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\\n\\nLaunching 2021
\\n\\nArtificial Intelligence, ISSN 2633-1403
\\n\\nVeterinary Medicine and Science, ISSN 2632-0517
\\n\\nBiochemistry, ISSN 2632-0983
\\n\\nBiomedical Engineering, ISSN 2631-5343
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\\n\\nDentistry (Coming Soon)
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\\n\\nNote: Edited in October 2021
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\n\nDesigned to cover fast-moving research fields in rapidly expanding areas, our Book Series feature a Topic structure allowing us to present the most relevant sub-disciplines. Book Series are headed by Series Editors, and a team of Topic Editors supported by international Editorial Board members. Topics are always open for submissions, with an Annual Volume published each calendar year.
\n\nAfter a robust peer-review process, accepted works are published quickly, thanks to Online First, ensuring research is made available to the scientific community without delay.
\n\nOur innovative Book Series format brings you:
\n\nIntechOpen Book Series will also publish a program of research-driven Thematic Edited Volumes that focus on specific areas and allow for a more in-depth overview of a particular subject.
\n\nIntechOpen Book Series will be launching regularly to offer our authors and editors exciting opportunities to publish their research Open Access. We will begin by relaunching some of our existing Book Series in this innovative book format, and will expand in 2022 into rapidly growing research fields that are driving and advancing society.
\n\nLaunching 2021
\n\nArtificial Intelligence, ISSN 2633-1403
\n\nVeterinary Medicine and Science, ISSN 2632-0517
\n\nBiochemistry, ISSN 2632-0983
\n\nBiomedical Engineering, ISSN 2631-5343
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\n\nDentistry (Coming Soon)
\n\nWe invite you to explore our IntechOpen Book Series, find the right publishing program for you and reach your desired audience in record time.
\n\nNote: Edited in October 2021
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This crystalline structure is classified by several items; e.g., single and poly-crystals, grain size, grain boundary characteristics, crystallographic orientation, and so forth [1]. These items are controllable by mechanical and chemical interaction with internally and externally straining [2]. For examples, a single crystal changes itself to polycrystalline state by introduction of dislocations with sufficiently high density [3]. The original grains are much refined by intense rolling [4] and by high shear straining [5]. The initial grain boundaries are also tunable by shuffling process through their interaction with dislocations [6]. That is, the crystalline structure is tailored by metal forming and materials processing to have preferable grain size, crystallographic orientations, and grain boundaries [7, 8, 9].
Let us consider how to control the crystallographic structure of metals and metallic alloys by the technology of plasticity in metal forming and surface treatment. Figure 1 depicts three case studies on the crystallographic structure change by rolling, shearing, and nitrogen supersaturation. Even when the initial grains are equiaxial, some of them are forced to partially align along the rolling direction. The skew distortion by intense rolling drives to shear the grains with spin-rotation as shown in Figure 1a. In case of embossing and piercing the sheet metals, the grains are distorted to plastically flow along the shearing plane with grain size refinement as depicted in Figure 1b. In the low temperature plasma nitriding, the plastic straining is induced by the nitrogen supersaturation into metallic lattices to form the slip-line system as shown in Figure 1c. Without externally applied stresses, the slip-line network is formed from the surface to the depth together with the nitrogen interstitial diffusion. In particular, the low temperature plasma nitriding process [10, 11, 12, 13, 14, 15] works as a powerful means to demonstrate that austenitic and martensitic stainless steel substrates are hardened and modified to have two-phase structure with the average grain size of 0.1 μm. These previous studies proved that grain size as well as crystallographic structure should be significantly controlled by the materials processing other than the shearing process in metal forming.
Three types of crystallographic evolution during metal forming and plasma nitriding. (a) Intense rolling, (b) embossing and piercing, and (c) low temperature plasma nitriding.
In the present chapter, the crystallographic structure evolution of stainless steels during the rolling, the piercing, and the plasma nitriding at 623 K for 14.4 ks is first described to deduce the mechanism of microstructure evolution during metal forming and materials processing. Next, the uniaxial loading test of plasma nitrided work at 623 K is performed to investigate the possibility of further microstructure evolution during posterior metal forming. Through these experiments, the effect of the interstitial element concentration as well as the plastic straining on the crystallographic evolution is discussed to search for the materials science model to describe the interaction between the interstitial mobility and the plastic straining. In the following, EBSD (Electron Back Scattering Diffraction) is employed to make crystallographic analyses. This technique is based on the automatic analysis of the Kikuchi pattern by the excitation of the electron beam on the surface of the sample in SEM (Scanning Electron Microscope) [16]. Among several analytical tools in EBSD, the crystallographic orientation for each grain is described by IPF (Inverse Pole Figure) and the strain induced phase transformation is also analyzed by phase mapping. In addition, the equivalent plastic strain distribution is estimated by KAM (Kernel Angle Misorientation) mapping.
Intense rolling with heat treatment has been utilized to fabricate the fine-grained stainless steel plates and sheets [4, 7, 17]. In the following, AISI304 sheet was employed as a work to reduce its thickness from 10 mm down to 1 mm by intense rolling. EBSD analysis was used to describe the crystallographic change in the rolled AISI304 sheet.
A typical rolling system was illustrated in Figure 2a. AISI304 sheet with the initial thickness of 10 mm was compressed and sheared between two work rolls in a single reduction. Since the reduction of thickness was 10% in this single rolling, nine steps were utilized to reduce the thickness down to 1 mm through this intense rolling. Figure 2b shows the rolled sheet by 90% reduction in thickness. This rolling is effective to reduce the average grain size of stainless steel works for embossing and piercing to be discussed in later.
Intense rolling process with high reduction in thickness. (a) Illustration on the rolling process in multi-steps for reduction of thickness by 90% and (b) rolled AISI304 sheet.
The original AISI304 sheet was characterized by three high intensity peaks in XRD analysis. As depicted in Figure 3, three peaks were detected in correspondence to γ (111), γ (200), and γ (220) planes in the austenitic phase. This microstructure changes to nearly full-martensitic phase; as also depicted in Figure 3, three martensitic peaks were detected as α’ (211), α’ (200), and α’ (110) besides for γ (220) and γ (111). This proves that original austenitic grains massively transform to martensitic ones in AISI304 sheets during the intense rolling. These martensitic grains can be inversely transformed back to austenite by heat treatment. This technique is useful to reduce the average grain sizes to be stated later.
Variation of XRD diagrams from the original AISI304 sheet before rolling to AISI304 after intense rolling and furthermore to rolled AISI304 sheet after low temperature plasma nitriding.
In addition to the strain-induced phase transformation in Figure 3, the intense rolling has much influence on the crystallographic structure in AISI304. Figure 4 depicts the inverse pole figure mapping, the KAM distribution, and the phase mapping on the cross-section of rolled AISI304 sheet, analyzed by EBSD. As stated before, the average grain size is reduced down to 1 μm in most of AISI304, since the plastic strains are applied to these regions as shown in Figure 4a and b. Low plastic strained regions in Figure 4b corresponds to assembly of larger grains laterally aligned in the rolling direction. Through comparison between Figure 4b and c, these textures are classified into two zones. One is thin and long full-martensitic textures. The shorter and dotted textures are corresponding to the retained austenitic textures.
EBSD analysis on the cross-section of rolled AISI304 sheet. (a) IPF mapping, (b) KAM distribution, and (c) phase mapping.
These textured crystallographic structures are further controlled by the low temperature plasma nitriding as well as the heat treatment to be discussed in later.
Fine piercing is an essential process in metal forming for accurate drilling of holes and for fine blanking. In the last decade, metastable austenitic stainless steel type AISI304 with fine grains has been developed by [18, 19, 20]; the effects of fine crystallographic structure on the elasto-plastic deformation have been closely studied in [21, 22, 23]. Figure 5 shows three metastable austenitic stainless steel AISI304 sheets with the thickness of 100 μm, where the grain size was reduced by rolling process from the normal-grained sheet with the average grain size (D) of 7.5 μm. Two fine-grained AISI304 sheets were yielded to have D = 3.0 and 1.5 μm, respectively, by reverse transformation of the strain-induced martensitic phase. In this thermo-mechanical treatment, near-fully martensitic grains in Figure 4c are inversely transformed together with reduction of grain sizes [4, 7].
IPF map and intensity map in the ND direction analyzed by EBSD. (a) Normal-grained AISI304 sheet with F = 7.5 μm, (b) fine-grained AISI304 sheet with D = 3.0 μm, and (c) fine-grained sheet with D = 1.5 μm.
Their IPF maps analyzed by EBSD were shown on the cross section in the sheet width direction. When D = 7.5 μm, most of grains have a preferred orientation to [111] direction, as shown in Figure 5a. The crystallographic structure for D = 3 and 1.5 μm became nearly the same as D = 7.5 μm as depicted in Figure 5b and c.
These AISI304 sheets with different grain sizes were pierced by CNC stamper under the same conditions; e.g., the punch diameter was 100 μm, the die diameter, 110 μm, and the clearance, 5% of sheet thickness. Figure 6 compares IPF mapping as well as phase mapping on the cross-section of punched hole among three AISI304 sheets with the different grain size by D = 1.5, 3.0, and 7.5 μm. As commonly seen in Figure 6a–c, most of grains along the side surface of hole are distorted and refined in size. The shearing of AISI304 sheet by piercing leaves the process-affected zones along the shearing plane. The phase mapping in Figure 5d–f reveals that these process-affected zones are just corresponding to the martensitic phase [24]. That is, the original austenitic matrix to AISI304 sheets is forced to transform to martensite by the shearing strain during the piercing process. This strain-induced martensitic transformation is a non-diffusive shear transformation; each grain in the affected-process zone massively makes transformation [25, 26].
EBSD analyses of IPF mapping in the ND direction and phase mapping on the cross-section of punched AISI304 sheet. (a) IPF map at D = 1.5 μm, (b) IPF map at D = 3 μm, (c) IPF map at D = 7.5 μm, (d) phase map at D = 1.5 μm, (e) phase map at D = 3 μm, and (f) phase map at D = 7.5 μm.
