2.3.4. Electrical Conductivity

Electrical conductivity at 20 ◦C was determined with the aforementioned portable pH/conductivity device, following the method of the IHC, except for the amount of honey which was reduced to 5 g (from 20 g in the initial method) as it was found to give the same results (data not shown). Results are expressed in mS/cm [38].


*4.4. Microstructure of Fireplace Concrete*

Scanning electron microscope(SEM) microphotographs with the energy dispersive spectroscopy (EDS)system of the tested samples are shown in Figures 9–13. Based on the conducted research, it is possible to determine the compact microstructure of all of the concrete materials, except for sample 4, which is characterized by the presence of large spherical pores (Figure 12). A compact structure was also present in the concrete materials containing glass fibers (Figure 9) or polypropylene fibers (Figures 11–13). Generally, a homogeneous microstructure was found in the analyzed concrete materials; however, in the case of sample 5, the uneven distribution of fibers was noted (Figure 13). There were also visible pores (except for sample 4), which may have resulted from inappropriate selection of the aggregate graining (filler) of the concrete material or deaeration. This is an important factor because the presence of pores lowers the density and thermal properties of the concrete material. EDS measurements confirmed the presence of cementderived aluminates and aluminosilicates, as well as the presence of fibers, aggregate grains, and pigments.

Quartz grains and zirconium were present in the microstructure of sample 1 (Figure 9). Sample 3 (Figure 11) contained increased amounts of calcium aluminosilicate and strengthening organic fibers. Sample 4 (Figure 12) is a foamed concrete featuring the addition of calcium carbonate. In the composition of sample 2 (Figure 10), increased amounts of magnesium, zirconium, and calcium can be seen. In particular, in micro-area b, dolomite crystals formed with the addition of titanium white reinforced with organic fibers were found. Concrete no. 5 (Figure 13) is a typical concrete reinforced with organic fibers and aggregate, where the presence of iron comes from the minerals of the aggregate.

**Figure 9.** SEM microphotograph and energy dispersive spectroscopy (EDS) analysis of sample 1.

**Figure 10.** SEM microphotograph and EDS analysis of sample 2.

**Figure 11.** SEM microphotograph and EDS analysis of sample 3.

**Figure 12.** SEM microphotograph and EDS analysis of sample 4.

**Figure 13.** SEM microphotograph and EDS analysis of sample 5.

From an application perspective, concrete systems can be reduced to a mixture of three components: cement, fine aggregate, and water. Cement is a composite system in which aggregate grains are surrounded by a hardened cement slurry. Concrete formation is determined by the cement's hydration reactions. Cement is a mixture of solid phases and mainly contains various types of aluminates and calcium aluminosilicates: allite C3S-(3CaO·SiO2), bellite C2S-(2CaO·SiO2), tricalcium aluminateC3A-(3CaO·Al2O3), brownmilleriteC4AF- (4CaO·Al2O3·Fe2O3), calcium oxide (free)-CaO, and gypsum-(CaSO4·0.5H2O).

Cement hydration is a multi-stage process. However, it can be assumed that the main phases of hydration are as follows:

The hydration of tricalcium aluminate, however, is more complicated. There are three basic stages in this process. In stage I, under the initially high concentration of gypsum, ettringiteis formed according to the following reaction:

$$\text{\textbullet\text{CaO}\cdot\text{Al}\_2\text{O}\_3 + \text{\textbulletCaSO}\_4\cdot2\text{H}\_2\text{O} + 2\text{6H}\_2\text{O} \rightarrow \text{\textbulletCaO}\cdot\text{Al}\_2\text{O}\_3 \cdot \text{\textbulletCaSO}\_4\cdot32\text{H}\_2\text{O}.\tag{7}$$

In stage II, when gypsum is lacking, a transitional phase is formed:

$$\text{\textbullet\text{CaO}\cdot\text{Al}\_2\text{O}\_3\cdot\text{Ca(OH)}\_2\cdot12\text{H}\_2\text{O}.\tag{8}$$

In stage III, the second stage phase reacts with ettringite, giving the low-sulfate form of aluminum sulphate according to the following reaction:

3CaO·Al2O3·3CaSO4·32H2O + 3CaO·Al2O3·Ca(OH)2·12H2O → 3CaO·Al2O3·CaSO4·12H2O + 2Ca(OH)2 + 20H2O. (9)

The hydration of braunmillerite is similar to that of tricalcium aluminate. In the presence of gypsum and water, the following structures are formed:

4CaO·Al2O3·Fe2O3 + CaSO4·2H2O + Ca(OH)2 + aq → 3CaO·(Al2O3, Fe2O3)·3CaSO4·32H2O. (10)

After braunmillerite is exhausted, the structure becomes 3CaO·(Al2O3,Fe2O3)·CaSO4·12H2O [42,43].

The hardened cement slurry is largely dominated by two phases: the CSH gel phase and the portlandite (Ca(OH)2) phase. There are also additional phases resulting from the hydration of tricalcium aluminate and braunmillerite. It should be noted that these phases contain large amounts of crystalline water, and this water is the main destructive factor for cement mortars at high temperatures, which accompanies the use of fireplaces and heating stoves. However, the content of elements such as magnesium, aluminum, calcium, and silicon significantly affects the thermal properties of the concrete [44].

#### **5. Discussion**

The addition of fibers to concrete improves the mechanical properties of mortars and may improve heat flow in concrete materials. At elevated temperatures, polypropylene melts and is absorbed by the surrounding cement matrix, creating a network of channels through which moisture and the resulting water vapor can flow [45]. Hardened cement mortar can also be treated as a fibrous composite in which the matrix is the hardened cement mortar. In the case of such a composite, the addition of a small amount of fibers, even with an elasticity modulus lower than that of the matrix, significantly improves the mechanical properties of the mortar. This is because during the hydration process of the paste components, a number of reactionsoccur, leading to the formation of mechanical stresses. The resulting stresses, which are related to internal contractions, are leveled until the structure stiffens. However, in the initial stage of hardening, the hardening paste has too low a mechanical strength to compensate for the stresses accompanying the hydration process (as confirmed by the results presented in Table 9).

The results of the mechanical strength after 28 days confirm this explanation. The highest bending strength was found for the samples with the highest amount of glass and polypropylene fibers, i.e., sample 1 (0.266 wt % of glass fibers) and sample 3 (0.209 wt % of glass fibers and 0.02 wt % of polypropylene fibers). The addition of fewer of these fibers or the addition of steel fibers (even in combination with glass fibers) caused more than a twofold decrease in mechanical strength in the described case, from 13.2 MPa for sample 3 to 4.7 MPa for sample 4.

When the proportion of organic fibers is so large that the fibers form a self-connected network of channels, it is known as a parallel transport model. That is, the transport of liquids and gases at temperatures above 400 ◦C takes place both through the network formed after melting the fibers and through the pores and cracks existing in the material. When there are fewer fibers, transport can be described by the so-called series-parallel model, where the incompletely connected network of channels is supplemented by the existing defects in the material [46,47].

The introduction of short polypropylene fibers, in addition to preventing detonation splashes during rapid heating or contact with fire, also has a very positive effect on the mechanical properties of mortars. Hardened cement mortar can be treated as a fibrous composite in which the matrix is hardened cement paste. In the case of such a composite, the addition of a small amount of fibers, even with a modulus of elasticity lower than that of the matrix, results in a significant improvement in the composite's mechanical properties. This is because several stress reactions take place during the hydration of the cement slurry components. In the subsequent period, the stresses arising from internal contraction are leveled until the structure is stiffened. However, in the initial stage of hardening, the hardening cement slurry still has too low a mechanical strength to compensate for the stresses accompanying the hydration. This yields the formation of a micro-fracture network, leading to material defects and a loss of continuity at the microscale. The addition of fibers causes bridging of the resulting cracks, which reduces their size and improves the material's resistance to brittle fractures [48].

However, the addition of short fibers to the cement matrix may regulate more than the matrix's hydration and hardening properties. Through the addition of fibers with an elasticity modulus higher than the matrix modulus, the strength properties of the material can be directly modulated. The addition of such fibers ensures that the material does not disintegrate when the cement matrix breaks, allowing the matrix to still transfer some loads.

The reactions taking place in the cement during heating can be divided into five stages:

The effect of high temperature, i.e., that above 100 ◦C, on hardened cement material is very specific and causes the rapid desorption of moisture from the outer layers. The resulting water vapor flows towards the colder inner layers, where it is reabsorbed. As the temperature increases, the thickness of the heated layer increases. When the moisturesaturated layer does not move quickly enough, it is overtaken by the wandering temperature front, which leads to the evaporation of water at the front border and an increase in internal pressure. This creates tensile forces perpendicular to the temperature front, which in turn leads to rapid removal of the surface layers in the form of microcracks and spatters. This phenomenon mayoccur cyclically, destroying increasingly deeper layers of the material.

To counteract this phenomenon, it is necessary to improve the heat flow, which will entail a reduction in the pressure inside the heated element. The best way to do this is to create an interconnected network of channels in the material through which moisture and the resulting water vapor can flow, or toimprove the material's thermal conductivity.

This can be achieved via the introduction of a fibrous material that melts at an elevated temperature and is easily absorbed by the surrounding cement matrix. Polypropylene is perfect for this purpose. This material melts at 170 ◦C, which is below the temperature at which water vapor is rapidly released and does not damage the concrete matrix. At low temperatures (<100 ◦C), all forms of water in concrete (capillary, adsorptive, and crystalline) become very good heat accumulators (the heat of water evaporation is approximately 80 kcal/mol). Exterior cladding concrete shapes used in furnaces are a good choice for this purpose.

Another type of thermal accumulation involves using the excess heat energy generated inside the fireplace or the stove to heat rooms over a longer timeframe. Such furnaces are made of natural soapstone (a greenish-gray rock containing mainly magnesium aluminosilicates), which has a very good heat accumulation capacity. In these furnaces, due to the accumulation capacity of soapstone and various technical solutions (e.g., the multiple circulation and swirling of exhaust gases), the temperature in the furnace can reach about 1200 ◦C (normally about 600 ◦C), which allows the so-called afterburning of exhaust gases (soot combustion). This results in high efficiency of the furnace and, above all, has a positive impact on the environment.

