Effect of acid-base biomass hydrolysates on cell growth and PHB formation
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In moving towards sustainable manufacturing with reduced carbon footprint, bio-based fuels, chemicals and materials produced from renewable resources have attracted great interest. Microbial cells, in working with other chemical and enzymatic catalysts, are often used in the conversion of feedstocks to desired products, involving different species of bacteria, yeast, filamentous fungi, and microalgae. A substantial amount of microbial biomass is generated in the industrial fermentations and often discarded as a waste. Because of a high cost associated with growth and disposal of the cell mass, reusing the microbial biomass may be an attractive alternative to waste disposal. In contrast to the biomass as energy storage (e.g. starch and oil) or plant structure (e.g. cellulose and hemi-cellulose), microbial biomass is biologically active, consisting primarily of proteins (10-60 wt%), nucleic acid (1-30 wt%), and lipids (1-15%) [1]. Few cases of reusing microbial biomass exist in industrial processes.
Poly(3-hydroxybutyrate) (PHB) is a representative polyhydroxyalkanoate (PHA) that is formed by many bacterial species as carbon and energy reserve [2,3]. Although the biopolyesters made from renewable feedstocks have the potential of replacing petroleum-based thermoplastics in many environmentally friendly applications, they are not widely accepted in the markets because of the high production cost [4]. Extensive research has been conducted to use cheap feedstocks [5,6], develop high cell density fermentation technology for high PHA productivity [7,8], and improve microbial strains that exhibit good performance under high osmotic pressure and environmental stress [9-11]. One major cost factor of PHA production is the recovery and purification of biopolyester for desired purity and material properties [4, 12]. Depending on strains and culture conditions, biopolyester may account for 50-80 wt% of cell mass [13]. They are stored in microbial cells as tiny amorphous granules [0.2-0.5 μm in diameter] and need a sophisticated treatment to separate them from the residual cell mass [14-16]. Two technologies, based-on solvent extraction or biomass dissolution, are usually adopted in PHA recovery. With solvent extraction, the PHA granules are dissolved in appropriate organic solvents, leaving cells or residual biomass intact [17,18]. With cell mass dissolution, the PHA granules are left intact while the non-PHA cell mass is decomposed and dissolved in aqueous solutions with help of biological and/or chemical agents [19,20]. Following either treatment, PHA and non-PHA cell mass can be separated with conventional solid/liquid separations. Separating biopolyester from the cells would generate a substantial amount of residual microbial biomass, 0.25 to 1 kg dry mass per kg of PHA resin, depending on the initial PHA content. As a mixture of proteins, nucleic acids, lipids, and wall fragments, the residual biomass has no market value and is discarded at an extra disposal cost.
According to the microbial structure and cellular composition [1], the residual microbial biomass is actually consisting of true biological compounds formed during cell growth while PHA is just a carbon storage material. In a conventional PHA fermentation, sufficient substrates and nutrients (C, N, P, minerals and some organic growth factors) are supplied to grow enough cell mass that in turn or simultaneously synthesize biopolyester from carbon substrates. A large portion of organic carbons and nutrients are therefore consumed to generate new cell mass that is going to be discarded as a solid waste after polymer recovery. Ideally, the residual biomass should be reused by microbial cells to generate new cell mass and/or PHA polymers. This would not only reduce the cost of waste treatment and disposal, but also save the cost of nutrients in PHA fermentation. The bacterial cells, however, cannot assimilate their cell mass because they lack appropriate enzymes to break down various biological macromolecules and their complex structure such as cellular walls and membranes [19]. If the cells or mutants from genetic engineering could easily assimilate their structural components, they might not be suitable to industrial PHA production because the cells would undergo autolysis under high environmental stress. It is highly possible, however, to make the residual biomass reusable during PHA recovery in which the non-PHA cell mass is decomposed and hydrolyzed in aqueous solutions [20]. Integration of PHA recovery with reusing of residual biomass in microbial PHA fermentation is a novel and challenging technology. This work shows the generation of biomass hydrolysates in a downstream PHB recovery and the beneficial utilization of the hydrolysates in cell growth and PHB formation.