Let us consider the effect of average grain size on this formation of affected-process zones with phase transformation. When D = 7.5 μm, the zone boundary between the austenitic and martensitic phases is shaped to be irregularly jagged in Figure 6d. With decreasing the grain size, this phase boundary gradually is homogenized to be smooth as seen in Figure 6e and f. To be noticed, the volume of strain-induced martensitic phase in Figure 6f becomes larger than that in Figure 6e. This increase of strain-induced martensitic phase volume ratio causes the variation of the fracture length of the hole at the punch stroke direction [24].
The strain induced phase transformation in the process-affected zones by piercing is commonly observed in the metastable austenitic stainless steel sheets. Among them, the chemical components in each class of stainless steels have influence on the microstructure change by the piercing process. Two types of metastable austenitic stainless steel sheets were prepared as a work sheet with the thickness of 100 μm for piercing experiments under the same conditions as shown in Figure 6.
Figure 7 compares the phase mapping on the cross-section of punched holes between the stainless steel AISI316L and AISI304. When punching out the AISI316L sheet, the strain-induced martensitic phase hardly occurred along the shearing plane; no martensitic phase was present in Figure 7a. Instead of martensitic phase maps, the ferritic phase was strain-induced even by this piercing process as predicted by [27]. In fact, much ferrite was detected at the vicinity of die corner in Figure 7a. Let us consider why no martensitic phase but ferritic phase is induced by piercing the AISI316L sheet. As stated in [27], the nominal strain to induce the martensitic phase into AISI316L is two times larger than that to induce the ferrite phase. Hence, when piercing AISI3016L sheet, the ferritic phase is much easier to be induced than the martensitic phase. Therefore, even when the ferrite phase is induced along the shearing plane by the piercing process, each grain deformation is not hindered by shearing. As a result, the process-affected zone area in AISI316L becomes larger along the shearing plane in Figure 7a than that in AISI304 in Figure 7b [28].
Comparison of phase mapping on the cross-section of fully pierced AISI316L and AISI304 austenitic stainless steel sheets. (a) Punched-out AISI316L sheet and (b) punched-out AISI304 sheet.
The difference of crystallographic structure between pierced AISI304 and AISI316L sheets reveals that the process-affected zone formation could be controlled by the strain-induced phase transformation during the piercing process. EBSD analysis is really a well-defined means to describe the relationship between the strain-induced phase transformation by piercing and the crystallographic structure of pierced stainless steel sheets.
Lower temperature plasma nitriding process of austenitic stainless steels than 700 K is governed by the nitrogen supersaturation with nitrogen interstitial occupation of octahedral vacancy sites of fcc-structured supercells as well as the nitrogen diffusion through refined grain boundaries and slipping lines [13, 14, 15]. FGSS316 plates and wires are employed to describe the nano-structuring process with grain size refinement by this plasma nitriding.
High density RF (Radio Frequency) – DC (Direct Current) discharging plasma nitriding system is utilized to generate the nitrogen-hydrogen plasmas. Figure 8 illustrates a typical hollow cathode device for homogeneously nitriding a single FGSS316 wire. RF-nitrogen/hydrogen plasma is ignited to surround the wire surface by a cylindrical plasma sheath. The activated nitrogen atoms (N*) and ions (N+) as well as the NH radicals are enriched in this sheath to increase the nitrogen ion density up to 1 × 1018 ions/m3. Under this plasma processing condition, the nitrogen solute diffuses into the depth of FGSS316 matrix in wire to form the nitrided layer.
Experimental setup for low temperature plasma nitriding of steel wires with use of the hollow cathode device. The same DC-bias was applied to the hollow and the FGSS316 wire.
The inner nitriding process with nitrogen supersaturation is described by the multi-dimensional relation in Figure 9. When some nitrogen interstitial atoms diffuse and supersaturate the fcc-structured lattices in Figure 9a, their original lattice constant (Λ0) increases to Λ by occupation of nitrogen interstitials into the octahedral vacancy sites in them. Other nitrogen atoms diffuse to further depth through the grain boundaries. Then, the nitrogen supersaturated (NS) zone expands with the elastic strain (εe) while unsaturated (US) zone does not deform; the strain incompatibility occurs on the boundary between NS and US zones. The misfit distortion (βmisfit) is induced along this zone boundary as depicted in Figure 9b; e.g., βmisfit = ωspin + εp, where ωspin is a spin tensor to rotate the NS zone and to generate the crystallographic misorientation into a current zone, and εp, a plastic strain tensor to compensate for the strain incompatibility across the zone boundary. Nitrogen solutes further diffuse into the depth of grains through these zone boundaries, as shown in Figure 9c.
Multi-dimensional relation in the nitrogen interstitial atom diffusion and supersaturation in the low temperature plasma nitriding.
Through this multi-dimensional inner nitriding, high elastic strain energy density in NS zones drives the phase transformation from austenite to martensite. The spin rotation of zones and sub-grains advances with nitrogen diffusion and supersaturation to refine the crystallographic structure. NS zones accompany with the plastic strain distribution along the NS-US zone boundaries.
This theoretical model is experimentally demonstrated in the following. As stated in [13, 14, 15], the inner nitriding advances homogeneously from the surface to the nitriding front end; it is rather difficult to experimentally describe each fundamental process separately from other processes in Figure 9. A normal-grained AISI316 plate is employed as a work material and nitrided at 623 K for 14.4 ks to decelerate the nitrogen diffusion rate and to describe the synergetic relation among the nitrogen supersaturation, the phase transformation, the plastic straining, the grain size refinement and the local nitrogen diffusion.
Figure 10 depicts the homogeneous and heterogeneous inner nitriding processes in the nitrided AISI316 at 623 K for 14.4 ks. Under this nitriding condition, the high nitrogen solute content, [N], around 5 mass% is present down to the nitriding front end (NFE) at the depth of 30 μm. To be noted, [N] remains to be 1 mass % even below NFE, as shown in Figure 10a. This implies that homogeneous nitriding advances to NFE and changes to be heterogeneous by localization in nitrogen diffusion below NFE. In fact, the phase mapping as well as the crystallographic structure changes drastically across this NFE, which was indicated by the gray two-dots chain line in Figure 10.
Experimental demonstration on the inner nitriding process. (a) Nitrogen content depth profile, (b) IPF mapping, phase mapping and KAM distribution, and (c) inner nitriding mechanism.
Due to EBSD analysis, the synergetic relation is described by the inverse pole figure, the phase mapping and the plastic strain distribution below NFE in Figure 10b. The coarse grains near the grain boundary were only nitrided to change their microstructure; e.g., the austenitic and martensitic zones exclusively distribute in an A-grain. The retained austenitic zones in this grain had higher plastic strains (or higher KAM) and different crystallographic orientations from the martensitic zones, the plastic strains of which were much lower than these austenitic ones. This formation of γ- and α’-zone mixture with plastic straining and crystallographic rotation just corresponds to the multi-dimensional inner nitriding process. As depicted in Figure 10c, the nitrogen diffusion localizes across NFE so that the nitrogen selectively diffuses along the original grain boundary below NFE. This nitrogen main stream branches into the neighboring grains to this gran boundary. In each grain, the nitrogen further diffuses through the zone boundaries to drive the phase transformation, the plastic straining, and the refinement of zone sizes. Next, more precise EBSD analysis is made on this A-grain.
As compared between Figure 11a and b, little plastic strains were detected in the α’-zone of A-grain while the surrounding γ-zones were much plastically strained. This proves that some of NS-zones massively transform to α’-zones since the elastic strain energy density reaches to the critical level and that plastic strains are induced only in the neighboring γ-zones to α’-zones to compensate for misfit strain between NS-zones and surrounding unsaturated γ-zones. Comparing Figure 11b and c, these highly strained zones are just corresponding to crystallographically refined zones. This assures that spin rotations are induced together with the plastic strains by misfit distortion on the distributed zone boundaries to refine each zone size. Owing to this synergetic mechanism, the refined zones have mutual boundaries with high misorientation angles. This grain size refinement reflects on the high strength and hardness of the nitrided layer by the low temperature plasma nitriding.
Synergetic relation in the inner nitriding at 623 K, locally observed in the grain-A. (a) Phase mapping, (b) KAM distribution, and (c) IPF mapping.
As had been discussed in [29, 30], the initial grain size of AISI316 has influence on the inner nitriding behavior; homogeneity in nitriding process is enhanced with reduction of initial grain size. This finding suggested that fine-grained AISI316 or FGSS316 structural components and parts could be homogeneously nitrided on the surface with a little effect of heterogeneous nitriding on the microstructure below NFE. In the following, a FGSS316 wire is nitrided at 623 K for 14.4 ks and its microstructure is precisely analyzed to describe the formation of nitrided layer as well as the microstructure evolution at the surface and below NFE during this low temperature nitriding. Figure 12 depicts the nitrogen solute mapping, phase mapping, and crystallographic structure on the lateral and longitudinal cross-sections of nitrided FGSS316 wire, respectively. The wire surface is surrounded by two-phase fine-grained layer with the thickness of 30 μm and the average nitrogen content of 5 mass% as seen in Figure 12a and d. The retained austenitic phase grains with larger size than nano-structured grains are present in the inside of nitrided layer as seen in Figure 12b, c, e, and f, respectively. This reveals that inner nitriding process advances almost homogeneously but that its synergetic relation is retarded in some parts to leave the nitrogen supersaturated γ-grains without phase transformation and grain size refinement. In the following section, these retained γ-zones are employed as a marker to investigate the effect of uniaxial loading on the nitrided surface layer.
Microstructure and nitrogen mapping across NFE on the lateral and longitudinal cross-sections of nitrided FGSS316 wire at 623 K for 14.4 ks. (a) Nitrogen mapping, (b) phase mapping, and (c) IPF mapping in the nitrided layer, on the lateral cross-section. (d) Nitrogen mapping (e) phase mapping in the nitrided layer, and (f) IPF mapping in the nitrided layer, on the longitudinal cross-section.