The temperatures in storage furnaces can reach 400 ◦C and higher. Ordinary concrete is rarely used as a material for furnaces due to its low resistance to such temperatures (the safe limit temperature is 450 ◦C). This is why, as a rule, ceramics that are much more resistant to high temperatures are used for this application, most often fireclay. In our case, sample 5 showed the highest thermal power (Table 8) (19.70 W), whereas sample 2 showed the lowestthermal power (8.55 W). In addition to the significant effect of density (sample 5—2.16 g/cm3; sample 2—1.76 g/m3), composition itself plays an important role. Sample 2 consisted mainly of cement, sand, and refractory aggregate, whereas sample 5 consisted mainly of magnesium silicates and sodium–calcium feldspar. Therefore, it is worth analyzing, in each case, the physicochemical processes thatoccur during the formation of hydration materials and their destruction under the influence of high temperatures.

#### **6. Conclusions**

Concrete elements can be successfully used in the construction of domestic fireplaces, thus taking advantage of the good heat accumulation properties of these elements. Both thermal and strength properties can be modified significantly via the addition of various aggregates as well as propylene and steel fibers. This modification consists mainly in changing the mechanisms of the heat flow and the decomposition products of concrete elements during heating—mainly water, although the density of the materials also has a significant impact.

From the perspective of thermal energy use, our study showed the clear advantage of concrete materials containing magnesium and sodium–calcium aggregates (feldspars). The highest values were recorded for sample 5 (which contained over 70% of the listed components). In turn, from the perspective of mechanical properties, samples 1 and 3 showed the highest mechanical strength and were reinforced with glass and polypropylene fibers, respectively. The results, however, depend on the applied temperature range. Therefore, in the case of fireplaces, it is suggested to use different multi-layer systems of concrete materials to enable the long-term heating of rooms via heat accumulation.

**Author Contributions:** Conceptualization, A.S., J.M.-P. and P.I.; methodology, J.M.-P., Ł.W. and T.G.; software, A.S.; validation, A.S., P.I. and J.M.-P.; formal analysis, J.M.-P., Ł.W. and T.G.; investigation, A.S. and J.M.-P.; writing—original draft preparation, A.S.; writing—review and editing, J.M.-P.; visualization, A.S.; supervision, M.K. and P.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to the agreement with Northstar Poland.

**Acknowledgments:** We would like to thank Northstar Poland for its cooperation and for providing research materials.

**Conflicts of Interest:** The authors declare no conflict of interest.


*2.2. Material Properties*

According to GB17671-1999 [17], the average compression strength of CGM-340 highstrength non-shrinkage grout after 28 days was 84.33 MPa, which is established from the compression testing of three specimens, as shown in Table 1. Similarly, the average compression strength of concrete from compression testing was 59.30 MPa according to GB17671-1999 [17], as shown in Table 2. Tensile testing was also performed to obtain the material property of rebar according to GB1499.2-2007 [18], as shown in Table 3. For example, the average yield strength and the ultimate strength of rebar with the diameter of 12 mm were 430.08 MPa and 574.43 MPa, respectively.

**Table 1.** Compressive strength of high-strength grouted mortar.

**Table 2.** Compressive strength of concrete.

**Table 3.** Mechanical properties of rebar.

**Table 3.** *Cont.*


Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic Synthesis: A Metal-Free Greener Approach

*Suresh Rajamanickam and Bhisma K. Patel*

#### **Abstract**

Thermally activated delayed fluorescent (TADF) molecules undergo efficient intersystem crossing (ISC) and reverse intersystem crossing (RISC) processes, making them as third-generation emitters in organic light-emitting diodes (OLEDs), photodynamic therapy (PDT) and time-resolved luminescence imaging. Apart from these applications, recently, TADF molecules have been used extensively as photocatalysts in light-mediated synthesis. In general, highly expensive complexes of Rh, Ir, Ru and organic dyes (Eosin Y, Rose Bengal, 9-mesityl-10-methylacridinium perchlorate [Acr-Mes]+ ClO4 ) are commonly used in the photocatalysis process. Organic-TADF based molecules help to avoid these costly metal catalysts and frequently used organic dyes, making the reaction economical and greener. This chapter will briefly summarize the photocatalytic properties of organic-TADF compounds in organic synthesis.

**Keywords:** thermally activated delayed fluorescence (TADF), photocatalysis, 4CzIPN, organo photoredox catalysis, radical chemistry, single electron transfer (SET), halogen atom transfer (XAT), cross dehydrogenative coupling (CDC), Minisci reaction, cyclopropanation, cyclization reaction, ring opening reaction, deuteration reaction, hydroformylation reaction

#### **1. Introduction**

The term 'photocatalysis' is derived from the concepts of photochemistry. Previously, ultraviolet (UV) irradiation was commonly applied in classical photochemical reactions. The use of high energy ultraviolet light has selectivity issues and requires a designer reaction setup. However, the recent photochemical reaction uses low energy and selective wavelength of visible light from Light-Emitting Diodes (LED). Due to low energy usage, modern photochemical reactions are highly selective. In general, visible light has low absorptivity, so it can not drive the organic reaction competently. A secondary substrate, usually a photocatalyst is introduced to enhance the light

absorptivity. This photocatalyst absorbs visible light and provides stable and long photoexcited states, which induces the substrates or reagents to participate in the chemical reaction. The photoexcited catalyst either donates or removes a single electron from the reacting partners, which triggers further reaction *via* oxidative or reductive quenching. The current need of modern organic chemistry is the effective utilization of raw materials, energy resources. Further, elimination of waste, toxic, hazardous solvents, reagents and replacement of expensive and less efficient processes are the basic tenets of Green Chemistry. In this regard, visible-light assisted photocatalyzed reactions provides a smooth pavement for sustainable chemical synthesis. In addition, the visible-light assisted photo-catalyzed reaction received much attention in the synthetic community due to the simplicity of reaction setup and broad applicability to various reactions. However, most visible light-mediated reactions use expensive metal-based Ru or Ir based photoredox complexes making the process economically unviable. Nevertheless, the use of bench-stable inexpensive Thermally Activated Delayed Fluorescence (TADF) organic material as photocatalysts obviate many of the problems.

Polish physicist Aleksander Jabłoński studied the molecular absorbance and emission of light. He developed the famous Jablonski diagram to explain the spectra and kinetics of fluorescence and phosphorescence. This diagram illustrates the excited states energy level of a molecule and their radiative and non-radiative transitions. A typical Jablonski diagram is shown in **Figure 1a**. Under appropriate light irradiation, the molecules excite to an excited singlet state (Sn), and then the excitons migrate to the lowest excited singlet state (S1) *via* internal conversion (IC). The lowest excited singlet state (S1) excitons subsequently migrate from the S1 state to the ground state

**Figure 1.** *Simplified Perrin-Jablonski diagram and commonly used TADF motif in photocatalysis.*

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

(S0) by releasing radiative light energy. This process is known as fluorescence or prompt fluorescence. Meanwhile, the singlet state excitons (S1) can further migrate to the lowest triplet excited state (T1) through intersystem crossing (ISC), which can undergo a radiative decay transition process from the lowest triplet excited state (T1) to the ground state (S0), (T1 ! S0 transition) which is termed as phosphorescence.

Followed by Jabłoński, French physicists Jean Baptist Perrin, the winner of 1926 Nobel Prize in Physics, and his son Francis Perrin rationalized a third type of radiative transition known as delayed fluorescence. This occurs when a molecule in the lowest triplet excited state (T1) transitions to the lowest excited singlet state (S1) *via* reverse intersystem crossing (RISC) followed by a radiative transition to the ground state (S0). The reverse intersystem crossing (RISC) from T1 to S1 is only possible when the molecule has a very small singlet-triplet energy gap ΔEST (upto <0.6 ev) between the lowest-lying singlet S1 and triplet excited states T1. When the ΔEST is very small, the environment heat helps to achieve the reverse intersystem crossing (T1 ! S1), resulting in delayed fluorescence. The overall process is termed as Thermally Activated Delayed Fluorescent (TADF). These TADF molecules have huge applications in the third-generation Organic Light-Emitting Diode (OLED) display technology. According to Transparency Market Research, the global OLED displays market is expected to reach \$100 billion by 2030 from \$4.9 billion in 2012. Recently, the TADF process is also incorporated in the Jablonski diagram and the diagram is termed as Perrin-Jablonski diagram.

The TADF proceeds *via* the result of multiple cycles between intersystem crossing (ISC) from S1 to T1 and the reverse intersystem crossing (RISC) from T1 to S1. So a delayed fluorescence is observed after the short-lived fluorescence. TADF emission is identical in wavelength to prompt fluorescence, but it occurs on a longer timescale. The emission lifetime of fluorescence or prompt fluorescence is shorter (nanosecond scale). On the other hand, the delayed fluorescence has a longer emission lifetime (microsecond or even millisecond scale).

Eosin Y is the first organic compound identified to show Thermally Activated Delayed Fluorescence (TADF) property. This inexpensive organic dye is widely used in photocatalysis (PC) due to its moderate redox potentials (in 1:1 ratio of acetonitrile and water ratio the ground oxidation and reduction potentials of Eosin Y are *E*ox = 0.78 V and *E*red = �1.06 V respectively. The excited state reduction and oxidation potentials are *E*ox\* = �1.11 V and *E*red\* = 0.83 V) and visible region absorption (λabs = 520 nm in MeOH). Followed by Eosin Y, other organic TADF molecules such as Rose Bengal, 9-mesityl-10-methylacridinium perchlorate [Acr-Mes]+ ClO4 � and polypyridyl metal complexes Ir(ppy)3, (Ir[dF(CF3)ppy]2(dtbpy))PF6 and [Ru(bpy)3] (PF6)2) were extensively studied in photocatalysis process (**Figure 1**).

