider that the low N-fixation and symbiotic performance of some strains were not due to the restrictions from the Rj-genotypes of the plants but could be attributed to the strains’ intrinsic capabilities. These observations might explain the reason for low yield of soybean in the Philippines. It was reported that many strains of B. elkanii were relatively inefficient microsymbionts of soybean and can induce chlorosis in soybean plants [52]. In a previous report [53], the high temperatures in tropical regions can limit the nodulation which could explain the low soybean yield.
It was expected that the strains which were classified as B. diazoefficiens could provide a better symbiotic performance than the other strains that were collected. However, the data showed that B. elkanii might establish a better symbiosis with local soybean cultivars in the Philippines. This result is crucial in order to devise strategies on how to increase the local production of soybean by inoculation with the indigenous strains.
Upon considering these results with the N-fixation and symbiotic performance ability of the strains, the number of nodules that can be formed from the single-strain inoculation does not seem to influence the amount of N that each strain can fix nor their symbiotic ability.
In this report, we have revealed that the distribution of tropical soybean bradyrhizobia seemed to be different than those of temperate bradyrhizobia in terms of population dominance of B. elkanii on higher temperature region like the Philippines. Additionally, it is proposed that for the Philippines, the most efficient N-fixer and symbiotically efficient species of bradyrhizobia would be B. elkanii. Yet, our results were made under the laboratory conditions only, so the results that were obtained here might not be as expected when done in field condition. For future research, utilization of more local soybean varieties with different soil types both in a controlled environment and on natural field condition would be beneficial to target the development of a site-specific and useful potential soybean inoculant. The data generated in this report would be beneficial for the augmentation of inoculation strategies in the country.
The authors would like to acknowledge the contributions of John Philip Tanay, Emmanuel Victor Buniao, Mary Joy Portin, and Maria Leah Sevilla of Central Luzon State University for their help on some laboratory experiments. This work was supported by the JSPS Grant-in-Aid for Scientific Research (KAKENHI Grant Number: 18K05376).
The authors declare no conflict of interest.
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\\n"}]'},components:[{type:"htmlEditorComponent",content:'IntechOpen’s Retraction and Correction Policy has been developed in accordance with the Committee on Publication Ethics (COPE) publication guidelines relating to scientific misconduct and research ethics:
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\n\nPolicy last updated: 2017-09-11
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