Let us analyze the effect of inner nitriding on the FGSS316 matrix below NFE. Figure 13a shows the initial microstructure of FGSS316 wire. This microstructure of FGSS316 before nitriding has equiaxial crystallographic structure with the average grain size of 2 μm. Figure 13b and c shows the crystallographic microstructure after nitriding on the lateral and longitudinal cross-sections of wire, respectively. The original microstructure is modified to form the skewed linear zones with finer grains as pointed by “b” in Figure 13c. These nitrogen-supersaturated zones consist of the transformed α’-zones and their surrounding plastic-strained. Just as discussed in Figures 10 and 11, the inner nitriding process advanced heterogeneously in the depth below NFE even when nitriding the FGSS316 wire.
Comparison of the crystallographic structure before and after plasma nitriding. (a) Initial microstructure of FGSS316 wire, (b) microstructure of FGSS316 matrix on its lateral cross-section, and (c) microstructure of FGSS316 matrix on its longitudinal cross-section.
Different from the metal forming, the plastic strains are induced along the network of subgrain and zone boundaries, which are newly generated in the inside of original matrix by nitrogen diffusion and supersaturation. Since this network also works as new nitrogen diffusion paths, further nitrogen diffusion and supersaturation advances into the depth of original grains. This concurrent co-working of nitrogen diffusion and supersaturation sustains the synergetic relation among the phase transformation, the plastic straining, and the grain size refinement. In particular, fine zone network results in refined nano-sized crystallographic structure after plasma nitriding.
The nitrided FGSS316 wire is further uniaxially loaded in tensile. The austenitic grains are continuously linked between its nitrided layer and inner matrix. This nitrided layer had influence on the mechanical response of wire since the same elasto-plastic strains are applied to these two regions. Precise microstructure analysis is also made to describe the microstructure evolution of this nitrided wire during the uniaxial loading.
The uniaxial tensile loading test was performed by using the precision universal testing machine AUTOGRAPH AGS-X 10 kN (SHIMADZU Co. Ltd.). This uniaxial loading was terminated when the maximum applied load reached 6 kN before fatal ductile fracture for microstructure analyses. The applied load and stroke were in situ monitored by the load cell and linear scaler, respectively. The bare FGSS316 wire without nitrided layer has an ultimate strength (σU) of 1.18 GPa at the stroke (δ) of 4.9 mm, or, at the nominal strain (ε) of 0.17.
A normal FGSS316 wire elongates in the tensile direction at room temperature (RT) without significant change of microstructure except for the formation and coalescence of voids near the fatal ductile fracture of wire [31]. Microstructure of high carbon steel wire is sensitive to drawing process since it has a multi-dimensional microstructure, where each austenitic grain boundary houses pearlite blocks and each block consists of pearlite colony with the same lamellar structure of cementite (or θ-phase) and lamellar ferrite [32]. However, little microstructure change occurs at the RT in its uniaxial tensile loading. A composite wire also has no microstructure evolution before pop-out of fibers from core matrix [33]. Let us investigate the microstructure at the nitrided layer and in the matrix of wire, respectively, and describe the effect of posterior elasto-plastic straining to nitriding on the microstructure evolution in wire.
Figure 14 depicts the plastic strain distribution and phase mapping on the lateral and longitudinal cross-sections of nitrided FGSS316 wire, respectively, after uniaxial loading. The retained austenite regions with larger size than fine grained two-phase nitrided layer in Figure 12 completely disappear at the nitrided layer. Every nitrided layer has homogeneous two-phase microstructure with fine grain sizes. Compared with the plastic strain distribution before uniaxial loading, the whole nitrided layer is uniformly subjected to high plastic straining. This reveals that the applied plastic strains by uniaxial loading drives the synergetic process of inner nitriding at the retained austenitic zones and induces the phase transformation from the retained γ-phase to γ−/α’-phase fine mixture.
Plastic straining and phase mapping on the lateral and longitudinal cross-sections of nitrided FGS316 wire after uniaxial loading. (a) Plastic strain lateral distribution, (b) phase mapping in lateral, (c) plastic strain longitudinal distribution, and (d) phase mapping in longitudinal.
The microstructure as well as the phase in the matrix below NFE is modified by this uniaxial loading. Nearly full austenitic phase of matrix before loading changes to mixture of γ-fibers and transformed α’-fibers. As shown in Figure 14b and d, these α’-fibers with its lateral size of 0.5 μm are aligned along the loading direction to form a bundle structure together with γ-fibers. The volume fraction of these α’-bundles increases from the vicinity of NFE to the depth in matrix. This suggests that the nitrided FGSS316 wire fractures of α’-bundles in ductile at its center when this fraction reaches to the critical maximum.
Let us compare the above phase mapping in matrix with the plastic strain distribution in Figure 14. The high plastic straining zones are just corresponding to the α’-fiber zones. This proves that this γ to α’-phase transformation during the uniaxial loading is induced by the high plastic straining of γ-zones. That is, the highly strained γ-grains are plastically strained and aligned along the loading direction to form the γ-fibers. During this microstructure evolution, some of γ-fibers transforms massively to α’-fibers.
The phase transformation and formation of bundle microstructure in the above reflects on the crystallographic structure after uniaxial loading. Figure 15 depicts the IPF mapping at the nitrided layer and in the matrix on the lateral and longitudinal cross-sections of wire, respectively. The γ-phase matrix before uniaxial loading has crystallographic structure with the average grain size of 2 μm and without preferred orientations. After loading, these γ- and α’-grains are aligned along the loading direction respectively to form α’- and γ-bundles. In particular, most of α’-bundles have unique (111) directions. This might be because the transformed bundles are aligned in the tensile directions under the constrained conditions by the nitrided layer, surrounding the matrix. Each initial γ-grain is forced to deform elasto-plastically only in the tensile direction and to form γ-fibers and γ-bundles since no plastic deformation is allowed in the lateral direction under the confinement of nitrided layer. Some of strained γ-fibers makes massive transformation to α’-fibers; these α’-fibers are assembled into a single α’-bundle with the preferred orientation to (111). After [26, 34], the easiest crystallographic orientation to form this martensitic fibers and bundles is thought to be (111) tensile direction.
Nitrogen mapping and crystallographic structure on the lateral and longitudinal cross-sections of nitrided FGSS316 wire after uniaxial loading. (a) Nitrogen mapping in lateral, (b) lateral IPF mapping in ND, (c) nitrogen mapping in longitudinal, and (d) longitudinal IPF mapping in TD.
Figure 15 reveals that the nitrided layer surrounding the matrix has homogeneous super fine-grained two phase structure after uniaxial loading. No cracks and defects are seen on the nitrided layer surface and in the inside of layer; the fatal fracture of this nitrided FGSS316 wire occurs as the ductile fracture of matrix as explained before. The fine continuous interface between the nitrided layer and the matrix also suggests that elasto-plastic compatibility is preserved across this interface.
The polycrystalline materials are generally described by the grain boundary characteristics and crystallographic orientation of each constituent grain as well as its grain size [35]. Each grain boundary energy is determined by the misfit orientation angle between adjacent grains. The compatible grain boundary has low energy enough to stack some amount of dislocations; while the incompatible one has high energy enough to interact with dislocations [36]. In the metal forming of these polycrystalline materials or in the nitrogen supersaturation process, the dislocations as well as the slipping lines and planes interact with their grains and grain boundaries. In intense rolling, the grains are sheared and deformed to align their crystallographic orientation along the rolling direction and to form the textured microstructure. The applied elastoplastic distortion by rolling works to induce the phase transformation during rolling by high elastic strain energy density, to shear and elongate the grains by the plastic strains, and to spin the grain orientations toward the preferred one along the rolling direction. As reported in [37], the grain size is reduced down to sub-microns by repetitive rolling; due to small misorientation angles between adjacent grains, they are easy to agglomerate into larger grains by heat treatment.
In piercing, the grains near the shearing plane are affected by the applied elastoplastic distortion [38]. The increase of elastic strain energy density during piercing induces the phase transformation. The grains are sheared and fractured along the shearing plain. The original crystallographic structure of work materials is changed to align along the shearing plane by severe spinning with piercing. Although the grains are refined at the vicinity of shearing plain, the misorientation angles among them are small enough to be identified as nearly the same grain. Even in other metal forming processes than two in the above, their mechanical interactions of elastoplastic distortion with crystallographic structure is described by the strain-induced phase transformation, the shear deformation, the grain size refinement as well as the elastic recovery from the elasto-plastically strained state in unloading.
On the other hand, no elastoplastic distortion was directly applied to granular structure by the low temperature plasma nitriding. Instead of this direct straining, a large elastic distortion is induced into the nitrogen supersaturated zones in the work materials by lattice expansion. This distortion reaches to 10%, enough to drive the phase transformation in zones as well as the plastic distortion to compensate for the misfit on the zone boundaries between the nitrogen supersaturated and unsaturated ones. The symmetric component of this distortion works as a shearing strain tensor to form new slipping lines network across the original grain boundary. The asymmetric one drives spin-rotation in each zone to form the zone boundaries with high misorientation angles and to significantly refine the original grain size of work materials. Since those newly built-up zone boundaries play as a nitrogen diffusion path, this process advances concurrently with the nitrogen solute diffusion from the surface to the depth of materials. Since the zone size ranges in the nanometer order, the stainless steel work materials are covered by two-phase, nano-grained, nitrogen steel surface layer. The smallest zone size is determined by the mechanical balancing between the elastic straining in each material supercell and the slip-line formation surrounding it in the nitrogen supersaturation [39]. Precise TEM analyses down to the atomic scale are useful to describe the nitrogen interstitial atom distribution in the supercell [40].