A laboratory strain of
A large amount of PHB-containing cell mass for biopolyester recovery was produced with a fed-batch culture in a 3L bench-top bioreactor (BioFlo 110, New Brunswick Scientific Co. NJ). The temperature, pH and dissolved oxygen were controlled at 30 oC, 6.8, and 10% air situation, respectively. A feed solution prepared with a sugar manufacturing byproduct containing about 50% sucrose was introduced into the bioreactor till the cell density reached 130 g/L and PHB concentration 94 g/L (72% PHB of cell mass). The cell mass was harvested with centrifugation and re-suspended in an acidic water (0.2M H2SO4) to make a cell slurry of 278 g dry mass/L. The slurry was reserved for later use.
The biopolyester was recovered and purified by dissolving the non-PHA cell mass (28% w/w) in sequential treatments consisting of acid pretreatment, base treatment, hypochlorite whitening, washing and drying [22].
Acid pretreatment: The cell mass in acidic solution was heated to boil and maintained for one hour under ambient conditions. The pretreated cellular solids were cooled to room temperature and separated from acid solution with centrifugation at 5,000 g for 10 min. The dissolved microbial biomass in the supernatant solution is referred to acid hydrolysates. The insoluble wet pellets were subjected to next base treatment.
Base treatment: The insoluble solids from the acid pretreatment were re-suspended in an equivalent volume of water to form slurry of about 200 g DM/L. The solution pH was raised to 10 to 11 with 5 M NaOH solution and stirred under ambient conditions for 30 to 60 min. A small amount of surfactants such as sodium dodecylsulfate (SDS, CH3(CH2)11OSO3Na) might be added to a concentration of 5 to 10 g/L. The slurry was then heated to boiling and maintained for 10 min under ambient conditions. After centrifugation, the dissolved biomass in the supernatant solution is referred to base hydrolysates. The sequential acid and base treatments above could also be performed without separation of the acid hydrolysates. In this case, sodium hydroxide was directly added into the acidic cell slurry and the pH was raised to 10-11 for base treatment. After centrifugation, the supernatant solution contained the hydrolysates generated from both acid and base treatments and is refereed to acid-base hydrolysates.
Whitening and washing: The insoluble wet pellets from base treatment were re-suspended in a commercially available bleaching solution containing 6% w/w of hypochlorite. The slurry was stirred for 1 to 2 hours under ambient conditions. The white PHB pellets were recovered with centrifugation. A small amount of biomass was dissolved and mineralized in the bleaching solution because of chemical oxidation, which is not considered for reuse in this work. The wet PHA pellets were washed two times with water and dried in oven. The final PHB product is a white powder.
The dissolution of non-PHA cell mass was monitored by measuring the characteristic absorption of amino acid residues at 280 nm with a UV/VIS spectrophotometer (Beckman Coulter DU530, Fullerton, CA). The concentration of proteins that can be stained in Bradford assay was measured with the spectrophotometer after protein-dye binding [23]. The content of PHB in original cell mass, in sequential treatments, and final product were determined via methanolysis of the biopolyester in methanol (3 wt% sulfuric acid) at 100 oC for 8-10 hours [24]. The 3-hydroxybutyric methyl ester was hydrolyzed into 3-hydroxybutyric acid when the solution pH was raised to 11 with a 10N NaOH solution. The liquid samples were analyzed using an HPLC equipped with a UV detector (Shimadzu, Japan) and an organic acid column (OA-1000, Alltech, Deerfield, IL). The column was maintained at 65 oC and eluted with a water-sulfuric acid solution (pH 2) at 0.8 mL/min. The monomeric acid and crotonic acid, a trace byproduct formed in methanolysis, were detected at 210 nm. For data quality control, the biopolyester was also extracted from the freeze-dried cell mass in hot chloroform followed by precipitation with methanol [21]. The PHB content was calculated from the purified PHB and compared with the results of HPLC analysis.