In 2012, Adachi group prepared a conformationally twisted electron-donor and acceptor TADF material, 2,4,5,6-tetra(carbazol-9-yl)benzene-1,3-dicarbonitrile (4CzIPN) by single-step reaction between 2,4,5,6-tetrafluoroisophthalonitrile and carbazole *via* nucleophilic aromatic substitution (SNAr) reaction (**Figure 2**) [1]. This molecule has a very small energy gap (ΔEST = 0.08 eV in toluene) between S1 and T1 levels. This small energy difference allows reverse intersystem crossing (RISC) from T1 to S1 to qualify it as a TADF molecule [1]. 4CzIPN is a poor single-electron oxidant or reductant in the ground states. On the other hand, it is a potent single electron transfer reagent in the excited states under visible-light irradiation. This TADF molecule has high photoluminescence quantum yield (94.6%) and a long lifetime at an excited state (5.1 μs). One of the primary advantages of 4CzIPN is its low synthetic cost [2].

**Figure 2.**

*Synthesis of 2,4,5,6-tetra(carbazol-9-yl)benzene-1,3-dicarbonitrile (4CzIPN).*

In 2016, Ollivier and Fensterbank group used 4CzIPN as a photocatalyst for an organic transformation [3]. Under blue LEDs irradiation, 4CzIPN undergo photoexcitation, the photoexcited 4CzIPN\* generated benzyl radical from benzyl *bis* (caecholato)silicates, the generated radical is trapped by (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) (**Figure 3**). The author further extended the scope of this methodology for the preparation of alkynylation, vinylation and Giese-type products by treating alkylsilicates with a variety of radical acceptors (**Figure 3**). This work open-up a new window for many organic transformations using 4CzIPN as an economically cheaper, greener organo photoredox catalyst.

Followed by Ollivier and Fensterbank radical-mediated synthesis [3], various organic transformations were documented using 4CzIPN as a photocatalyst under the irradiation of visible light or blue LEDs. Besides this, many reactions were reported using 4CzIPN in combination with a transition metal. This chapter excludes transition metal assisted (synergic catalysis) synthesis and mainly focuses on 4CzIPN as an independent photocatalyst without any transition metals.

#### **2. Cyclopropanation reactions**

In 2018, a group of Gutierrez and Molander demonstrated a redox-neutral photocatalytic cyclopropanation of olefins with triethylammonium *bis*(catecholato) iodomethylsilicate using a combination of 4CzIPN and blue LEDs light. Triethylammonium *bis*(catecholato)iodomethylsilicate serve as an iodomethyl radical precursor (**Figure 4**) [4].

From mechanistic aspects, the photocatalytically generated halomethyl radical is trapped by the alkene and generate a stable tertiary radical. This radical accepts a

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

**Figure 4.**

*4CzIPN-catalyzed cyclopropanation of alkene using iodomethylsilicate.*

**Figure 5.** *4CzIPN-catalyzed cyclopropanation of alkene using iodomethylsilicate.*

single electron from the 4CzIPN˙ , to form an anion and regenerate the catalyst. It underwent an anionic *3-exo-tet* ring closure that leads to a cycopropylation product. With the support of both computational and control experiments performed, the authors concluded that the reaction proceeds *via* an anionic *3-exo-tet* ring closure (**Figure 5**) [4].

In the same year, the Molander group further extended their aforementioned cyclopropanation methodology [4] to the homoallylic tosylates system (**Figure 6**) [5]. In their previous report, the leaving group (iodo) is attached to the radical precursor motif itself [4]. In their follow-up work, the Molander group incorporated the leaving group into the alkene core. They treated three different alkyl radical precursors such as *bis*(catecholato)alkylsilicates, alkyltrifluoroborates and 4-alkyl dihydropyridines, with an array of linear homoallylic tosylates systems [5]. All leads to 1,1-disubstituted cyclopropanes derivatives in moderate to good yields *via in-situ* generated anionic cyclization (**Figure 6**). The classical electrophilic carbenoid Simmons-Smith

**Figure 6.** *4CzIPN-catalyzed cyclopropanation of linear homoallylic tosylates using alkyl radical precursors.*

**Figure 7.** *4CzIPN-catalyzed cyclopropanation of exocyclic homoallylic tosylates using bis(catecholato)alkylsilicates.*

cyclopropanation reaction is incompatible for Lewis basic heterocycle and free amine functional groups. However, this radical-polar annulation reaction (RPARs) protocol is highly comfortable with those substrates (Lewis basic heterocycles and free amines substrates). α-Heteroatom, secondary, tertiary and benzylic radical generated from trifluoroborate reagents all smoothly delivered cyclopropanation derivatives [5].

In successive work, the reaction between *bis*(catecholato)alkylsilicates and exocyclic homoallylic tosylates in the presence of photocatalyst 4CzIPN under blue LEDs irradiation afforded polycyclic cyclopropanes (**Figure 7**) [6]. This reaction proceeds smoothly with five (indanone) and six-membered (tetralone) carbocylic tosylate alkenes. But, seven-membered *i.e*. cycloheptanone-based tosylate failed to provide a cyclopropanation product. However, the introduction of heteroatoms (nitrogen and oxygen) in the benzylic position of seven-membered ring *i.e*. benzoazepines and

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

benzoxepines-derivatives, smoothly afforded Radical-Polar Crossover (RAC) annulation products (**Figure 7**) [6].

Around the same time, Noble and Aggarwal's group jointly documented 4CzIPNcatalyzed cyclopropane reaction by treating aliphatic carboxylic acids with electrondeficient internal and external chloro alkenes (**Figure 8**) [7]. The reaction proceeded *via* decarboxylative radical addition followed by a polar cyclization cascade pathway (**Figure 8**). This methodology shows broad substrate scope for both acids (including cyclic, acyclic carboxylic acids, amino acids and dipeptides) and electronwithdrawing groups (carboxylate esters, nitriles, primary amides, sulfones and phosphonate esters) attached chloro alkenes.

Homoallyl chlorides provided good yields of 1,1-disubstituted cyclopropanes. On the other hand, allyl chlorides lead to vicinal substituted cyclopropanes with moderate yields. The slightly lowered yield obtained is due to the formation of an allylic ester by-product *via* S*N*2 reaction between allylic chloride and carboxylate (**Figure 8**) [7]. It is noteworthy to mention that, this mild organic photocatalyst protocol is applied for the late-stage cyclopropylation of a variety of acid-containing bioactive natural products namely, dehydroabietic acid (terpenes), biotin (vitamin B7), trolox (vitamin E analogue), cholic acid (bile acid) and gemfibrozil (fibrate drug) (**Figure 8**) [7].

From a mechanistic perspective, the excited photocatalyst 4CzIPN\* underwent SET with the carboxylate to form a carbon center radical by reduction of the excited photocatalyst to radical anion (4CzIPN˙ ). The carbon center radical underwent Giese-type addition into the homoallyl chloride to generate the stabilized alkyl radical. This stabilized alkyl radical accept a single electron from 4CzIPN˙ leading to a stabilized carbanion. Polar 3-exotet cyclization of stabilized carbanion afforded cyclopropane product (**Figure 9**) [7].

The above cyclopropanation reactions have considerable advantages over other reagents such as diazomethane (respiratory irritant) and highly pyrophoric diethylzinc (C2H5)2Zn, used in the Simmons-Smith reaction.

**Figure 8.** *4CzIPN-catalyzed cyclopropanation of allyl and homo allyl chlorides with carboxylic acid.*

#### **Figure 9.**

*A mechanism for 4CzIPN-catalyzed decarboxylative cascade radical addition–polar cyclization reaction towards substituted cyclopropanes synthesis.*

Subsequently, various research groups generated 4CzIPN photo catalyzed decarboxylative carbon-centered radicals from a carboxylic acid and added it into alkenes (*via* Giese-type addition) [8–11] or aromatic heterocyclic (Minisci reaction) [12, 13].

For example, Wang group generated a carbon-centered radical (**10B**) *in-situ via* a decarboxylative path from the anionic form of diethoxyacetic acid (**10A**) by photoirradiation of 4CzIPN. The generated radical (**10B**) adds regioselectively into aryl olefins (styrene derivatives), at the less substituted site which leads to a stable benzylic radical (**10C**). This benzyl radical then accepts a single electron from the radical anion of photocatalyst 4CzIPN˙ to form a benzyl anion (**10D**). Protonation of benzyl anion followed by acid hydrolysis of the acetal provided hydroformylation product (**10E**) (**Figure 10**) [8]. In sum, Wang group developed a 4CzIPN catalyzed hydroformylation of aryl olefins with diethoxyacetic (**Figure 10**). In this reaction, diethoxyacetic acid acts as the formylation reagent (formyl radical equivalent). It is noteworthy to mention that this is the first radical-based hydroformylation strategy. The author avoided competitive radical polymerization reaction by performing the reaction in a continuous flow system for electron-deficient olefins, and batch method applied for electron neutral and rich olefinic systems. This radical formylation protocol was successfully extended towards late-stage formylation of biologically relevant complex olefins (**Figure 10**) [8].

The authors studied the competitive reaction between aryl and alkyl olefin in both inter and intra-molecular manners. The reaction showed higher chemoselective at the aryl olefin site. Alkyl olefin site remains intact in both continuous flow and batch method (**Figure 11**) [8].

Schubert group employed 4CzIPN-mediated decarboxylative radical conjugate addition to C]C bonds of dehydroalanine (Dha) and its derivatives peptides (**Figure 12**) [9]. This protocol opens up new avenues to a diastereoselective synthesis of unnatural amino acids and the late-stage derivatization of a tripeptide (**Figure 12**) [9].