Even when the rolled and pierced steel specimens are uniaxially loaded, their microstructure never changes themselves at room temperature before fracture. Their uniaxial stress-strain curves are determined by the original microstructure of as-rolled and as-pierced materials since no mechanical interaction occurs between the controlled grains by previous metal forming and the applied strain by uniaxial loading. On the other hand, the in situ microstructure evolution takes place during the uniaxial loading of the nitrided FGSS316 wire. The nitrogen supersaturated layer works a double role under this uniaxial tensile loading. The original FGSS316 matrix inside the wire is elastically supported by this nitrided layer. Since the austenitic grains are continuously linked with those in the nitrided layer, the matrix inside the wire is intensely elongated to change its microstructure to two-phase fibrous grains by the uniaxial loading. The microstructure in the as-nitrided layer is also affected by uniaxially applied plastic strains. The retained austenitic grains with large size in the nitrided layer change themselves to two-phase, fine grains, which are the same as homogeneously nitrided nanostructure before uniaxial loading. This in situ refinement of granular structure in the nitrided layer reflects on the hardness profile. The original hardness of as-nitrided layer is 1400 HV; this is further enhanced up to 1600 HV after uniaxial loading. This proves that nitrogen could diffuse locally to the retained austenitic zones along the slip-lines by externally applied plastic straining and drive the nitrogen supersaturation process in them for refinement of their microstructure. Owing to the elastic constraint by the nitrided layer, the work-hardening process during the uniaxial loading is enhanced in the wire matrix to attain higher ultimate stress (σU); e.g., σU = 1.23 GPa in the nitrided FGSS316 wire at δ = 5.7 mm or at ε = 0.19.
This in situ microstructure evolution by uniaxial loading posterior to nitriding, suggests further possibility of crystallographic control to improve the mechanical properties of metallic works, takes place in the nitrided layer and in the matrix inside without mutual interactions. The microstructure evolution of low temperature nitrided members and parts in the above must be enhanced during warm and hot processing. After recent work on the high carbon steel wire during drawing [41, 42], significant reduction of lamellar ferrite distance as well as free carbon dissociation from the cementite lamellar structure in the perlite colony and block are responsible for high strengthening of high carbon steel wires. This implies that further carbon supersaturation is a key process to drive the in situ evolution to the preferred crystallographic microstructure to higher strength of wire. Owing to the equivalent role between carbon and nitrogen solutes to be working as an interstitial atom in steel [43], local nitrogen mobility from nitrogen supersaturated zone with high nitrogen content to NS-zones with lower nitrogen content could drive the in situ nitrogen alloying process of wire matrix during warm-/hot-drawing and rolling.
In this nitriding a priori to metal forming, FGSS316 wire was first nitrided and then uniaxially loaded. How about the plasma nitriding of the rolled AISI304 plate? As shown in Figure 3, the nearly full-martensitic phase of rolled AISI304 plate changed to a mixture of nitrided austenitic and martensitic phases. Since the original martensitic and austenitic peak positions shift to the low angle of 2θ and their peak widths become significantly broad, this mixture composes of the fine grained austenitic and martensitic zones with nitrogen supersaturation.
EBSD was also employed to describe this microstructure change of rolled AISI304 plate after nitriding. As shown in Figure 16a, the textured structure of rolled AISI304 completely disappeared and changed to fine-grained structure without preferred crystallographic orientation. This change is driven by high plastic straining in Figure 16b; every original grains with and without textures by rolling is plastically strained and spin-rotated by the nitrogen supersaturation to form homogeneous fine-grained structure. As depicted in Figure 16c, this fine microstructure consists of two phase with the fraction of martensite by 70%. This dramatic crystallographic structure evolution proves that posterior nitriding to metal forming is useful to further control the microstructure of stainless steels.
Crystallographic structure on the cross-section of the rolled AISI304 plate after plasma nitriding at 673 K for 14.4 ks. (a) IPF mapping in the normal direction, (b) KAM distribution, and (c) phase mapping.
In the metal forming like intense rolling and fine piercing, the microstructure of work materials is changed by the applied plastic distortion with less influence to tool materials. In the rolling process, the original austenitic phase of stainless steels changes to be nearly full martensitic and to have textured microstructure with the preferred orientation to the rolling direction. This crystallographic structuring is intrinsic to the microstructure change by shearing with the reduction of thickness. In the piercing process by shear localization, the austenitic work material after piercing has new sheared and fractured surfaces including the affected zones. These zones consist of the phase-transformed martensite, the work-hardened austenite, and the elastically recovered zones. This crystallographic structure change is precisely described by EBSD on the cross-section of pierced work materials. In addition, various factors influence on this structure change including the grain size of work materials and the shear localization control as well as the chemical components in stainless steels.
In the low temperature plasma nitriding, no plastic strains are externally applied to work materials but nitrogen interstitial atoms are distributed from their surface to their depth with high concentration. Owing to the synergetic process in this inner nitriding process, the plastic distortion is concurrently induced by nitrogen diffusion and supersaturation. Since the nitrogen solute is homogeneously distributed in the nitrided layer with high content, the plastic distortion tensor also uniformly distributes in this layer. This homogeneous plastic distortion changes the normal crystallographic structure of AISI316 plates and wires; e.g., fine-grained AISI316 (FGSS316) microstructure of wires with the average grain size of 2 μm changes to the super-fine grained, two phase structure with the average grain size less than 0.1 μm. During this homogeneous nitriding, the retained austenitic zones distribute in the nitrided layer.
A priori nitriding to cold metal forming is a way to significantly control the microstructure and mechanical properties. The nitrided FGSS316 wire is elasto-plastically strained in the uniaxial direction so that the whole nitrided layers have fine-grained two-phase structure without retained austenite. This microstructure evaluation in local reflects on the homogeneous increase of hardness in the nitrided layer. This local interaction between nitrogen solute mobility and externally applied plastic strains at room temperature reveals that the microstructure and mechanical properties of nitrided work materials could be modified and improved by the metal forming posterior to the nitriding. In particular, the warm and hot post-treatment by drawing, rolling, forging, and stamping has capability to control the crystallographic structure of nitrided parts and components.
Posterior nitriding to metal forming is another way to refine the microstructure and to improve the mechanical properties. In case when the rolled AISI304 plate is further nitrided at 673 K, its textured microstructure completely changes to super-fine grained, two phase structure. The intense plastic straining by nitrogen supersaturation also plays a role to control the crystallographic structure.
The authors would like to express their gratitude to Mr. T. Inohara (LPS-Works, Co., Ltd.), Mr. T. Yoshino, and Y. Suzuki (Komatsu-Seiki Kosakusho, Co., Ltd.) for their help in experiments. This study was financially supported by the METI-Program on the Supporting Industries at 2019.
The authors declare no conflict of interest.
Gene multiplicity or redundancy is a characteristic of microorganisms and means there are two or more genes coding for proteins that perform the same function. Inactivation of any of these genes does not affect or has little relevance to the biological phenotype [1]. Gene redundancy has been observed in all organisms, including prokaryotes and eukaryotes, and is particularly important for actinobacteria that produce metabolites of industrial interest [2]. The most significant property of
One little-studied area is the carbon metabolism in these organisms. Few studies have examined the presence of genes that participate in the glycolytic pathway, TCA cycle, or phosphoenolpyruvate-pyruvate-oxaloacetate (PEP-PYR-OXA) node. In general, microorganisms metabolize glucose through the glycolysis and hexose monophosphate pathways [6]. Many of the intermediates in these metabolic pathways are used to synthesize other essential bacterial compounds (amino acids, polysaccharides, nucleic acids, lipids, fatty acids, and antibiotics).
The EMP pathway consists of nine reactions, in which the final product is pyruvate. The first reaction consists of the isomerization of glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. Another phosphate with adenosine triphosphate (ATP) as the donor is incorporated into fructose 6-phosphate by phosphofructokinase. The next step is the cleavage of fructose 1,6-diphosphate by aldolase, generating dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which can be interconverted by triose phosphate isomerase. Glyceraldehyde 3-phosphate is oxidized to 1,3-diphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase to generate NADH. The phosphoglycerate kinase-catalyzed reaction generates an ATP molecule, an example of substrate-level phosphorylation. The 3-phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase and is subsequently dehydrated by enolase. Phosphoenolpyruvate (PEP) is used to generate another molecule of ATP and PYR through a reaction catalyzed by pyruvate kinase. Then, four ATP molecules are generated, and two high-energy phosphate bonds are used; thus, the net gain is two ATPs per oxidized glucose molecule [6].
The TCA cycle is one of the most important metabolic pathways, not only as part of catabolism but also as an important intermediate for amino acid biosynthesis and synthesizing secondary metabolites. Generally, the citric acid cycle is the main oxidation pathway for carbon chains of carbohydrates, fatty acids, and many amino acids to CO2 and water. At each turn of the cycle, two molecules of CO2 are released. Most of the energy generated during oxidation is stored as NADH, FADH, or ATP (or GTP).
Because the intermediates of the TCA cycle are used as precursors in other pathways, they are replenished through anaplerotic reactions. Under normal conditions, the reactions that take intermediates in the cycle and those that replace them are kept in dynamic equilibrium; therefore, their concentrations remain constant. The most common anaplerotic reactions are those in which PYR or PEP are converted to Oxaloacetic acid (OXA) or malate. For example, this first reaction can be mediated by PEP carboxylase in some plants, yeasts, and bacteria or by the malic enzyme (ME), which is widely distributed in prokaryotes and eukaryotes [7].
Streptomycetes are bacteria that produce the largest amount of commercially used antibiotics worldwide. To date, many
Many antibiotics have precursors that act as intermediates for different metabolic pathways. Gunnarsson et al. (2004) described how central carbon metabolism is linked to producing many different antibiotics [9]; however, little has been achieved to improve the synthesis of inermediates and influence the biosynthesis of antibiotics or other commercially important compounds.