The purified PHB and non-PHB cell mass were examined with a Nicolet Avatar 370 FTIR spectrometer (Thermo Electron Co., Madison, WI). The solids were pressed on a germanium crystal window of micro-horizontal attenuated total reflectance (ATR) for measurement of single-reflection and absorption of infrared radiation by the specimens. The thermal properties of PHB powder were examined with a differential scanning calorimeter (DSC). A Modulated 2920 instrument (TA Instruments, New Castle, DE) equipped with a refrigerated cooling system was run in heat-cool-heat mode at a rate of 5 oC/min under nitrogen. The selected temperature range was 30oC – 210 oC with sample weights of 4.5 – 5.5 mgs. Images of cell and PHB granules were obtained with an energy-filtering transmission electron microscopy (120 kV LEO 912, Carl Zeiss SMT Inc. MA). The instrument has an in-column electron energy loss spectrometer, allowing analysis of light element in thin sections.
Figure 1 elucidates the sequential treatment of PHB-containing cells in a process of PHB recovery and purification. Starting with 100 dry cell mass, the cells in a slurry of 278 g DM/L were first treated in an acidic solution (0.2M H2SO4). A substantial amount of microbial proteins was released from the damaged cells, depending on temperature and time as shown in Figure 2.
Sequential treatment of PHB-containing biomass (100 g dry mass) and generation of biomass hydrolysates. Surfactant SDS is optional for high PHB purity.
Effect of temperature and time on release of proteins from microbial cells in acidic solution.
During the acid pretreatment, the original amorphous PHB granules became partially crystallized (data not shown here), which improved the granule’s resistance to abiotic degradation in the following treatments [20]. The biomass hydrolysates (13.4 g dry mass) dissolved in supernatant solution was discharged as acid hydrolysates. The residual PHB-containing biomass was further subjected to a base treatment by raising the slurry pH to 10.5 with a 10M NaOH solution. About 15.4 g dry mass was dissolved in the supernatant solution and discharged as base hydrolysates. After a small amount of residual biomass (2 g dry mass) was removed via oxidation with hypochlorite, the final PHB powder (69.2 g dry mass) contained 96.4 wt% PHB. The overall PHB recovery yield was 92.6%, or 7-8% PHB was lost in repeated hydrolysis and solid/liquid separations. When a small amount of surfactant such as sodium dodecylsulfate (SDS) was added in the base treatment, the PHB purity of the final biopolyester resin could be increased to 99.4%.
Transmission electron microscope images: microbial cells containing native PHB granules (top left), cells with damaged walls in acid pretreatment (top right), PHB granules with attached residual cell mass (bottom left), and purified PHB granules (bottom right)
Figure 3 is the electronic microscopy images of the original cells with PHB inclusion bodies, the cells with damaged porous cell walls in acid pretreatment, the PHB granules with residual cellular mass in base treatment, and the purified PHB granules after whitening and washing. It is interesting to see that the cell walls became porous in the acid pretreatment, which allowed release of proteins and other biological components in cytoplasm. The original cell structure, however, was maintained to keep the PHB granules within the damaged cells. After base treatment, the cell walls were almost completely decomposed, and a small amount of residual cell mass, probably some hydrophobic cellular components, was attached to PHB granules. After whitening and washing, the non-PHA cell mass was removed to give purified PHB granules.
Figure 4 compares the FTIR spectra of the purified PHB granules and the original oven-dried PHB-containing cell mass. The peaks of amide I band at ~1650 cm-1 and amide II band at ~1540 cm-1 (N-H bend) are characteristic infrared radiation absorption of the proteins in cell mass [25,26]. They disappeared in the spectrum of purified PHB granules. This was confirmed with a pure PHB prepared with solvent extraction (the spectrum not shown here). It was also noticed that the native amorphous PHB granules became crystallized during the process of purification, which will be further discussed. This structural change in PHB matrix was also reflected in the absorption of infrared radiation at wave numbers of 1180, 1210, and 1280 nm-1 [27].