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

**Figure 10.** *4CzIPN-photoredox catalyzed hydroformylation of olefins.*

*4CzIPN-catalyzed intra- and intermolecular chemoselectivity hydroformylation reactions.*

#### **3. Three-component C**d**C and C**d**N bond formation reaction**

So far, we discussed 4CzIPN catalyzed two compound reactions [3–9]. For the first time in the year 2019, the Studer group developed a 4CzIPN photocatalyzed threecomponent reaction for 1,2-amidoalkynylation of unactivated alkenes (**Figure 13**) [10]. Photoexcited TADF (4CzIPN\*) generated an amidyl radical (**14B**) *in-situ* from the anionic form of Troc-protected *α*-amido-oxy acid (**14A**) *via* a single electron transfer followed by decarboxylation and extrusion of acetone (**Figure 14**). An amidyl radical (**14B**) is added into alkene, which generated an alkyl radical (**14C**), which then couples with an alkyne radical produced from ethynyl benziodoxolones (EBX) to form 1,2-amidoalkynylation product (**14D**) [10].

This three-component reaction showed broad substrate scope for mono, di and tri substituted terminal alkene and substituted benziodoxolones. In addition to these, vinyl ethers, esters and enamides are also compatible with these reaction conditions (**Figure 13**). The reaction provided a high level of chemo-selective product. The polar effect plays a major role in chemo-selective product formation. An amidyl radical is attached at the less substituted site of alkene and the alkyne radical is attached at the more substituted site of alkene (**Figure 13**) [10]. For a particular note, this is the first

**Figure 13.**

*4CzIPN-catalyzed 1,2-amidoalkynylation of unactivated alkenes.*

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

**Figure 15.**

*Radical clock experiments in the synthesis of 1,2-amidoalkynylation of unactivated alkenes.*

transition metal-free alkene aminoalkynylation. Before this report, alkene aminoalkynylation reactions are restricted to disubstituted alkenes.

The author concluded that the reaction proceeded through a radical pathway by performing two different radical clock experiments using 1,6-diene and vinylcyclopropane (**Figure 15**) [10].

#### **4. Photoinduced C**d**Si bond formation via decarboxylation of silacarboxylic acids**

All the aforementioned examples deal with photoinduced CdC bond formation *via* decarboxylation of carboxylic acids. In 2020, Uchiyama group generated a silyl radical by photoirradiation of silacarboxylic acids in the presence of 4CzIPN. This silyl radical is successfully trapped by an alkene, leading to silyl alkane *via* CdSi bond formation (**Figure 16**) [11]. Substrate scopes of the reaction were demonstrated with three silacarboxylic acids (Ph2MeSiCOOH, Ph2 *t* BuSiCOOH and PhMe2SiCOOH) and a broad range of electron-withdrawing substituted alkenes (**Figure 16**). Interestingly, 1,1,2,2-tetraphenyldisilane-1-carboxylic acid, (having SidH and SidCOOH) also provided decarboxylative silyl radical coupled product, without affecting the SidH group. (**Figure 16**, product no. 16.8) [11].

#### **5. TADF-photocatalyzed Minisci reactions**

The addition of nucleophilic radical to electron-deficient nitrogen-containing heteroarenes bases followed by a formal hydrogen atom loss is known as **Minisci reaction** [14, 15]. The major problem associated with the Minisci reaction is regioselectivity. For example, classical Minisci reaction on quinoline often provides a mixture of C2 and C4 addition products. In 2020, Phipps group developed a TADFphotocatalyzed protocol for regioselective C2 and C4 functionalization of quinoline with phenylalanine-derived redox-active esters (**Figure 17**) [12]. The authors systematically studied the effect of acid, solvents and photocatalyst in the regioselective decarboxylative functionalization (alkylation) of quinolone. The Brønsted acids, photocatalyst and solvent all play major roles in selectivity. The TADF-photocatalyst 4CzIPN with *p*-toluenesulfonic acid (PTSA) in a more polar solvent (DMA) favor C4-functionalized product (5.4:1 ratio of C4/C2). On the other hand, another TADFphotocatalyst 3DPAFIPN with structurally bulky 2,4,6-triisopropylbenzenesulfonic acid (TIPBSA) in a less polar solvent (dioxane) leads to the highest C2 selectivity (C4/C2 = 1:7.3) (**Figure 17**) [12].

Sherwood and co-workers employed a 4CzIPN-photocatalyzed Minisci reaction between a variety of electron-deficient *N*-containing heterocycles and the intermediate of an *in-situ* generated *N*-(acyloxy)phthalimides (NAP) from aliphatic carboxylic acid (**Figure 18**) [13]. Sherwood used various substituted heteroarenes namely

*4CzIPN-photocatalyzed one-pot Minisci reaction between hetreoarenes and in-situ generated* N*-(acyloxy) phthalimides (NAP).*

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

pyridine, quinoline, isoquinoline, quinoxaline, quinaldine, quinazolinone, phthalazine, purine, azaindole, benzimidazole, benzothiazole, benzoxazole, indazole and caffeine to test the efficiency of the reaction protocol. Among these, azaindole, benzoxazole and indazole provided the least amount of product (5%), all other substrates afforded moderate to excellent yields of Minisci functionalized products. In addition, this one-pot reaction protocol showed a high degree of functional group tolerance towards late-stage functionalization (LSF) of nucleosides (nebularine and peracetylated nebularine, adenosine), alkaloids (quinine, camptothecin) and anticancer marketed drug scaffolds namely vemurafenib, imatinib (**Figure 18**) [13].

From mechanistic aspects, when blue LEDs light is exposed to organophotocatalyst 4CzIPN and *in-situ* synthesized *N*-(acyloxy)phthalimides (NAP) (**19A**), the latter underwent reductive fragmentation to generate an alkyl radical (**19B**) and phthalimide (by extrusion of carbon dioxide). This is followed by SET from excited 4CzIPN\* to a phthaloyl radical. The alkyl radical (**19B**) was added to the electrondeficient *N*-containing protonated heteroarenes (**19C**) to form adducts (**19D**, **E**). The adduct (**19E**) further underwent a second SET with the oxidized form of the organophotocatalyst 4CzIPN˙ + to give a Minisci product (**19F**) and regenerated the catalyst (**Figure 19**) [13].

In the afore-mentioned Minisci protocols (**Figures 17** and **18**), acid additives were used in the reaction medium [12, 13]. In 2019, Graham and Noonan demonstrated an acid additive-free, large-scale (67 g) photoredox catalyzed Minisci reaction towards the synthesis of 2,4-dichloro-6-[1-(methylsulfanyl)cyclopropyl]pyrimidine (**Figure 20**) [16]. This protocol reduces four reaction steps in the classical production

**Figure 19.**

*Proposed mechanism of 4CzIPN-photocatalyzed one-pot Minisci reaction between hetreoarenes and in-situ generated* N*-(acyloxy)phthalimides (NAP).*

**Figure 20.** *3DPA2FBN-photocatalyzed one-pot Minisci reaction towards ceralasrtib synthesis.*

**Figure 21.** *4CzIPN-photocatalyzed decarbonylative Minisci reaction.*

of cancer's phase II clinical trials molecule ceralasertib [16]. Compared to 4CzIPN (50% yield), 3-DPA2FBN (70%) is a more effective photocatalyst for the abovementioned transformation.

In general most of the Minisci reactions proceeds through the decarboxylation (dCO2) pathway [12, 13, 15, 16]. Large amounts of oxidants are generally required when aldehydes are used as the radical precursors. In 2019, Huang and Zhao groups disclosed a visible-light-induced photoredox decarbonylative (dCO) Minisci type CdC bond formation (alkylation) between aldehydes and *N*-heteroarenes (**Figure 21**). The reaction proceeded at room temperature and air (O2) was used as the sole oxidant (**Figure 21**) [17].

This reaction is highly compatible with secondary and tertiary aldehydes. However, primary alkyl aldehydes and aromatic aldehydes failed to deliver decarbonylative Minisci-type alkylated products. The substrate scope of this aerobic photoredox decarbonylative alkylation reaction is decorated by various mono *N*heteroarenes (quinolines, isonicotinonitrile, isoquinoline, phenanthridine, quinoxyfen, hydroquinine, and benzothiazole) and multi-nitrogen containing heteroarenes such as 1,10-phenanthroline, phthalazine, quinoxaline and imidazo[1,2 a]pyridine. Mono-nitrogen heteroarenes provided a good yield of Minisci-type alkylation products. On the other hand, multi-nitrogen-containing heteroarenes delivered a lower yield of products (**Figure 21**) [17].

The author proposed a plausible reaction mechanism, as shown in **Figure 22**. Visible light-induced photoexcited catalyst 4CzIPN\* underwent SET with O2 to form a superoxide radical anion (O2˙ ). This superoxide radical anion abstracts a hydrogen atom from aldehyde to produce an acyl radical (**22A**). Decarbonylation of an acyl radical (**22A**) generates an alkyl radical (**22B**). The alkyl radical (**22B**) addition to the protonated *N*-heteroarene (**22C**) leads to *N*-heteroarene radical cation (**22D**). Further, sequential deprotonation of radical cation (**22D**) and SET between alpha alkyl radical and [4CzIPN]+ afforded final alkylated quinolone product (**22E**) and regeneration of photocatalyst 4CzIPN (**Figure 22**) [17].

#### **6. Cross-dehydrogenative Minisci type reactions**

Cross dehydrogenative coupling (CDC) reaction is step and atom economical reaction. It plays a vital role in the construction of a diverse array of CdC and Cdheteroatom bonds, by functionalizing CdH bonds of all types sp, sp2 , sp3 [18–23]. *Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

**Figure 22.**

*A plausible mechanism for 4CzIPN-photocatalyzed decarbonylative Minisci reaction.*

**Figure 23.** *A plausible mechanism for 4CzIPN-photocatalyzed decarbonylative Minisci reaction.*

In 2020 Li and An group demonstrated an acid-free, 4CzIPN photocatalyzed, Minisci reaction between diverse Csp3 dH sources and *N*-heteroarenes (**Figure 23**) [24].