Genome sequencing of important antibiotic-producing
There are many databases, such as KEGG, to identify how many genes code for the same activity. It is known that nine enzymes participate in the glycolytic pathway, as shown in Table 1, and it was found that three genes encode phosphofructokinase, which catalyzes the phosphorylation of fructose 6-phosphate in most antibiotic-producing
SALS | SAVERM | SCLF | SCO | SGR | SHJGH | SKA | SLAV | SNOUR | SPRI | SVEN | |
---|---|---|---|---|---|---|---|---|---|---|---|
Antibiotic | Salinomycin | Avermectin | Clavulanic acid | Actinorhodin, Undecylprodigiosine | Streptomycin | Rapamycin | Kanamycin | Streptothricin | Nystatine | streptogramin | Chloramphenicol |
Phosphoglucomutase | SAVERM_803 (556) | SCO7443 (546) | SGR_6728 (547) | SHJGH_1760 (546) | |||||||
Phosphoglucose isomerase | SLNWT_5962 (550) | SAVERM_1770 (549) SAVERM_6302 (550) | BB341_22345 (550) | SCO1942 (551) SCO6659 (550) | SGR_5578 (552) | SHJGH_3162 (550) SHJGH_7334 (550) | CP970_33250 (550) | SLAV_27865 (553) | SNOUR_30680 (558) | SPRI_5587 (552) | SVEN_1571 (556) |
Phosphofructokinase | SLNWT_1861 (341) SLNWT_5764 (345) SLNWT_7260 (341) | SAVERM_2822 (341) SAVERM_6083 (342) SAVERM_7123 (341) | BB341_07185 (341) BB341_21445 (342) BB341_25335 (341) | SCO1214 (341) SCO2119 (342) SCO5426 (341) | SGR_2110 (341) SGR_6306 (341) | SHJGH_2417 (341) SHJGH_3367 (342) SHJGH_6262 (341) | CP970_13335 (341) CP970_32070 (342) CP970_41110 (341) | SLAV_12675 (341) SLAV_26605 (343) SLAV_31335 (342) | SNOUR_05765 (341) SNOUR_14515 (341) SNOUR_29760 (342) | SPRI_2465 (341) SPRI_5380 (342) SPRI_6334 (341) | SVEN_0823 (341) SVEN_5078 (341) |
Aldolase | SLNWT_0350 (285) SLNWT_3143 (343) | SAVERM_1445 (301) SAVERM_4523 (340) | BB341_12300 (283) BB341_15265 (340) | SCO3649 (343) SCO5852 (282) | SGR_285 (289) SGR_3418 (343) | SHJGH_2271 (286) SHJGH_5149 (340) | CP970_20370 (343) CP970_37965 (278) | SLAV_00910 (278) SLAV_20325 (340) SLAV_32740 (281) SLAV_38480 (278) | SNOUR_09120 (293) SNOUR_22110 (340) | SPRI_0508 (299) SPRI_3596 (340) | SVEN_3414 (340) SVEN_7329 (278) |
Triose phosphate isomerase | SLNWT_5959 (259) | SAVERM_6298 (258) | BB341_22330 (258) | SCO0578 (259) SCO1945 (258) | SGR_5575 (258) | SHJGH_3166 (258) | CP970_33235 (261) | SLAV_27850 (262) | SNOUR_30660 (258) | SPRI_5584 (262) | SVEN_1574 (259) |
Glyceraldehyde 3P-dehydrogenase | SLNWT_5957 (335) | SAVERM_2990 (334) SAVERM_6296 (335) | BB341_01000 (481) BB341_22320 (335) | SCO1947 (336) SCO7040 (481) SCO7511 (332) | SGR_5573 (336) SGR_936 (481) | SHJGH_1334 (337) SHJGH_1590 (332) SHJGH_1800 (481) SHJGH_3168 (335) | CP970_08935 (481) CP970_33225 (336) | SLAV_27840 (334) SLAV_31635 (481) SLAV_35550 (332) | SNOUR_30650 (334) SNOUR_35775 (481) | SPRI_5582 (336) SPRI_6547 (481) | SVEN_0459 (461) SVEN_1576 336 SVEN_7344 (331) |
Phosphoglycerate kinase | SLNWT_5958 (403) | SAVERM_6297 (403) | BB341_22325 (403) | SCO1946 (403) | SGR_5574 (403) | SHJGH_3167 (403) | CP970_33230 (403) | SLAV_27845 (403) | SNOUR_30655 (403) | SPRI_5583 (403) | SVEN_1575 (403) |
Phosphoglycerate mutase | SLNWT_3230 (511) | SAVERM_3979 (253) | BB341_12505 (252) BB341_19285 (217) | SCO4209 (253) SCO6818 (511) | SGR_4005 (253) | SHJGH_4634 (253) | CP970_23555 (253) | SLAV_17715 (252) | SNOUR_07945 (218) SNOUR_18235 (253) | SPRI_4058 (253) | SVEN_3958 (252) |
Enolase | SLNWT_1793 (427) | SAVERM_3533 (428) | BB341_17295 (426) | SCO3096 (426) SCO7638 (434) | SGR_4439 (426) SGR_6721 (434) | SHJGH_4327 (431) | CP970_25900 (428) | SLAV_05265 (435) SLAV_22580 (426) | SNOUR_24895 (426) | SPRI_0700 (432) SPRI_4452 (428) | SVEN_2899 (428) SVEN_6040 (433) |
Pyruvate kinase | SLNWT_1865 (474) SLNWT_5886 (478) | SAVERM_2825 (476) SAVERM_6217 (478) | BB341_07200 (474) BB341_22070 (477) | SCO2014 (478) SCO5423 (476) | SGR_2113 (476) SGR_5516 (479) | SHJGH_3253 (478) SHJGH_6259 (457) | CP970_13350 (476) CP970_32715 (478) | SLAV_12690 (476) SLAV_27525 (475) | SNOUR_14530 (475) SNOUR_30335 (480) | SPRI_2468 (475) SPRI_5515 (474) | SVEN_1640 (475) SVEN_5075 (476) |
Gene multiplicity in glycolytic pathway genes.
The conversion of fructose 6-phosphate to fructose 1,6-bisphosphate with the concomitant hydrolysis of adenosine triphosphate represents the first irreversible step specific to glycolysis. This reaction catalyzed by phosphofructokinase (PFK; EC 2.7.1.11) is subjected to tight control, thus rendering it a critical regulatory point of the glycolytic flux [10]. The genes that encode PFK present a high multiplicity, and in these 11 antibiotic-producing
Partial multiple alignments of fosfofructokinase proteins from antibiotic producing
The next reaction is performed using aldolase (EC 4.1.2.13), which catalyzes the conversion of fructose 1-6-diphosphate to glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. Most
Triosephosphate isomerase (EC 5.3.1.1) is an enzyme that converts dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Of all the antibiotic-producing
Another
Partial multiple alignment of glyceraldehyde 3-phosphate dehydrogenase proteins (A) in antibiotic producing
In all antibiotic-producing
Phosphosphoglycerate mutase (EC 5.4.2.11) performs the internal transfer of a phosphate group from the C-3 carbon to the C-2 carbon, resulting in the isomerization of 3-phosphoglycerate to 2-phosphoglycerate. Only two genes are involved in this activity in
Enolase (EC 4.2.1.11), also known as phosphopyruvate hydratase, is a metalloenzyme responsible for converting 2-phosphoglycerate to PEP.
Pyruvate kinase (EC 2.7.1.40) is a key enzyme involved in the last step of glycolysis that catalyzes the transfer of a phosphate group from PEP to ADP, yielding one molecule of PYR and one molecule of ATP. There are two types: type I and type II, and both enzymes show positive cooperative effects concerning PEP. The type I enzyme is activated by fructose 1,6-bisphosphate (F1,6BP) and the type II by AMP [13]. According to the amino acid sequences of PYKF and PYKA enzymes from
The TCA cycle is a central metabolic pathway of all aerobic organisms and synthesizes many important precursors and molecules [14] and eight reactions are performed for complete glucose oxidation.