FTIR spectra of purified PHB granules and PHB-containing oven-dried cell mass (ODCM).
The purified PHB granules were subjected to repeated heating and cooling in a differential scanning calorimeter (DSC), and the results are presented in Figure 5. In the first heating (solid blue line), two melting peaks were observed, around 156 oC and 167 oC, respectively, indicating that the PHB powder had two types of crystalline structures. The melted polymer was re-crystallized again during the first cooling (dotted blue line), starting at 100 oC and ending at 80 oC. When the crystallized PHB was subjected to the second heating (solid black line), it was noticed that the relatively small melting peak at 156 oC (or crystalline structure) observed in the first heating disappeared. Only one melting peak (crystalline structure) was observed, starting at 160 oC and ending at 181 oC with a peak around 174 oC. This phenomenon indicates that the first melting peak in the first heating represents a type of crystalline structure that could not be formed at a cooling rate of 5 oC/min. The whole endothermic event in the second heating absorbed 87J/g PHB. Based on a theoretical melting enthalpy of 100% crystalline PHB (146 J/g) [28], it can be estimated that about 60% of PHB matrix was crystallized during the first cooling (Eq. 1).
Where Xc is the PHB crystallinity, ΔHm and ΔHt are the melting enthalpies of PHB powder and a theoretical PHB crystal [28].
Differential scanning calorimetry (DSC) measurement of purified PHB granules in repeated heating and cooling: the first heating (solid blue line) followed by the first cooling (dotted blue line) and the second heating (solid black line) following by the second cooling (dotted black line).
In contrast to the thermal plastic behavior of PHB powders, the oven-dried cell mass containing 73% PHB could not be melted till carbonization. This fact reveals a complicated interaction between the biopolyester and the residual biomass. It also shows that a composite of 73% PHB and 27% cellular mass is not a thermoplastic material, but a rigid composite. The role of cellular mass in the PHB composite is not clear yet.
As shown in Figure 1, about 94 wt% of residual microbial biomass was decomposed and hydrolyzed in the acid pretreatment (~44% biomass) and base treatment (~50% biomass). The remaining small amount (~6 wt %) of residual cell mass is most likely mineralized via oxidation with hypochlorite, a strong oxidation agent. The acid hydrolysates are primarily the cytoplasm proteins released from the damaged cells (Figures 2 and 3). The released biological macromolecules were subjected to further hydrolysis in the thermal acidic solution. The acid hydrolysates solution had a clean brownish color and contained 30-45 g/ of soluble biomass, depending on the density of cell slurry and treatment conditions. The base hydrolysates solution with a dark color contained the hydrolysis products of hydrophobic cell components including lipids and membrane proteins. After centrifugation, the concentration of soluble biomass in the supernatant solution was 30 to 50 g/L. The sequential treatments disrupted and dissolved the structural components so that they could be removed from PHB granules. Equally important, the biomass and biological macromolecules were decomposed into small molecule hydrolysates such as amino acids and organic acids. These hydrolysates could become appropriate substrates that can be assimilated by microbial cells in microbial PHB production.