Sun group developed 4CzIPN and quinuclidine-catalyzed direct CdH silylation of quinoxalinones or electron-deficient heteroarenes *via* Cross Dehydrogenative Coupling (CDC) between quinoxalinones Csp2 dH and silane SidH bond (**Figure 24**). The reaction proceeded with the combination of photoredox (4CzIPN) and hydrogen atom transfer (HAT) catalyst (quinuclidine) (**Figure 24**) [25]. From a mechanistic perspective, a single electron transfer (SET) between photoexcited catalyst 4CzIPN\* and quinuclidine (**24.A**) generates 4CzIPN˙ radical anion and quinuclidinium radical cation (**24.B**). The hydrogen atom transfer between silane and quinuclidinium radical cation (**24.B**) produced silyl radical (**24.C**) and protonated quinuclidine (**24. D**). Pyridine base accepts hydrogen from protonated quinuclidine (**24.D**) to regenerate hydrogen atom transfer (HAT) catalyst quinuclidine (**24.A**). Meanwhile, the *insitu* generated silyl radical couple with the quinoxalinones (**24.E**) to form a radical adduct (**24.F**). Parallelly, superoxide radical anion (O2˙ ) is formed by another single electron transfer (SET) between 4CzIPN˙ and 1 O2. Finally, the intermediate **24.F** underwent direct hydrogen atom transfer (HAT) with superoxide radical anion (O2˙ ) to give the desired CDC silylated product **24.G** (**Figure 24**) [25].

#### **Figure 24.**

*4CzIPN and quinuclidine catalyzed silylation of quinoxalinones and electron-deficient heteroarenes with alkyl silane via CDC approach.*

**Figure 25.**

*4CzIPN-photocatalyzed cascade oxidative aryl-trifluoromethylations and aryl-methylcyanation of* N*-aryl acrylamides.*

#### **7. Cyclization reactions**

Cai group established a 4CzIPN-photocatalyzed intramolecular cascade oxidative aryl-trifluoromethylations [26] and aryl-methylcyanation [27] of *N*-aryl acrylamides for the synthesis of functionalized oxindole (**Figure 25**). Cai used Langlois reagent (sodium triflinate, CF3SO2Na) and acetonitrile as trifluoromethyl and methylcyanation sources, respectively. The aryl-trifluoromethylations reaction proceeded without a strong oxidant. Oxygen present in industrial-grade nitrogen (with 0.5 mol% of oxygen) acts as the oxidant. The use of high purity argon (>99.999%) completely failed to provide any trifluoromethylations product (**Figure 25**). On the other hand, aryl-methylcyanation reaction proceeds in the

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

presence of strong oxidant, phenyliodine *bis*(trifluoroacetate) (PIFA) and an additive 1,3,5-trimethoxybenzene (**Figure 25**). Both aryl-trifluoromethylations [26] and arylmethylcyanation [27] reactions show broad substrate scope with *N*-aryl, alkyl acrylamides. However, substrates **25.3**, **25.4** and **25.5** readily decomposed and failed to provide their desired product (**Figure 25**).

From a mechanistic perspective, the photoexcited 4CzIPN\* catalyst decomposes sodium triflinate (CF3SO2Na) into CF3 radical and SO2. This CF3 radical is added into alkene of *N*-aryl acrylamide to generate a tertiary carbon center radical (**26.A**) or its resonance oxygen radical (**26.B**). Radical cyclization of **26.A** or **26.B** followed by sequential oxidation and deprotonation steps leads to trifluoromethylated oxindole product **26.C** (**Figure 26**) [26].

The author proposed a plausible reaction mechanism of aryl-methylcyanation of *N*-aryl acrylamides, as shown in **Figure 27**. The additive 1,3,5-trimethoxy benzene reacts with phenyliodine *bis*(trifluoroacetate) PIFA to delivered the diaryliodonium salt **27.A**. Which underwent single electron transfer (SET) with excited photocatalyst 4CzIPN\*, generated active iodanyl radical **27.B** species. Which abstract acetic hydrogen from acetonitrile or acetone or dimethylsulfoxide to provide the corresponding

#### **Figure 26.**

*A plausible mechanism for 4CzIPN-photocatalyzed cascade oxidative aryl-trifluoromethylations and arylmethylcyanation of* N*-aryl acrylamides.*

#### **Figure 27.**

*A plausible mechanism for 4CzIPN-photocatalyzed cascade oxidative aryl-methylcyanation of* N*-aryl acrylamides.*

alkyl radical **27.C**. This alkyl radical underwent radical addition into *N*-aryl acrylamide alkene, which leads to stable tertiary carbon center radical **27.D**. A sequential radical cyclization, followed by deprotonation, afforded alkyl functionalized oxindole product (**27.E**) (**Figure 27**) [27].

Cai group further extended the reaction protocol to *N*-benzoyl acrylamides to synthesize cyano, acetone, dimethylsulfoxide and trifluoromethyl substituted isoquinolinediones (**Figure 28**) [26, 27].

In 2021, Yu group demonstrated a 4CzIPN catalyzed cascade cyclization of *N*arylpropiolamides to 3-phosphorylated, trifluoromethylated or thiocyanated azaspiro [4.5]trienones (spiro-γ-lactam derivatives). In this radical-initiated cascade annulation reaction, diphenylphosphine oxide or diethyl phosphite, 1-trifluoromethyl-1,2 benziodoxol-3(*1H*)-one (Togni's reagent II) or NH4SCN have been used as phosphoryl, CF3 and SCN sources respectively (**Figure 29**) [28]. The reaction showed broad substrate scope for *N*-(4-methoxyphenyl)propiolamides bearing different *N*-substituents (R1 ) and Ar substituents (**Figure 29**). Interestingly *N*-arylpropiolamide

**Figure 28.**

*4CzIPN-photocatalyzed cascade oxidative functionalization of* N*-benzoyl acrylamides.*

**Figure 29.** *4CzIPN-photocatalyzed cascade oxidative functionalization of* N*-arylpropiolamides.*

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

**Figure 30.** *Proposed mechanism for 4CzIPN-photocatalyzed cascade oxidative functionalization of* N*-arylpropiolamides.*

containing a piperidine ring reacts with diphenylphosphine oxide, providing polyfused heterocycle product **29.5** in very good yield (80%) [28].

This cyclization reaction proceeds *via* the combination of energy transfer (EnT) and single electron transfer (SET) mechanism. The photo-excited catalyst 4CzIPN\* transfer its energy to *N*-arylpropiolamide **30.A**, to form a high-energy-level **30.A\***. Then a SET between lauroyl peroxide (LPO) and **30.A\*** generated *N*-arylpropiolamide radical cation (**30.B**) and dodecanoate anion. Afterward, the second SET between diphenylphosphine oxide (**30.C**) and **30.B** provided *N*-arylpropiolamide **30.A** and diphenylphosphoryl radical (**30.D**). This phosphoryl radical (**30.D**) regioselectively added into alkyne of **30.A**, generated alkenyl radical intermediate (**30.E**). Subsequently it underwent intramolecular radical cyclization to form an azaspiro radical (**30.F**). The third single electron transfer reaction between **30.F** and lauroyl peroxide (LPO) leads to an azaspiro cation (**30.G**) and dodecanoate anion. Sequential addition of H2O, followed by elimination of methanol and deprotonation, provided the desired phosphorylated azaspiro[4.5]trienones product (**30.H**) (**Figure 30**) [28]. A similar reaction mechanism was adopted for trifluromethylated and thiocyanated azaspiro [4.5]trienones synthesis (**Figure 30**) [28].

#### **8. Ring opening reaction**

He and co-workers demonstrated a 4CzIPZ catalyzed aerobic oxidative cleavage of unstrained Csp3dCsp3 bonds of morpholine derivatives using visible light as the energy source and O2 as an oxidant (**Figure 31**) [29]. The author proposed that the

**Figure 31.** *4CzIPN-photocatalyzed oxidative cleavage of morpholine derivatives.*

photoexcited 4CzIPN\* was reduced by *N*-aryl morpholine *via* a single electron transfer to generate 4CzIPN and aminium radical cation (**31.A**). Simultaneously, oxidation of 4CzIPN by O2 regenerate photocatalyst 4CzIPN and superoxide anion (O2˙ ). This superoxide anion (O2˙ ) abstracts a hydrogen atom from the aminium radical cation (**31.A**) to form an iminium intermediate (**31.B**) and HOO. Afterwards, the HOO abstracts a proton from the iminium intermediate (**31.B**) and form an enamine species (**31.C**). Concurrently, singlet oxygen (1 O2) is produced by sensitization with 4CzIPN\* *via* energy transfer (ET). This singlet oxygen (1 O2) is reacted with enamine (**31.C**) to form a dioxetane intermediate (**31.D**), which readily decompose and provide the desired oxidative cleaved product (**31.E**) [29].

#### **9. Deuteration reaction**

In 2020, Leonori and co-workers incorporated deuterium in unactivated 1°, 2° and 3° alkyl iodide using a combination of synergistic photoredox 4CzIPN catalyst and Bu3N as the halogen atom transfer (XAT)-agent precursor and methyl thioglycolatedD2O as the D-atom donor (**Figure 32**) [30].

From mechanistic aspects, the excited photocatalyst 4CzIPN\* oxidize Bu3N followed by deprotonation leads to α-aminoalkyl radical (**33.A**) and 4CzIPN˙ . The αaminoalkyl radical (**33.A**) abstract a halogen-atom from the alkyl halide to generate an alkyl radical (**33.B**) and α-iodoamine (**33.C**) *via* XAT. This α-iodoamine (**33.C**) species dissociate into the iminium iodide (**33.D**). Meanwhile, HAT, between deuteriated methyl thioglycolate (**33.E**) and alkyl radical (**33.B**) provided desired deuteriated product (**33.F**) and thiol radical (**33.G**). Finally, a single electron transfer between 4CzIPN˙ and thiol radical (**33.G**) regenerate photocatalyst 4CzIPN and methyl thioglycolate (**Figure 33**) [30].