The first reaction is citrate synthase (CS, EC 2.3.3.1), which catalyzes the irreversible conversion of OXA and acetyl-CoA into citrate. The proteins are classified into two types: type I and type II, and are encoded by four genes in eight of the selected antibiotic-producing
SALS | SAVERM | SCLF | SCO | SGR | SHJGH | SKA | SLAV | SNOUR | SPRI | SVEN | |
---|---|---|---|---|---|---|---|---|---|---|---|
Citrate synthase | SLNWT_1427 (439) SLNWT_1428 (422) SLNWT_4294 (369) SLNWT_5026 (434) | SAVERM_2427 (388) SAVERM_2428 (418) SAVERM_3859 (366) SAVERM_5330 (429) | BB341_05380 (375) BB341_05385 (432) BB341_11620 (363) BB341_18800 (429) | SCO2736 (429) SCO4388 (366) SCO5831 (421) SCO5832 (390) | SGR_1691 (381) SGR_1692 (432) SGR_3076 (367) SGR_4826 (432) | SHJGH_4003 (433) SHJGH_4518 (221) SHJGH_6697 (421) SHJGH_6698 (388) | CP970_11090 (395) CP970_11095 (417) Cp970_24400 (366) CP970_28100 (451) | SLAV_10725 1 (383) SLAV_10730 2 (416) SLAV_17155 I (368) SLAV_24055 I (433) | SNOUR_17670 (367) SNOUR_26625 (433) | SPRI_4216 (366) SPRI_4812 (429) | SVEN_2535 (433) SVEN_4205 (366) SVEN_5584 (377) |
Aconitase | SLNWT_0836 (904) | SAVERM_2258 (905) | BB341_04765 (908) | SCO5999 (904) | SGR_1506 (911) | SHJGH_6830 (905) | CP970_10270 (904) | SLAV_10035 (904) | SNOUR_12030 (904) | SPRI_1818 (905) | SVEN_5812 (910) |
Isocitrate dehydrogenase | SLNWT_6831 (739) | SAVERM_7214 (739) | BB341_23905 (739) | SCO7000(739) | SGR_1224 (740) | SHJGH_7521 (739) | CP970_35840 (739) | SLAV_07110 (739) | SNOUR_17135 (406) SNOUR_33110 (740) | SPRI_3067 (409) SPRI_6612 (739) | SVEN_0436 (741) |
2-oxoglutarate dehydrogenase E1 | SLNWT_2008 (1276) | SAVERM_2972 (1276) | BB341_08105 (1287) | SCO5281 (1272) | SGR_2226 (1267) | SHJGH_6150 (1170) | CP970_14080 (1294) | SLAV_13205 (1305) | SNOUR_15075 (1262) | SPRI_2599 (1272) | SVEN_4966 (1266) |
2-oxoglutarate dehydrogenase E2 (dihydrolipoamide succinyltransferase) | SLNWT_2008 | BB341_08105 | SCO1268 (372) SCO7123 (417) | SGR_2226 | SHJGH_6150 | SLAV_13205 | SNOUR_15075 | SPRI_2599 | SVEN_4966 | ||
2-oxoglutarate dehydrogenase E2 (dihydrolipoamide dehydrogenase) | SLNWT_5161 (467) | SAVERM_2154 (478) SAVERM_6024 (462) | BB341_21130 (462) | SCO2180 (486) | SGR_5330 (468) | SHJGH_6150 | CP970_31745 (462) CP970_41240 (467) | SLAV_26305 (462) SLAV_35195 (467) | SNOUR_29490 (462) SNOUR_37990 (466) | SPRI_0819 (466) SPRI_1688 (469) SPRI_5319 (462) | SVEN_1842 (501) SVEN_3731 (467) |
2-oxoglutarate ferredoxin oxidoreductase subunit beta | SLNWT_4515 (373) | SAVERM_4876 (359) | BB341_10730 (333) | SCO4594 (352) SCO6269 (350) | SGR_2930 (357) | SHJGH_5497 (353) | CP970_17695 (363) | SLAV_16235 (356) SLAV_39690 (467) | SNOUR_22995 (364) | SPRI_3245 (349) | SVEN_4303 (353) |
2-oxoglutarate/2-oxoacid ferredoxin oxidoreductase subunit alpha | SLNWT_4516 (644) | SAVERM_4877 (642) | BB341_10725 (597) BB341_25540 (597) | SCO4595 (645) SCO6270 (630) | SGR_2929 (654) | SHJGH_5498 (642) | CP970_17690 (643) | SLAV_16230 (649) SLAV_39695 (621) | SNOUR_23000 (650) | SPRI_3244 (648) | SVEN_4304 (653) |
Succiny CoA synthetase subunit β | SLNWT_4730 (392) | SAVERM_1818 (376) SAVERM_3452 (392) | BB341_10035 (391) | SCO4808 (394) SCO6585 (383) | SGR_2723 (393) | SHJGH_5672 (392) SHJGH_7288 (376) | CP970_05375 (374) CP970_16770 (392) | SLAV_15400 (392) | SNOUR_17190 (392) | SPRI_1230 (375) SPRI_3081 (393) | SVEN_4485 (424) |
Succiny CoA synthetase subunit α | SLNWT_4731 (305) | SAVERM_1817 (299) SAVERM_3451 (294) | BB341_10030 (294) | SCO4809 (294) SCO6586 (308) | SGR_2722 (294) | SHJGH_5673 (294) SHJGH_7289 (293) | CP970_05370 (290) CP970_16765 (294) | SLAV_15395 (295) | SNOUR_17185 (294) | SPRI_1229 (296) SPRI_3080 (294) | SVEN_4486 (294) |
Succinate dehydrogenase, iron-sulfur protein | SLNWT_4174 (248) SLNWT_4779 (244) | SAVERM_3182 (258) SAVERM_3398 (257) SAVERM_7309 (249) | BB341_01735 (248) BB341_08890 (255) BB341_09850 (252) | SCO0922 (248) SCO4855 (257) SCO5106 (259) | SGR_2690 (255) SGR_705 (248) | SHJGH_5739 (259) SHJGH_5963 (257) SHJGH_7644 (249) | CP970_03035 (248) CP970_15135 (256) CP970_16460 (255) | SLAV_06165 (246) SLAV_14090 (260) SLAV_15205 (252) | SNOUR_15960 (265) SNOUR_16955 (257) SNOUR_30130 (247) | SPRI_0753 (248) SPRI_2773 (256) SPRI_3031 (252) | SVEN_4531 (252) SVEN_4752 (257) SVEN_6837 (249) |
Succinate dehydrogenase, flavoprotein subunit | SLNWT_4173 (652) SLNWT_4780 (584) | SAVERM_3181 (667) SAVERM_3397 (584) SAVERM_7308 (649) | BB341_08885 (667) BB341_09845 (584) | SCO0923 (649) SCO4856 (584) SCO5107 (653) SCO7109 (576) | SGR_2689 (584) SGR_706 (649) | SHJGH_5740 (584) SHJGH_5964 (651) SHJGH_7643 (649) | CP970_03040 (652) CP970_15130 (656) CP970_16455 (584) | SLAV_06170 (650) SLAV_14085 (633) SLAV_15200 (584) | SNOUR_15955 (640) SNOUR_16950 (584) SNOUR_30135 (648) | SPRI_0754 (647) SPRI_2772 (647) SPRI_3030 (584) | SVEN_4532 (584) SVEN_4753 (636) SVEN_6836 (648) |
Succinate dehydrogenase hydrophobic membrane anchor protein | SLNWT_4782 (161) | SAVERM_3396 (160) | BB341_09840 (158) | SCO4857 (160) | SGR_2688 (160) | SHJGH_5741 (160) | CP970_16450 (163) | SLAV_15195 (160) | SNOUR_16945 (154) | SPRI_3029 (158) | SVEN_4533 (159) |
succinate dehydrogenase cytochrome β-556 subunit | SLNWT_4172 (223) SLNWT_4781 (110) | SAVERM_3395 (126) SAVERM_7307 (235) | BB341_01745 (207) BB341_09835 (144) | SCO0924 (243) SCO4858 (126) | SGR_707 (234) SGR_2687 (126) | SHJGH_5742 (110) SHJGH_7642 (223) | CP970_03045 (235) CP970_16445 (126) | SLAV_06175 (241) SLAV_15190 (126) | SNOUR_16940 (126) SNOUR_30140 (278) | SPRI_0755 (234) SPRI_3028 (126) | SVEN_4534 (126) SVEN_6835 (208) |
Fumarate hydratase Class I | SLNWT_2307 (554) | SAVERM_3218 (558) | BB341_08995 (562) | SCO5044 (558) | SGR_2481 (558) | SHJGH_5903 (534) | CP970_15380 (558) | SLAV_14315 (559) | SNOUR_16110 (558) | SPRI_2823 (555) | SVEN_4713 (556) |
Fumarate hydratase, class II | SLNWT_2312 (473) | SAVERM_3221 (467) | SCO5042 (461) | SGR_2491 (471) | SHJGH_5902 (461) | CP970_15390 (470) | SLAV_14340 (467) | SNOUR_16150 (464) | SVEN_4708 (469) | ||
Malate dehydrogenase | SLNWT_4746 (329) | SAVERM_3436 (329) | BB341_09975 (329) | SCO4827 (329) | SGR_2711 (329) | SHJGH_5712 (329) | CP970_16700 (329) | SLAV_15340 (329) | SNOUR_17130 (329) | SPRI_3065 (329) | SVEN_4498 (329) |
Gene multiplicity in TCA cycle genes.
Partial multiple alignment of citrate synthase proteins (A) in antibiotic producing
Aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), the E1 component of 2-oxoglutarate dehydrogenase (EC 1.2.4.2), and malate dehydrogenase (EC 1.1.1.37) are encoded by unique genes in all
The 2-oxoglutarate dehydrogenase complex is a central enzyme in aerobic metabolism that catalyzes the oxidative decarboxylation of oxoglutarate, generating NADH [16]. 2-oxoglutarate dehydrogenase is composed of three subunits, E1 (EC 1.2.4.2), E2 (EC 2.3.1.61, dihydrolipoamide succinyltransferase), and E3 (EC 1.8.1.4; dihydrolipoamide dehydrogenase). As previously mentioned, there is a single gene coding for component E1 in all selected antibiotic-producing
Multiple alignments of the amino acid sequences of the E2 subunit of 2-oxoglutarate dehydrogenase showed close resemblance, with many highly conserved amino acids within the conserved domains, such as the YDHR region, which is part of the 2-oxoacid dehydrogenase acyltransferase catalytic domain. The proteins encoded by
Multiple alignment of 2-oxoglutarate dehydrogenase component E2 (A) in antibiotic producing
There is an alternative way to perform the synthesis of 2-oxoglutarate via 2-oxoglutarate ferredoxin oxidoreductase (EC 1.2.7.11) formed by two subunits called a and b.
The next reaction is catalyzed by succinyl-CoA synthetase (EC 6.2.1.5), which is also composed of two protein subunits: α and β.
Succinate dehydrogenase (SDH; EC 1.3.5.1, EC 1.3.5.4), which catalyzes the oxidation of succinic acid to generate fumaric acid is a four-subunit multimeric enzyme (iron-sulfur protein, flavoprotein subunit, hydrophobic membrane anchor protein, and cytochrome b-556 subunit). The number of genes that code for each subunit varies, and even the same genome
The genes that code for iron-sulfur protein have great redundancy, finding three copies in all antibiotic-producing
It has been found that there are three different flavoprotein proteins, some with 649–652 amino acids with identities greater than 90%, another group of proteins with 584 amino acids and identities between 91 and 93%, and the last group with 633–667 residues and identities greater than 80%. The flavoproteins SCO7109 and SHJGH_5964 do not resemble each other or the proteins of the previous groups.
The hydrophobic membrane anchor proteins are smaller than the previous ones, around 17 kDa, and present identities between 77% and 82%, while there are two types of cytochrome b subunit, one of around 223–243 amino acids with lower identities than the previous ones, between 58% and 71%. The second type includes proteins of smaller size and identity between 74% and89%.