FTIR spectra of acid-base biomass hydrolysates (black line, cell debris) and cellular components extracted with acetone (blue line)
In addition to the two types of biomass hydrolysates described above, a mixed hydrolysates of residual biomass was generated when the acid pretreatment and base treatment were performed sequentially without solid/liquid separation. It eliminated one operation of solid/liquid separation, but the acid hydrolysates (primarily proteins) were subjected to additional hydrolysis in the base solution. Figure 6 shows the FTIR spectrum of an acid-base hydrolysates. As observed in the IR spectrum of the original cell mass in Figure 4, a major component of the hydrolysates was the amino acids or proteins with infrared radiation absorbance at 1500 to 1700 cm-1 [25]. Another major component in the acid/base hydrolysates had the IR absorbance at 1000 to 1200 cm-1, which was attributed to cell lipids and/or similar compounds. This was confirmed with the spectrum of cell mass extract in acetone. Acetone is a common solvent used to remove hydrophobic lipids, steroids and pigments from PHB-containing cell mass [17]. It does not dissolve and extract PHB and proteins. Based on the observations above, it was concluded that the major cellular components in the acid hydrolysates were derived from cytoplasm proteins and in the base hydrolysates from cell walls, lipids and membrane proteins. In the acid-base hydrolysates, the products were derived from both groups, i.e. amino acids or peptides derived from proteins, and lipids derived from cell walls and membranes. It should be pointed out that the composition of acid-base hydrolysates is not a simple mixture of acid- and base-hydrolysates because the acid hydrolysates were further hydrolyzed in base treatment.
Because of the hydrophobic properties of PHB granules, the residual hydrophobic impurities of cell mass might be attached to the granules and difficult to remove by washing with water. A surfactant such as SDS in the base treatment can remove most of the impurities to a high PHB purity (>99 % w/w). A large portion of the surfactant, however, may be left in the hydrolysates solution and may have an adverse effect on the reuse of the hydrolysates in PHB production.
An acid hydrolysates solution containing 38 g/L of soluble solids was added into a glucose medium to give a predetermined percentage of residual biomass to glucose at 0, 10, 20 and 25% of sugar, respectively. The initial glucose concentration was controlled at a constant level of 9.6 g/L. The flask cultures of no biomass hydrolysates were run in parallel as controls. As shown in Figure 7, the acid biomass hydrolysates were beneficial to both cell growth and PHA formation. Because the residual biomass might also contain some insoluble solids and PHB granules lost in PHB recovery, both cell density and PHB concentration were compared at 24 hours and 48 hours to show the net gains. The benefits of biomass hydrolysates were statistically significant based on the deviations of duplicates.
The acid hydrolysates might have two positive effects on microbial PHA formation. First, the hydrolysates promoted cell activity on glucose utilization, giving higher cell densities than the controls in the first 24 hours. This nutritional effect was similar to those of organic nutrients such as yeast extract and peptone, which are widely used in microbial cultures to provide nutrients and growth factors to the cells. A fast cell growth can reduce the cultivation time, resulting in a high PHB productivity. Second, the biomass hydrolysates might also be used as an extra carbon source to generate more cell mass than the controls in 48 hours. This carbon source effect, however, might play a minor role because the cell density did not increase with cell debris load. In fact, too much acid hydrolysates deteriorated the gains as shown in Figure 7. The reason is not clear yet. A load of acid biomass hydrolysates to glucose from 10 to 20 wt% seems appropriate for both cell growth and PHB formation.
An acid-base biomass hydrolysates solution containing 48.5 g/L of soluble solids was added into a glucose medium at predetermined percentage of cell debris to glucose from 0 to 40%. The glucose medium without biomass hydrolysates was run in parallel as controls. As shown in Table 1, the concentrations of both cell mass and PHA content, after 48 hours cultivation, were substantially higher than those of the control. The overall cell growth yield (Yx/s) and PHA formation yield (Yp/s) are calculated from the amounts of cell mass and PHA formed in 48 hours based on the initial concentration of glucose. The relative yields (Yx’ and Yp’) based on the controls were increased by 100 – 300%. More interestingly, the inhibitory effect of acid biomass hydrolysates was not observed in the use of acid-base hydrolysates, and the load of cell debris to glucose can be increased to about 39 wt%. Most likely, the unknown inhibitors in acid hydrolysates were further decomposed into less inhibitory hydrolysates in the base treatment. Since the acid biomass hydrolysates contained primarily the cytoplasm proteins released from the damaged cells, the soluble proteins might adversely affect cell growth at high concentrations. After being hydrolyzed in base solution into peptides and amino acids, the small molecule hydrolysates become less inhibitory and more usable to the cells.