In addition to deuteration reaction, Leonori and co-workers further utilized the *insitu* generated alkyl radical (**34.B**) towards cross-electrophile coupling between

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

**Figure 32.**

*4CzIPN-photocatalyzed deuteration of alkyl halides.*

**Figure 33.** *Proposed mechanism for 4CzIPN-photocatalyzed deuteration of alkyl halides.*

**Figure 34.** *4CzIPN-photocatalyzed deuteration of alkyl halides. Hydroalkylation and allylation.*

electron-deficient olefins or allyl chlorides following Giese-type hydroalkylation mechanism (**Figures 33** and **34**) [30].

## **10. Conclusions**

It is well-known that photocatalytic reactions are powerful tools for a wide range of organic transformations. In this regard, visible-light-induced metal complexes have gained huge attention in the last two decades. Recently TADF materials have been used as an alternative for metal photocatalyst. In this chapter, we summarized a few TADF materials, particularly 4CzIPN as photocatalyst for various radical-based organic transformation reactions. This inexpensive TADF photocatalyst is less toxic and greener. A large number of TADF materials are prepared and used in OLEDs applications. However, only very few TADF molecules are explored in visible light promoted organic transformations. This TADF catalyzed organic transformation reactions are still in its infancy. Many new organo photocatalysts should be discovered for milder organic transformation.

#### **Acknowledgements**

B.K. Patel. acknowledges the support of this chapter by SERB (EMR/2016/007042) and CSIR 02(0365)/19-EMR-II. R. Suresh acknowledges the support of this chapter by SERB for funding under the National Post-Doctoral Fellowship scheme SERB-NPDF (PDF/2021/002055) and MRC, IISc Bangalore.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Suresh Rajamanickam1,2 and Bhisma K. Patel1 \*

1 Department of Chemistry, Indian Institute of Technology Guwahati, Guwahati, India

2 Materials Research Centre, Indian Institute of Science, Bangalore, Karnataka, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Thermally Activated Delayed Fluorescence (TADF) Compounds as Photocatalyst in Organic… DOI: http://dx.doi.org/10.5772/intechopen.103960*

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#### **Chapter 8**


Biological Nitrogen Fixation

Nitrogen is an essential component of all living organisms including plants. It is an important constituent of amino acids, nucleic acids, proteins, and energy currency (ATP, GTP, ADP), etc. Therefore, it is known as a building block of cells. Although N2 is present in about 78% of the atmosphere, it is unavailable for plants and animals due to its complex structure, i.e., the triple bond between two nitrogen atoms [93]. PGPB can convert this gaseous form of nitrogen into a usable form, i.e., ammonia, by the use of an enzyme system, nitrogenase, and make it available to plants. Microorganisms that fix nitrogen belong to diverse genera such as *Azotobacter* spp. [94], *Bacillus polymyxa* [95], *Gluconoacebacter diazotrophicus* [96], and *Burkholderia* spp. [97]. There is also a report that inoculation of mixed inoculants of *Gluconacetobacter diazotrophicus, Burkholderia tropica, Azospirillum amazonense, Herbaspirillum rubrisubalbicans,* and *Herbaspirillum seropedicae* has been very effective in promoting N fixation in sugar cane [98]. *Azospirillum* is the most studied nitrogen fixer among these bioinoculants as reported by Calvo et al. [8]. It has been reported that there is a major increase in nitrogen content in plants when some plant species are inoculated with *Azospirillum* strains. For instance, *A. lipoferum* and *A. brasilense* show 7–12%, and *A. diazotrophicus* gives a 60–80% increase in wheat and sugarcane, respectively.

#### Solubilisation of Phosphate

Phosphorus is also a chief nutrient for plants next to nitrogen. In agricultural soils, the total concentration of phosphorus generally varies between 400 and 1200 mg/kg−1 but only 1 mg/kg−1 is available in the forms of dihydrogen phosphate (H2O4P−) and hydrogen phosphate (HO4P−2) [14]. Soil contains P in inorganic and organic forms which are insoluble [14]. The inorganic form of P contains about 20–50% of total P [99] and is generally available in the form of PO4− ions, which are sparingly soluble due to the adsorption of positively charged ingredients of soil and also are precipitated with some metals such as Al, Fe and Ca [99]. Insoluble organic P is also available in the form of inositol phosphates, phosphate esters, and uncharacterized large organic molecules and contains 50–80% of total P [99]. In agriculture systems, the low availability of P in soil is a significant problem [8]. PGPB can increase the nutrition of plants through the process of solubilization of P [14]. To solubilize insoluble inorganic and organic P, bacteria use several approaches. There are two mechanisms for the solubilization of P by bacteria that are predominant, i.e., through the production of organic acids or through the production of phosphatase and phytase enzymes [100,101]. Due to organic acid production, insoluble inorganic P gets transformed into soluble form due to the presence of carboxyl and hydroxyl groups that chelate the cations bound to phosphates [102] and transform them into soluble forms. The pH of immediate soil is also decreased by the production of organic acids and the solubility of P is improved by releasing phosphate ions [103]. PGPB are also known to solubilize

organic P by the production of phosphatase and phytase enzymes and converting them into soluble forms [104] which plants can easily uptake from the soil.

The predominant rhizospheric bacteria and others that have the ability to solubilize P belong to the genera *Burkholderia*, *Pseudomonas*, *Bacillus*, *Rhizobium*, *Agrobacterium*, *Achromobacter*, *Streptomyces*, *Micrococcus*, *Erwinia*, etc. Most phosphate-solubilizing bacteria that show good results under laboratory conditions, may not work well in soil conditions [99]. Therefore, rigorous field studies are ongoing to successfully characterize field-compatible phosphate-solubilizing potent bacteria.

#### Production of an Iron Chelating Compound

In the biosphere, iron is the fourth most abundant micronutrient. In aerobic conditions, iron is mostly found in ferric ions or Fe+3 which is insoluble and not easily accessible to plants and microorganisms [105]. In calcareous soil, Fe is not available for plants due to alkaline conditions making it less soluble [106]. Microorganisms, especially PGPB have a mechanism for producing low-molecular-weight iron-chelating compounds known as siderophore [93]. These compounds help in transporting iron into the bacterial cells and also make it available to plants. Siderophores also act as a biocontrol agent as they can create iron-deficient areas near the plant roots by inhibiting plant pathogens [105]. There is a significant increase in Fe uptake in some plants such as sunflower and maize in nonsterile calcareous soils as compared to sterile soil. This occurs due to the action of soil microorganisms that help in the uptake of Fe to plants [106]. It has been shown by Sharma et al. [107] that there is a significant increase in iron content in rice when inoculated with the strain of *Pseudomonas* because of the production of siderophores and also enhanced nutritive value of rice grains due to the increased levels of iron. Thus, siderophore production is an important trait of PGPB and enhances the iron uptake of plants.

### Phytohormone Production

Microbial inoculants such as PGPB are also known to produce a number of plant hormones or plant-growth regulators that alter the architecture of roots and the growth of plants [108–110]. These plant hormones are gibberellins, auxins, ethylene, cytokinins, and abscisic acid [111]. A number of physiological processes can be regulated by these hormones, including root elongation, formation of root hairs, and root initiation [8]. Indole-3-acetic acid (IAA) has been widely reported as a natural auxin produced by microbial inoculants [112]. Many plant functions are influenced by IAA such as root initiation, differentiation of vascular tissue, expression of many plant genes, and mediation of tropic responses [8]. Cytokinins also play an important role in plants, including delaying leaf senescence and promoting mitotic cell division in roots and shoots [16]. Flower and fruit production, seed germination, and dormancy of vegetative organs are affected by gibberellin hormones [113]. Furthermore, abscisic acid is mainly involved in responses to environmental stresses such as high salinity, and drought along with plant development [114]. In addition, ethylene is well known as a ripening hormone, but there are other roles that have been reported such as cell expansion, flower and leaf senescence, and seed germination. Ethylene is also known as a stress hormone because it is produced under abiotic as well as biotic stress [115]. An inhibitory effect on root growth has also been reported due to the production of high concentrations of ethylene, which ultimately reduces plant growth. To overcome this problem, PGPB are also known to produce a vital enzyme, L-aminocyclopropane—1-carboxylate deaminase (ACC deaminase), which catalyzes the formation of the intermediate precursor of ethylene, ACC (1-amino cyclopropane-1-carboxylic acid), into α ketobutyrate and ammonia and regulates the biosynthesis of ethylene [8]. Moreover, PGPB also produced some low-molecular-weight volatile organic compounds (VOCs) such as ketones, alcohols, hydrocarbons, and aldehydes, which have generally high vapor pressure and enter into the atmosphere [116]. These compounds are collectively termed microbial volatile organic compounds (mVOCs) [117]. Initially, Fernando et al. [118] and Vespermann et al. [119] reported some biocontrol activity of these

VOCs of some rhizospheric microorganisms, but later promotion of plant growth also reported the role of VOCs, for instance, the growth promotion in *Arabidopsis thaliana* by VOCs of PGPB strains containing acetoin and 2,3 butanediol [120]. PGPB also affects the morphology of roots and provides nutrition to plants.