The hydration reaction of fumarate to generate malate is catalyzed by fumarate hydratase (EC 4.2.1.2; fumarase). There are two classes of proteins: fumarase classes I and II. Most antibiotic-producing
The final TCA cycle reaction is catalyzed by malate dehydrogenase, which catalyzes the reversible conversion of malate to OXA using NAD+ or NADP+ as the coenzyme [17], which is only encoded by a single gene in all microorganisms that produce different antibiotics. The multiple sequence alignment of this enzyme showed a high degree of conservation between them, distributed in two main clades from a common ancestor, which was then subdivided into eight subclades (Figure 5). The identity is higher than 90%.
Partial multiple alignment of malate dehydrogenase proteins (A) in antibiotic producing
The PEP-PYR-OXA node is a major branch of central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle [7]. A large variety of enzymes involved in the node have been reported, such as PEP carboxylase (EC 4.1.1.31), pyruvate carboxylase (EC 6.4.1.1), PEP carboxykinase (EC 4.1.1.32), malic enzymes (EC 1.1.1.38), pyruvate kinase (EC 2.7.1.40), and pyruvate phosphate dikinase (EC 2.7.9.1). These enzymes are indispensable for distributing PEP, PYR, and OXA in
This anaplerotic pathway does not present multiplicity in any of the genes that encode the enzymes participating in the PEP-PYR-OXA node, except for malic enzymes and pyruvate phosphate dikinase as shown in Table 3. PYR carboxylase is an enzyme that is present only in
SALS | SAVERM | SCLF | SCO | SGR | SHJGH | SKA | SLAV | SNOUR | SPRI | SVEN | |
---|---|---|---|---|---|---|---|---|---|---|---|
Pyruvate carboxylase | SLNWT_3899 (1124) | SCO0546 (1124) | SHJGH_7997 (1124) | SPRI_1970 (1124) | |||||||
Phosphoenol pyruvate carboxylase | SLNWT_4462 (912) | SAVERM_3566 (910) | BB341_17140 (909) | SCO3127 (911) | SGR_4379 (909) | SHJGH_4361 (910) | CP970_25710 (917) | SLAV_22415 (920) | SNOUR_24730 (921) | SPRI_4413 (909) | SVEN_2951 (909) |
NAD-Malic enzyme | SLNWT_2497 (476) | SAVERM_1514 (587) SAVERM_3870 (573) SAVERM_5126 (477) | BB341_17990 (476) | SCO2951 (471) | SGR_4581 (478) | SHJGH_4187 (471) SHJGH_4528 (570) | CP970_26965 (477) | SLAV_23225 (474) | SNOUR_25740 (477) | SPRI_4590 (477) | SVEN_2718 (476) |
NADP-Malic enzyme | SLNWT_2020 (407) | SAVERM_2981 (407) | BB341_08155 (412) | SCO5261 (409) | SGR_2236 (413) | SHJGH_6136 (407) | CP970_14130 (407) | SLAV_13290 (400) | SNOUR_15125 (409) | SPRI_2612 (403) | SVEN_4951 (403) |
Phosphoenol pyruvate carboxykinase | SLNWT_4964 (609) | SAVERM_3287 (607) | BB341_09370 (621) | SCO4979 (609) | SGR_2556 (608) | SHJGH_5842 (607) | CP970_15680 (623) | SLAV_14715 (613) | SNOUR_16390 (605) | SPRI_2904 (606) | SVEN_4658 (607) |
pyruvate phosphate dikinase | SLNWT_5381 (902) | SAVERM_5654 (916) | BB341_01330 (608) BB341_19830 (905) | SCO0208 (898) SCO2494 (909) | SGR_5048 (903) | SHJGH_3733 (906) SHJGH_8556 (895) | CP970_29700 (896) | SLAV_24965 (933) | SNOUR_27780 (911) | SPRI_5017 (903) | SVEN_2286 (934) |
Gene multiplicity in PEP-PYR-OXA node.
PEP carboxylase, an enzyme that catalyzes the carboxylation of PEP to OXA, is encoded by a single gene and is present in all antibiotic-producing
PEP carboxykinase, an enzyme with the opposite action to the previous ones, decarboxylates OXA to form PEP. This enzyme could be considered gluconeogenic; however, it is also part of the PEP-PYR-OXA node for distributing the carbon flux between the different central pathways of metabolism [19]. Its molecular weight is approximately 67 kDa, some of which are a few amino acids long. In the same way as the previous enzymes,
MEs catalyzes the oxidative decarboxylation of l-malate to produce PYR and CO2, coupled with the reduction of NAD(P)+ cofactors (EC 1.1.1.38, EC 1.1.1.40). In general, NAD+- dependent MEs function to provide PYR for the TCA cycle, whereas NADP+- dependent MEs function to generate NADPH for anabolic reactions. Bacterial ME isoforms are comparatively understudied and collectively demonstrate greater structural and functional diversity (ranging from “minimal” 40 kDa subunits to much larger 85 kDa multidomain proteins). The greater bacterial ME complexity arises from the need for allosteric regulation owing to the non-compartmentalization of the bacterial cell [20].
The genes coding for MEs in these antibiotic-producing
Pyruvate phosphate dikinase (EC 2.7.11.32) converts PEP, inorganic pyrophosphate, and AMP into PYR, inorganic phosphate, and ATP. This protein is a gluconeogenic and anaplerotic enzyme and, in some bacteria, it plays other important roles. This protein is associated with virulence in
Finally, the genes that encode all enzymes of the glycolytic pathway, the TCA cycle, and the PEP-PYR-OXA node are distributed throughout the genome and represent up to 50 kb that might not be needed. Although many genes are found in the core, others are also found in the arms, and the question arises as to whether all genes are expressed and translated. For example, four genes that encode CS are transcribed to generate functional proteins? Viollier et al. (2001) reported that the deletion of the
As mentioned before, antibiotics are synthesized from precursors that are intermediates from carbon metabolism. By providing an overview of the gene multiplicity and determining which enzymes are encoded by a single gene, it will be possible to design strategies aimed at the sufficient biosynthesis of precursors for the metabolism of microorganisms to satisfy the demand for these same compounds to form antibiotics.
On the other hand, the growth of
Genetic multiplicity is present in all antibiotic-producing
This study was partially supported by grant IN214116 (DGAPA-UNAM).
The authors declare no conflict of interest.
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Catarina Guedes and F. Xavier Malcata",authors:[{id:"83136",title:"Prof.",name:"F. Xavier",middleName:null,surname:"Malcata",slug:"f.-xavier-malcata",fullName:"F. Xavier Malcata"}]},{id:"30642",doi:"10.5772/34423",title:"Meiofauna as a Tool for Marine Ecosystem Biomonitoring",slug:"meiofauna-as-a-tool-for-marine-ecosystem-monitoring",totalDownloads:3912,totalCrossrefCites:22,totalDimensionsCites:84,abstract:null,book:{id:"1689",slug:"marine-ecosystems",title:"Marine Ecosystems",fullTitle:"Marine Ecosystems"},signatures:"Maria Balsamo, Federica Semprucci, Fabrizio Frontalini and Rodolfo Coccioni",authors:[{id:"100075",title:"Prof.",name:"Maria",middleName:null,surname:"Balsamo",slug:"maria-balsamo",fullName:"Maria Balsamo"},{id:"104309",title:"Dr.",name:"Federica",middleName:null,surname:"Semprucci",slug:"federica-semprucci",fullName:"Federica Semprucci"},{id:"104311",title:"Dr.",name:"Fabrizio",middleName:null,surname:"Frontalini",slug:"fabrizio-frontalini",fullName:"Fabrizio Frontalini"},{id:"104313",title:"Prof.",name:"Rodolfo",middleName:null,surname:"Coccioni",slug:"rodolfo-coccioni",fullName:"Rodolfo Coccioni"}]},{id:"35136",doi:"10.5772/29571",title:"Transmission Biology of the Myxozoa",slug:"transmission-biology-of-the-myxozoa",totalDownloads:2726,totalCrossrefCites:35,totalDimensionsCites:64,abstract:null,book:{id:"2052",slug:"health-and-environment-in-aquaculture",title:"Health and Environment in Aquaculture",fullTitle:"Health and Environment in Aquaculture"},signatures:"Hiroshi Yokoyama, Daniel Grabner and Sho Shirakashi",authors:[{id:"78409",title:"Dr.",name:"Hiroshi",middleName:null,surname:"Yokoyama",slug:"hiroshi-yokoyama",fullName:"Hiroshi Yokoyama"},{id:"83562",title:"Dr.",name:"Daniel",middleName:"Stefan",surname:"Grabner",slug:"daniel-grabner",fullName:"Daniel Grabner"},{id:"122643",title:"Dr.",name:"Sho",middleName:null,surname:"Shirakashi",slug:"sho-shirakashi",fullName:"Sho Shirakashi"}]},{id:"24078",doi:"10.5772/26795",title:"Photobacterium damselae subsp. damselae, an Emerging Pathogen Affecting New Cultured Marine Fish Species in Southern Spain",slug:"photobacterium-damselae-subsp-damselae-an-emerging-pathogen-affecting-new-cultured-marine-fish-speci",totalDownloads:3795,totalCrossrefCites:19,totalDimensionsCites:45,abstract:null,book:{id:"612",slug:"recent-advances-in-fish-farms",title:"Recent Advances in Fish Farms",fullTitle:"Recent Advances in Fish Farms"},signatures:"A. Labella, C. Berbel, M. Manchado, D. Castro and J.J. Borrego",authors:[{id:"67855",title:"Prof.",name:"Juan J.",middleName:null,surname:"Borrego",slug:"juan-j.-borrego",fullName:"Juan J. 