Effect of acid hydrolysates to glucose loading ratio on cell growth (top) and PHB formation (bottom) in reuse of the residual biomass for PHB production.
Biomass hydrolysates (g/L) | Biomass /Glucose (wt%) | Cell density (g/L) | PHB (wt%) | Yx/s (g/g) | Yp/s (g/g) | Yx’ | Yp’ |
0.0 | 0 | 2.1 ± 0.5 | 45 ± 1.2 | 0.18 | 0.081 | 1.0 | 1.0 |
1.15 | 9.4 | 4.0 ± 0.2 | 55 ± 1.6 | 0.33 | 0.18 | 1.8 | 2.24 |
2.30 | 18.8 | 4.5 ± 0.3 | 60 ± 1.7 | 0.38 | 0.23 | 2.1 | 2.84 |
4.60 | 38.8 | 5.6 ± 0.4 | 61± 1.5 | 0.47 | 0.29 | 2.6 | 3.58 |
Effect of acid-base biomass hydrolysates on cell growth and PHB formation
In the process of PHB recovery (Figure 1), three types of biomass hydrolysates may be generated, depending on operations: acid, base, and acid-base biomass hydrolysates. They may have different nutrient values or inhibitory effects on cell growth and PHB synthesis. Solutions of three types of biomass hydrolysates were added into a glucose medium for pre-determined concentrations of cell debris. Controls without hydrolysates were run in parallel. The ratios of cell densities (g/L) to the controls were compared after 48 hours cultivation as shown in Figure 8. The nutritional value of acid hydrolysates in cell growth is similar to that of base hydrolysates. The nutrient value of acid-base hydrolysates, however, is significantly higher than those of hydrolysates from individual treatment.
Comparison of three types of biomass hydrolysates (acid, base, and acid-base) on cell growth in a glucose mineral medium. The relative cell gain is the ratio of cell density to the controls.
Based on an average cell yield (Yx/s = 0.45) of PHB fermentation on glucose [29], 45 kg of cell mass containing 70 wt% of PHB is generated from 100 kg of glucose consumed. A downstream recovery and purification as shown in Figure 1 can generate 31 kg PHB resin and 13 kg acid-base hydrolysis of residual microbial biomass. It is assumed that the acid hydrolysates are not separated, but hydrolyzed sequentially in the base treatment and discharged with the base hydrolysates together. If this amount of residual biomass is reused in next PHB fermentation, the percentage of biomass hydrolysates to glucose is 13% at maximum (13 kg for 100 kg glucose), a moderate load of biomass hydrolysates (Table 1). In real fermentations, more glucose is often added because of the residual glucose in the spent medium. This quick calculation indicates that most of residual biomass discharged from downstream separations can be reused in the next PHA fermentation. In addition to the elimination of a waste stream, the productivity and yields of PHA fermentation can also be significantly improved.
SDS is a popular surfactant used in PHA recovery to disrupt the cells or remove a small amount of hydrophobic impurities from PHB granules [30, 31]. The purity of PHB granules can be increased from 96.4% to above 99% when a small amount of SDS was added in the base treatment. The surfactant left in the base solution, however, may have an adverse effect when the cell debris is reused in microbial PHB fermentation. An acid-base biomass hydrolysates solution containing 48.5 g/L of soluble solids and SDS were added into a glucose medium. The cell debris concentration was kept at 1.94 g/L, and SDS concentration was increased from 0.2 to 0.8 g/L with SDS. Controls without biomass hydrolysates and SDS were run in parallel. Table 2 gives the results of cell growth and PHA formation at different surfactant levels at 24 and 48 hours, respectively.