#### 4.6.2. Arbuscular Mycorrhizal Fungi (AMF)

Fungi are also found in soil and are associated with plant roots in the following two ways: through mutualistic symbiosis and parasitism. In mutualistic symbiosis, both organisms live together and establish beneficial relationships, whereas, in parasitism association, one partner benefits and the other is harmed [121]. A beneficial and heterogeneous group of fungi that establishes symbiotic relationships with more than 90% of plant species is known as mycorrhizal fungi [1]. Mycorrhizal fungi can be categorized into different groups, but arbuscular mycorrhizal fungi are a prevalent type of endomycorrhiza and are commonly associated with horticultural and crop plants. Early in the history of land plants [122], arbuscular mycorrhizal fungi (phylum Glomeromycota) appeared first and were associated with diverse plant taxa [123]. A special branched structure formed during the penetration of fungal hyphae of Glomeromycota species in root cortical cells of plants is called arbuscules [121,124]. Today, there is great interest in the use of these mycorrhizal fungi in sustainable agriculture, which have been established to provide enhanced nutrients (macro and micronutrients) and water uptake and also help plants survive biotic and abiotic stress [125–130]. There is a recent report that not only is there interconnection between fungi and plants established by the hyphal network, but also connecting individual species of plants within a community and helping in signaling among interplant species [131,132]. AMF plays an important role in stimulating plant growth through several mechanisms [133]: (i) enhancing the uptake of water; AMF increases the surface area of the root through which plants can easily take up water; (ii) availability of nutrients, especially phosphorus, under nutrient-deficiency condition; (iv) modifications of root architecture; (v) changes in enzymatic and physiological activities, especially for plants that are involved in antioxidative responses; and (vi) induction of ABA plant hormones, which are mainly involved in stress conditions [134]. Auge, Brundertt, and Begum [125,135,136] reported some ameliorating effects of drought due to mycorrhizal symbiosis in some plant species, including wheat, onion, soybean, lettuce, and corn. This occurs due to increased root growth resulting in enhanced tolerance to drought. It also maintains high water efficiency and increased growth when plants are colonized by AMF [8]. Furthermore, the water potential of plants may also be affected by the changes in the structure of soil by the production of a soil-binding material such as glomalin, a glue-like substance that is insoluble in nature by the hyphae of AMF [125]. Protection of plant roots from the toxicity of heavy metals by the use of AMF has also been reported by Leyval et al. [137]. There are also some reports available and reporting that drought tolerance of plants is augmented by the application of co-inoculation with AMF and PGPR. For example, improved plant growth, stomatal conductance, the efficiency of water use, as well as increased photosynthetic rate, being reported in lettuce plants when co-inoculated with AMF *Glomus mosseae* and *G. intraradices* and *Bacillus* spp (PGPR). A better result was obtained in co-inoculation with AMF and *Bacillus* spp. as compared to individual organisms. This occurred due to PGPR, i.e., *Bacilus* spp., enhancing the growth and colonization of AMF [138]. But there are some limitations on the use of AMFs as biostimulants, which may result from their biotrophic character; they have difficulty for propagation on a large scale, and researchers have been unsuccessful in understanding the determinants of host specificities and other population dynamics of mycorrhizal fungi in agricultural ecosystems [139]. Some fungi which are distinct from mycorrhizal species are also reported such as *Trichoderma* spp and *Sebacinales*, which are able to colonize roots and provide nutrients to their hosts, but the mechanisms are not well studied [121]. However, these fungi can be used as bioinoculants to improve the nutritional status of plants. *Trichoderma* spp. is well known for its biocontrol and biopesticidal activities, but Colla et al. [40] and Shoresh et al. [140] also reported some

stimulatory effects on plants such as enhanced efficiency of nutrients, morphogenesis, and organ growth along with increased tolerance to abiotic stress. These fungal endophytes may be considered biostimulants as well as biopesticides based on these effects on plants as reported by researchers [40,140]. Therefore, microbial inoculants including beneficial bacteria as well as fungi are a promising tool in sustainable agriculture. They not only enhance plant nutrition but also assist plants in tolerating a number of environmental stresses. They improve our agriculture system without any deleterious effects. The overall mechanisms of action of different plant biostimulants in the plant are represented below (Figure 1):

**Figure 1.** Schematic representation of the different types of plant biostimulants and their beneficial mechanisms in plants [1,8].

#### **5. Risk Status of Microbial Inoculants (Plant Growth Promoting Bacteria)**

PGPB, which are considered potent candidates for plant growth, should be safe for mammals. Some of the microbial inoculants commonly used as biostimulants, and their risk groups are listed in Table 2. Despite their array of beneficial effects on plants (Table 2), they may pose a risk to other living organisms, especially human beings. Although most PGPB do not have a negative effect, some genera are involved in causing infections in animals and humans. Bacteria belonging to the genera *Serratia, Acinetobacter, Bacillius cereus*, *Stenotrophomona, Enterobacter, Herbaspirillum, Ochrobactrum,* and *Pseudomonas* are not only powerful candidates for plant-growth promotion but may also cause disease in humans [141,142]. *Pseudomonas,* besides being a potential candidate as PGPR, is also responsible for many types of opportunistic infections in humans who are aged, immunocompromised, or suffering from conditions such as cancer, severe burns, or cystic fibrosis. Some common pathogenic species of *Pseudomonas* are *P. cepacia, P. aeruginosa, P. putida, P. fluorescens,* etc [143]. Although *Bacillus* sp. is commonly known for its wide variety of applicability in agriculture, industry, and the pharmaceutical sector, it still is associated with many types of illness in humans and animals. It can cause disease in immunocompromised as well as in healthy individuals. Some species may cause minor infections, but some species may be associated with severe or lethal infections. *B pumilus, B licheniformis, B coagulans,* and *B thuringiensis* are examples of *Bacillus* species that are associated with various infections [144]. *Aeromonas* sp. *is* used as PGPR but also causes diseases in immunocompetent and immunocompromised people such as septicemia, gastroenteritis, and wound infections [145]. Another potent PGPR, *Comamonas* spp., is also associated with many life-threatening illnesses such as endocarditis, and septicemia in immunocompetent individuals [146]. *Streptomyes* sp. can cause changes in tissue structure in humans leading to diseases such as cancers, mycetomas, and actinomycetomas [147]. In spite of such immense positive impact of *Trichoderma* sp. on plant health, it is now emerging as a human pathogen causing diseases such as peritonitis, subcutaneous infections, and hematologic disorders [148]. Although *Enterobacter* sp. has a variety of uses as a plant growth stimulator, is also known to cause nosocomial infections and is involved in an array of ailments such as skin infections, inflammation in the respiratory system, and meningitis in neonates, immunocompromised individuals and hospitalized patients [149]. But there are no national or international rules or regulations to assess the risk associated with the commercial use of these plant-beneficial microbes [150]. Even commercial biofertilizers such as Biosubtilin, Nitrofix, and Bioderma. (Table 2) do not mention risks associated with the respective inoculants in their packets.

Risk groups and biosafety levels (BSL) are two terminologies used to describe and categorize microbes as per the level of hazards they can cause [93]. According to the World Health Organization (WHO 2015), microorganisms that are categorized under various risk groups (RG) are based on certain criteria such as their pathogenicity and virulence, host range, mode of transmission, availability of vaccines for effective prevention, availability of medications, etc. Thus, the classification, e.g., from RG-1 to RG-4 articulate the level of hazard a particular microorganism causes. RG-1 refers to a group of microbes that do not cause or are not associated with any type of illness in healthy animals (including humans). Microbes under RG-2 group are associated with a disease that is generally mild and there are medications readily available to treat the disease. RG-3 microbes are concomitantly associated with a serious and lethal disease that may or may not be treatable. Microbes belonging to RG-4 category have the ability to cause fatal and deadly diseases for which treatment is rarely found. Biosafety level (BSL), e.g., from BSL-1 to BSL-4, is a precautionary procedure and protocol used to avoid or prevent risks associated with these risk groups while handling them. Organisms belonging to BSL-1 are nonpathogenic in nature and can be easily handled in the laboratory through general laboratory guidelines. Microorganisms under BSL-2 have the ability to cause disease in a healthy individual, but there are ample medications and vaccines available to easily cure such diseases. Proper laboratory guidelines and special training are required to handle BSL 2 organisms. Specialized safety measures and containment facilities are required to handle microbes that come under the BSL-3 group because such microbes can cause fatal infections but do have effective remedies and anticipatory treatments available. BSL-4 encompasses high-risk-associated organisms, which have aerosol-transmission ability and for which effective treatment is not available. Laboratory personnel handling such organisms must have special training and should know the primary and secondary containment of BSL-4 organisms. The literature suggests that many bacteria isolated from the rhizosphere, soil, and water, besides having PGP activity, are also involved in causing diseases in immunocompromised and healthy individuals [151–153]. Hence there is an immense need to develop a systemic and polyphasic approach through which we can check the disease-causing ability of microbes isolated from an environmental niche in addition to checking their PGP activity and bioinoculant development [154]. A study by Vílchez et al. stabilized a polyphasic protocol called EHSI (environmental and human safety index) to check the biosafety level of plant-growthpromoting bacteria. EHSI articulates the overall effect of PGPB on soil microflora, beneficial macroflora and fauna, and animal and human health. In this study, according to EHSI, both being potent PGPB, *Pseudomonas putida* KT2440, is relatively safe as compared to *Burkholderia cepacia* CC-Al74. In another study by Kim et al. [155], it was suggested to assess or check for the presence of genes involved in the virulence or pathogenicity of novel bacteria isolates to determine their safety level concerning humans and plants. Keswani et al. [150] suggested that whole-genome sequencing of a bacterial isolate is the best way to obtain a complete understanding of its phylogenetic categorization and pathogenic behavior. Hence, research organizations and institutions which are involved in isolating novel microbial isolates and bioinoculant development, after thorough polyphasic characterization, should use isolates that belong to BSL-1 and Risk group-1 for bioformulation because they will pose minimum risk to the environment and human health [150].

**Table 2.** Microbial inoculants in agriculture and horticulture systems and their indicative risk status in the risk group databases.

Risk group \* (classification of prokaryotes into risk groups under Biological Agents Ordinance: RG 1 refers to prokaryotes that generally do not cause infectious disease in humans; RG 2 refers to those microbial groups which do not pose a significant risk to laboratory workers but may cause disease if there is exposure and for which there are therapeutic interventions available), RG—Risk group, BSL—Biosafety level as per ATCC; #—The exact name of the organisms in the original concerned database of risk group; (-) indicates that it has not been commercially formulated.