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This chapter will discuss an innovation in seaweed cultivation of the genus Eucheuma, which is the prime marine commodity in the tropical regions of the world. Research conducted during 2015-2017 and 2019 in Southeast Sulawesi Province, Indonesia, provided an overview of the use of floating cage that showed very significant growth results. The research result showed that the growth rates of Eucheuma denticulatum and Kappaphycus alvarezii in floating cage seemed faster and resulted in better thallus morphology. Daily production of E. denticulatum and K. alvarezii that were cultivated in floating cage was higher than daily production of E. denticulatum and K. alvarezii cultivated on longline. Specific growth rate (SGR) of E. denticulatum and K. alvarezii cultivated by using floating cage method was also higher than E. denticulatum and K. alvarezii cultivated by using longline method. Moreover, the cultivation by using floating cages produces good growth rates with no effect of herbivore attacks.",book:{id:"8928",slug:"emerging-technologies-environment-and-research-for-sustainable-aquaculture",title:"Emerging Technologies, Environment and Research for Sustainable Aquaculture",fullTitle:"Emerging Technologies, Environment and Research for Sustainable Aquaculture"},signatures:"Ma’ruf Kasim, Abdul Muis Balubi, Ahmad Mustafa, Rahman Nurdin, Rahmad Sofyan Patadjai and Wardha Jalil",authors:[{id:"309893",title:"Prof.",name:"Maruf",middleName:null,surname:"Kasim",slug:"maruf-kasim",fullName:"Maruf Kasim"},{id:"313040",title:"MSc.",name:"Abdul Muis",middleName:null,surname:"Balubi",slug:"abdul-muis-balubi",fullName:"Abdul Muis Balubi"},{id:"313041",title:"MSc.",name:"Wardha",middleName:null,surname:"Jalil",slug:"wardha-jalil",fullName:"Wardha Jalil"},{id:"313042",title:"MSc.",name:"Ahmad",middleName:null,surname:"Mustafa",slug:"ahmad-mustafa",fullName:"Ahmad Mustafa"},{id:"313043",title:"MSc.",name:"Rahman",middleName:null,surname:"Nurdin",slug:"rahman-nurdin",fullName:"Rahman Nurdin"},{id:"313044",title:"MSc.",name:"Rahmat Sofyan",middleName:null,surname:"Patadjai",slug:"rahmat-sofyan-patadjai",fullName:"Rahmat Sofyan Patadjai"}]},{id:"62842",title:"Integrated Rice and Aquaculture Farming",slug:"integrated-rice-and-aquaculture-farming",totalDownloads:1919,totalCrossrefCites:2,totalDimensionsCites:3,abstract:"The burning problems like scarcity of food for ever-growing human population in the present world are addressed by adapting various methods for production of protein, carbohydrate, oils and other food materials. One of the methods to produce high amount of food is integrated farming including rice-aquaculture farming, which produces protein and carbohydrate as major components besides others. Rice-aquaculture farming produces grain (carbohydrate) and animal protein without affecting the quality and quantity of rice yield on the same piece of land and renders additional financial gain besides main crop (rice) like conventional monoculture. The aquatic species grown in the integrated culture are mainly distinct types of fishes, selected crustaceans and other selected species. Profitable rice-aquaculture integrated farming is popular in Asian countries than in Western countries. However, the integrated rice-aquaculture farming has its own limitations. The type of methods, culture species, influencing factors, and pros and cons of rice-aquaculture integrated farming are discussed in the present chapter.",book:{id:"7229",slug:"aquaculture-plants-and-invertebrates",title:"Aquaculture",fullTitle:"Aquaculture - Plants and Invertebrates"},signatures:"Pamuru Ramachandra Reddy and Battina Kishori",authors:[{id:"242524",title:"Dr.",name:"Ramachandra Reddy",middleName:null,surname:"Pamuru",slug:"ramachandra-reddy-pamuru",fullName:"Ramachandra Reddy Pamuru"},{id:"255022",title:"Dr.",name:"Kishori",middleName:null,surname:"Battina",slug:"kishori-battina",fullName:"Kishori Battina"}]},{id:"24074",title:"Embryonic and Larval Development of Freshwater Fish",slug:"embryonic-and-larval-development-of-freshwater-fish",totalDownloads:7466,totalCrossrefCites:1,totalDimensionsCites:2,abstract:null,book:{id:"612",slug:"recent-advances-in-fish-farms",title:"Recent Advances in Fish Farms",fullTitle:"Recent Advances in Fish Farms"},signatures:"Faruk Aral, Erdinç Şahınöz and Zafer Doğu",authors:[{id:"25600",title:"Prof.",name:"Faruk",middleName:null,surname:"Aral",slug:"faruk-aral",fullName:"Faruk Aral"},{id:"29132",title:"Dr.",name:"Zafer",middleName:null,surname:"Dogu",slug:"zafer-dogu",fullName:"Zafer Dogu"},{id:"39952",title:"Dr.",name:"Erdinc",middleName:null,surname:"Sahinoz",slug:"erdinc-sahinoz",fullName:"Erdinc Sahinoz"}]},{id:"68966",title:"Novel Biofloc Technology (BFT) for Ammonia Assimilation and Reuse in Aquaculture In Situ",slug:"novel-biofloc-technology-bft-for-ammonia-assimilation-and-reuse-in-aquaculture-in-situ",totalDownloads:1951,totalCrossrefCites:2,totalDimensionsCites:8,abstract:"Ammonia is one of the most harmful risks for success of fish and shrimp culture. There is no effective solution for harmlessness of ammonia in traditional aquaculture operations except exchanging water, which would bring negative effects on environment, or fixing expensive equipment. Biofloc technology (BFT) that appeared in recent years supplies a novel solution for this issue without exchanging huge water and fixing equipment. This technology could assimilate ammonia almost in real time with many other supplemental benefits. Because of the very high nutritional value for fish and shrimp, bioflocs, the by-product of BFT, could also be reused as a complemented food in situ or a gradient for feedstuff to replace expensive fishmeal or be processed to pellet diet to feed fish and shrimp directly. However, some aspects with regard to the effective use of biofloc as a food source for fish and shrimp, such as high lipid content, productivity, and palatability, need to be further researched in detail.",book:{id:"8928",slug:"emerging-technologies-environment-and-research-for-sustainable-aquaculture",title:"Emerging Technologies, Environment and Research for Sustainable Aquaculture",fullTitle:"Emerging Technologies, Environment and Research for Sustainable Aquaculture"},signatures:"Hai-Hong Huang",authors:[{id:"305215",title:"Dr.",name:"Hai-Hong",middleName:null,surname:"Huang",slug:"hai-hong-huang",fullName:"Hai-Hong Huang"}]}],onlineFirstChaptersFilter:{topicId:"32",limit:6,offset:0},onlineFirstChaptersCollection:[],onlineFirstChaptersTotal:0},preDownload:{success:null,errors:{}},subscriptionForm:{success:null,errors:{}},aboutIntechopen:{},privacyPolicy:{},peerReviewing:{},howOpenAccessPublishingWithIntechopenWorks:{},sponsorshipBooks:{sponsorshipBooks:[],offset:8,limit:8,total:0},allSeries:{pteSeriesList:[{id:"14",title:"Artificial Intelligence",numberOfPublishedBooks:9,numberOfPublishedChapters:90,numberOfOpenTopics:6,numberOfUpcomingTopics:0,issn:"2633-1403",doi:"10.5772/intechopen.79920",isOpenForSubmission:!0},{id:"7",title:"Biomedical Engineering",numberOfPublishedBooks:12,numberOfPublishedChapters:107,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2631-5343",doi:"10.5772/intechopen.71985",isOpenForSubmission:!0}],lsSeriesList:[{id:"11",title:"Biochemistry",numberOfPublishedBooks:33,numberOfPublishedChapters:330,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2632-0983",doi:"10.5772/intechopen.72877",isOpenForSubmission:!0},{id:"25",title:"Environmental Sciences",numberOfPublishedBooks:1,numberOfPublishedChapters:19,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2754-6713",doi:"10.5772/intechopen.100362",isOpenForSubmission:!0},{id:"10",title:"Physiology",numberOfPublishedBooks:14,numberOfPublishedChapters:145,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-8261",doi:"10.5772/intechopen.72796",isOpenForSubmission:!0}],hsSeriesList:[{id:"3",title:"Dentistry",numberOfPublishedBooks:9,numberOfPublishedChapters:140,numberOfOpenTopics:2,numberOfUpcomingTopics:0,issn:"2631-6218",doi:"10.5772/intechopen.71199",isOpenForSubmission:!0},{id:"6",title:"Infectious Diseases",numberOfPublishedBooks:13,numberOfPublishedChapters:123,numberOfOpenTopics:4,numberOfUpcomingTopics:0,issn:"2631-6188",doi:"10.5772/intechopen.71852",isOpenForSubmission:!0},{id:"13",title:"Veterinary Medicine and Science",numberOfPublishedBooks:11,numberOfPublishedChapters:112,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2632-0517",doi:"10.5772/intechopen.73681",isOpenForSubmission:!0}],sshSeriesList:[{id:"22",title:"Business, Management and Economics",numberOfPublishedBooks:1,numberOfPublishedChapters:22,numberOfOpenTopics:3,numberOfUpcomingTopics:0,issn:"2753-894X",doi:"10.5772/intechopen.100359",isOpenForSubmission:!0},{id:"23",title:"Education and Human Development",numberOfPublishedBooks:0,numberOfPublishedChapters:11,numberOfOpenTopics:1,numberOfUpcomingTopics:1,issn:null,doi:"10.5772/intechopen.100360",isOpenForSubmission:!0},{id:"24",title:"Sustainable Development",numberOfPublishedBooks:1,numberOfPublishedChapters:19,numberOfOpenTopics:5,numberOfUpcomingTopics:0,issn:"2753-6580",doi:"10.5772/intechopen.100361",isOpenForSubmission:!0}],testimonialsList:[{id:"6",text:"It is great to work with the IntechOpen to produce a worthwhile collection of research that also becomes a great educational resource and guide for future research endeavors.",author:{id:"259298",name:"Edward",surname:"Narayan",institutionString:null,profilePictureURL:"https://mts.intechopen.com/storage/users/259298/images/system/259298.jpeg",slug:"edward-narayan",institution:{id:"3",name:"University of Queensland",country:{id:null,name:"Australia"}}}},{id:"13",text:"The collaboration with and support of the technical staff of IntechOpen is fantastic. 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