Biomass hydrolysates (g/L) | SDS (g/L) | 24 hours | 48 hours | ||
Cell mass (g/L) | PHB (wt%) | Cell mass (g/L) | PHB (wt%) | ||
0 | 0 | 2.08 ± 0.02 | 36.8 ± 1.1 | 2.7 ± 0.02 | 44.4 ± 0.6 |
1.94 | 0.2 | 3.69 ± 0.06 | 49.7 ± 2.1 | 4.99 ± 0.04 | 60.6 ± 1.1 |
1.94 | 0.4 | 3.27 ± 0.05 | 39.6 ± 1.2 | 4.58 ± 0.04 | 53.8 ± 0.8 |
1.94 | 0.6 | 2.70 ± 0.04 | 33.9 ± 1.5 | 3.51 ± 0.03 | 50.3 ± 0.7 |
1.94 | 0.8 | 2.59 ± 0.04 | 17.8 ± 1.3 | 3.54 ± 0.03 | 25.8 ± 1.2 |
Effect of surfactant SDS and biomass hydrolysates on cell growth and PHB formation
Compared with the controls of no biomass hydrolysates and surfactant, all the cultures containing the acid-base hydrolysates exhibited better cell growth. Particularly, the increase of cell concentration from 24 hours to 48 hours was the new cell mass formed in 24 hours from glucose and cell debris in the presence of SDS. The formation of PHB, however, was deteriorated at high SDS concentrations (0.6-0.8 g/L). At a low or moderate SDS concentrations (0.2-0.4 g/L), the positive effect from biomass hydrolysates was much higher than the negative effect of surfactant. The PHB concentrations, after 48 hours cultivation, were 2.4 to 3 g/l, in comparison with 1.2 g/L of the control. The results in Table 2 indicate that the dosage of SDS in PHA recovery should be controlled according to the amount of residual microbial biomass generated. The mass ratio of SDS to biomass hydrolysates should be less than 20% w/w or better at 10% w/w. In a typical PHB recovery process as shown in Figure 1, the amount of SDS used should be less than 2.9 g for 10% of acid-base cell debris or 5.8 g for 20% of acid-base cell debris. The consumption of SDS is therefore 4-8 % of PHB resin produced. It is much lower than the SDS dosages used in the conventional separations [30, 31].
Residual microbial biomass is an inevitable waste generated in downstream recovery of polyhydroxyalkanoates from microbial cells. With a separation technology based on sequential dissolution of no-PHB cell mass in aqueous solutions, the cell mass separated from the PHB-granules is decomposed and hydrolyzed into small molecule hydrolysates that can be assimilated by microbial cells as nutrients and/or carbon source. A type of biomass hydrolysates generated from continuing treatment in acid and base solutions exhibits the best nutrient value for cell growth and PHA formation. The acid-base hydrolysates contains two major water-soluble components derived from the cell proteins and lipids, respectively. When PHB-producing cells are fed with the hydrolysates in a glucose mineral solution, the cells grow faster and form more biopolyester in comparison with the controls that do not contain the hydrolysates. The glucose-based yields of cell mass and PHA bioplastics are significantly improved. SDS is an efficient surfactant to remove the small amount of hydrophobic residues for high PHB purity, but also a potential inhibitor to microbial PHA formation. When the amount of surfactant is less than 20% of an acid-base biomass hydrolysates, its negative effect is overwhelmed by the nutritional value of hydrolysates. Under these conditions, it is highly possible to reuse most of the residual biomass discharged from PHB recovery in the next microbial PHB fermentation. It therefore eliminates a waste stream from bioplastics production and saves the nutrients with improved PHA productivity and yield.
The authors acknowledge a support from Bio-On to this work. MP and MJ are graduate students of Molecular Bioscience & Bioengineering at UHM.