In addition to posing health risks to animals, unprecedented use of PGPR also affects other biotic communities of an ecosystem, especially soil resident flora. As it is already known that newly introduced microorganisms change the microenvironment of soil, creating their niche which can have an immense effect on the structure and composition of resident microbes [217]. The interaction of PGPR with soil flora may be negative, positive,

or neutral depending upon the nature of the PGPR introduced into the soil [218]. The main concern is the introduction of antimicrobial-producing PGPR in the soil milieu [219]. A study by Walsh et al. [220] revealed that there was a reduction in the diversity of the rhizobacterial population due to the introduction of 2,4-diacetylphoroglucino (an antibiotic substance) producing bacteria in the rhizosphere. Some type of perturbance in the resident-flora population is also possible as found in the study by Albright et al. [221].

#### **6. Safety Determination of Microbial Inoculants**

Several microbes belonging to *Pseudomonas, Bacillus, Acinetobacter, Burkholderia, Staphylococcus, and Stenotrophomonas* have been used as inoculants for plant-growth promotion and biocontrol of plant pathogens; however, these also include microbes identified as opportunistic pathogens and that cause human pathogenesis [150]. It has also been reported that the invasion and colonization mechanisms involved in the pathogenesis of PGPR on plant and human tissues are similar [151,222,223]. Therefore, the safe application of PGPR to protect human health and the environment is needed, which involves collaborative efforts of different expertise, and technological advancements. Microbial inoculants need to be identified and well characterized to unveil their hidden risks to humans and the environment. Several physiological and molecular approaches are now used to check the virulence and pathogenicity of infectious microbes. These methods can also be employed to detect the pathogenicity level of PGPB. The following are some important detection methods that can be taken into consideration.

#### *6.1. Morphological and Biochemical Methods*

To detect the pathogenicity level of bacteria, it is necessary to identify the species of bacteria which can be done through cultural studies and fast biochemical tests. For example, growth on blood agar will indicate that the bacteria are pathogenic in nature. The use of differential and selective media will enhance the probability of isolating microbes that have a pathogenic nature. Various biochemical tests such as tests for enzyme detection of catalase, urease, deaminase, decarboxylase, deaminase, β galactosidase, hydrolase, etc., are helpful in the polyphasic characterization of bacteria. These enzymes can also be detected using chromogenic media that contains specific chromogenic substrates which are hydrolyzed and produce a particular color in the media indicating the presence of enzymatic activity in bacteria. Nowadays various biochemical kits and their detecting instruments are available commercially, which enables the rapid detection of microbes [224–226].

### *6.2. Antibiotic Sensitivity Method*

Sensitivity to various antibiotics will indicate whether the given bacterial isolate is safe for release into the environment or not because multiple drug-resistant PGPB bacteria that somehow cause disease in humans and animals will be difficult to treat or cure such disease through prevalent antibiotics. In addition, antibiotic resistance is generally plasmidborne and most of the plasmid can be transferred from one bacterium to another, thereby spreading the antibiotic-resistant character in the soil microbiome [227].

#### *6.3. Protein Profiling Method*

Every genus has a particular set of proteins, and protein profiling will help in identifying the bacterial genus. Even various species in one genus can be differentiated through protein profiling as they have a particular set of proteins, i.e., they contain enzymes involved in a unique biochemical pathway. In addition, it may be possible that these unique biochemical pathways enable a particular microbe to thrive in a harsh climate making them a more favorable candidate for bioinoculant production [227].

#### *6.4. Molecular Level Detection Techniques*

Studying at the genetic level is the most precise, rapid, and sensitive technique in today's era to help in the proper understanding and identification of microbial species. Detection of ubiquitous and universal sequences (containing conserved and variable regions) such as 16s rDNA/18s rDNA is the most prominent and simple way to identify microbes at the species level. Techniques based on the hybridization process are used to detect genes of interest through the use of probes tagged with fluorescent dyes. For example, the fluorescence in situ hybridization (FISH) method uses universal probes to detect a particular microbe [228,229].

Amplification of genes conferring the virulence property of a particular microbe is also an effective way to check the pathogenic nature of bacteria. Quantitative polymerase chain reaction (qPCR) and reverse transcriptase real-time PCR (RT-qPCR) are employed as amplification techniques. One such example is an *invA* gene, which is a virulence gene found in *Salmonella* sp. that is detected through PCR using compatible primers [230].

Gene chip technology or DNA microarray is yet another efficient technique that can not only identify and differentiate among various species of microbes through a variety of probes and universal or consensus primers, but can also give information regarding different resistant measures adopted by a specific microbe [231]. With the advent of the Sanger method of sequencing, a first-generation sequencing technique, it is now possible to sequence the whole genome of a particular microorganism in a very rapid and efficient way. The sequence of the whole genome will not only identify the bacterium but also disclose its pathogenic nature and resistant profile. Whole-genome sequencing also helps in the rapid designing of primers [232,233]. Nowadays, NGS (next-generation sequencing) has proven to be a powerful method for the detection of virulence factors of infectious microorganisms within a few hours. In clinical microbiology, there are numerous methods available for the detection of human pathogens, which are compiled in Table 3. These technologies, in combination with the routine characterization and evaluation of potential microbial biostimulants, can be used to guide as per Figure 2 for the safe development and enrichment of microbial stimulants for use in agriculture.

**Table 3.** Comparative table of different technological approaches for the detection of pathogenic organisms.

**Figure 2.** A graphical abstract of isolation, characterization, and identification of microbial inoculants and their commercialization as per their respective categorization into different risk groups (compiled from) [150,227,237,238].

#### **7. Legal Framework of Biofertilizer Implementation in Different Countries**

In spite of having so many advantages over chemical pesticides, the biofertilizer industry faces too many legal obstacles to overcome before entering into commercial production. Earlier the legal regulations regarding biofertilizer use were very inadequate and weak. But in today's era, as researchers have shown the great potential of biofertilizers, many countries amended and developed strong policies and legal regulations to increase the usage of biofertilizers [240,241].

Having an appropriate legal definition is a crucial part of making biofertilizers an appealing commercial product to the producers. In the USA and European Union (EU), there is no proper definition of biofertilizers that can define their actual characteristics. In the EU, biofertilizer comes under e EU Commission Regulation n. 889/2008 on organic production, which states that biofertilizers can only be used as plant protectants against pests and diseases. Hence, biofertilizer comes under the legal agenda of plant protection products. The same outline is followed by the US National Organic Program which categorizes biofertilizers as biological organisms that can only be used as plant protectants [242].

Compared to other countries, India has the most comprehensive and defined legal regulation and framework for biofertilizer implementation. In India, biofertilizer comes under the Essential Commodities Act of 1955, Ministry of Agriculture, and can be defined as "the product containing carrier based (solid or liquid) living microorganisms which are agriculturally useful in terms of nitrogen fixation, phosphorus solubilization or nutrient mobilization, to increase the productivity of the soil and/or crop". Seven standard criteria have been set to formulate a biofertilizer that includes viable inoculum density, the physical form, level of contamination, pH, moisture content, the particle size of carrierbased products, and efficacy level. Four groups of microbes are mainly included under the biofertilizer category i.e., *Azotobacter, Rhizobium, Azospirillum*, mycorrizal fungi, and phosphate-solubilizing bacteria [242,243].

In Poland, Polish Law on Fertilizers and Fertilization 2007 includes biofertilizers under "growth stimulators" and groups them under plant conditioners. This law defines biofertilizer as "a positive impact on plant growth or other metabolic processes of plants in other ways than plant nutrients" and shall "pose no threat to [the] health of humans or animals or to the environment after their use and storage instructions" [242].

Spain, which is one of the leading countries in organic farming, does not have a separate category and definition of biofertilizer in its legal structure. It includes microorganisms as one of the components of compost and organic amendments under Real Decreto 506/2013 [242].

China has a strict and defined legal framework for biofertilizer implementation. It has set various parameters through which it can access the quality of biofertilizer including inoculum density, water, and carbon content, outer appearance, granule size, contamination, viability, and validity. Chinese standards mostly rely on the amount of inoculum to access the quality of biofertilizer, which should range between >1.5 × 109 CFU mL−1 or >0.2 × 109 CFU g−1 and >0.5 × 109 CFU mL−1 or >0.1 × 109 CFU g−1, for solid and liquid products, respectively. Seven categories of microorganisms are included in biofertilizers, i.e., fast- and slow-growing species of rhizobia, organic and inorganic phosphate-solubilizing bacteria, nitrogen-fixing bacteria, silicon-solubilizing bacteria, and various consortia containing multiple microorganisms [244].

### **8. Conclusions**

Plant biostimulants prove beneficial to plants by improving their growth. Microbial inoculants, single or consortia, naturally improve plant growth and performance without using any agrochemicals in the field. They can act as biofertilizers, soil improvers, growth regulators, stress relievers, and biocontrol agents. However, more research needs to explore and establish their biocontrol properties. Much research has been conducted to understand their properties and functions followed by their commercialization to promote eco-friendly and safe agriculture practices for the fortification of plants with nutrients. The global markets of biostimulants also need to be expanded in the near future so that farmers can easily buy these products at affordable prices. Furthermore, extensive characterization research emphasizing the safety issues of the inoculant microbes becomes inevitable to address recent reports of many inoculants belonging to either higher-risk groups or potential pathogens of human beings, such as *Pueudomonas*, *Klebsiella*, *Enterobacter*, *Acinetobacter,* etc., which may cause various kind of suffering, for example, septicemia, gastroenteritis, wound infections, inflammation in the respiratory system, meningitis, etc., of varied severity under different conditions of human-health status, such as immunocompromized and comorbidity with other diseases, etc. Advances in technologies including biochemical, immunological, proteomics, and genomics approach unraveling the characters and identification of microbes have enabled the research community to rapidly and accurately address safety concerns, such as pathogenicity, of biostimulant microbes following a suitable strategic plan before releasing the inoculant for field application.

**Author Contributions:** Conceptualization, M.K., P.S. and A.K.; resources, A.K.; writing—original draft preparation, M.K., P.S. and A.K.; writing—review and editing, K.K.K. and A.K.; visualization, K.K.K. and A.K.; supervision, K.K.K. and A.K.; project administration, A.K.; funding acquisition, K.K.K. and A.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.