Density functional theory (DFT) is a low-cost, time-saving quantum mechanical (QM) theory, used to compute many physical characteristics of solids with high precision. The research in this field ranges from the development of novel analytical approaches focused on the design of precise exchange-correlation functionals to the use of this technique to predict the molecular and electronic configuration of atoms, molecules, complexes, and solids in both gas and solution phases. The history to DFT’s success is the quest for the exchange-correlation functional, which utilizes density to represent advanced many-body phenomena inside one element formalism. If a precise exchange-correlation functional is applied, it may correctly describe the quantum nature of matter. The estimated character of the exchange-correlation functional is the basis for DFT implementation success or failure. DFT’s early breakthroughs concentrated on the most fundamental issues in chemistry, such as the opportunity to generate functionals that could describe both molecular geometries as well as dissociation energy. The fact that every feature of a system in ground state is a unique ground state density functional was demonstrated by Hohenberg-Kohn, laying the foundation for DFT, which is now used to explore novelty of materials. This chapter is aimed to present an overview of DFT by describing the theoretical foundations, widely used approximations, current advances, and issues addressed, as well as future horizons.
The Schrodinger Equation [1] for a many body system may be simplified to Kohn-Sham equation, which is a single particle independent Schrodinger equation, and can be numerically solved with density functional theory. This computational process produces physical characteristics of solids; however, this hypothesis is based on electron density rather than wave functions, for which scientist Walter Kohn was given the Nobel Prize in 1998 [2]. Despite the fact that no exchange-correlation effects had been documented at the time, Thomas and Fermi claimed in 1927 that total density is the essential parameter in many body problems [3, 4]. The theorems of Hohenberg, Kohn, and Sham laid the groundwork for DFT in 1964, stating that the functional of a many-body problem’s (non-degenerated) ground state electron charge density may completely characterize all properties in absence of magnetic field [5].
Hohenberg and Kohn [6] stated seemingly two simple theorems in 1964 that enabled the implementation of DFT.
In order to establish a mathematical relation, let us assume external potentials as
Where T, U, and Vext represents the K.E of electrons, coulomb interaction, and external potential respectively. Quantum mechanically the factors T, U, and Vext can be expressed as;
The solution of Hamiltonian for Eq. (1) can be expressed as;
The
Following a thorough exploration of the situation, established on
Employing essential property of ground state:
Alternatively, by swapping;
By adding above equations we get;
The Eq. (8) confirms clear disagreement, and two unlike potentials, v(r) as well as v′(r) will certainly provide different density ρ(r) and ρ′(r) respectively. As a result, details relating density and external potential are needed to determine the Hamiltonian information. Also, T and U are known for N-partials systems so
Consider,
Also,
In order to have minimum energy functional, the corresponding density
Assuming
As a result, provided the density functional is accurately described, one may easily compute the ground state density as well as energy in an identified external potential. Furthermore, it also demonstrates that
The theorems given by Hohenberg-Kohn are exact; however not very useful in real calculations [6]. The equation given by Kohn-Sham [7] turned DFT into an applied tool. They converted the difficult problem of electrons interacting together in external effective potential (Vext) into the electrons that are non-interacting in Vext, and the total energy for a ground state of interacting electrons in fixed potential,
Where universal density functional G[ρ] holds exchange-correlation, and is expressed as;
The kinetic energy for a many body system having non-interacting electrons is denoted by
and
The exchange correlation energy
and
The Ex term denotes the reduction in energy as an outcome of anti-symmetrization, and it may be represented through a single particle orbital as;
and
and
Where the single term in the summation refers to the energy of a molecule ‘j’ at site ‘r’ in relation to a molecule ‘k’ at ‘r′’. The system’s energy is further reduced owing to mutual avoidance of the interacting particles, such as electrons that are anti-parallel and lower their energy by evenly arranging their moments. Kohn-Sham mapping of interacting and non-interacting system is shown in Figure 1.
Kohn-Sham mapping of interacting and non-interacting system.
The energy of ground state may be obtained by differentiating Eq. (14) with respect to
By employing density ρs(r), the minimum state for a non-interacting many-body system is;
Equating Eqs. (26) and (27), the potential Vs can be obtained as;
The equation for a one-particle system that is non-interacting in potential vs(r) can be derived from the equation of interacting electrons of the system in the presence of v(r).
The ρ(r) of an original system is replicated by orbitals, where fk is the kth orbital occupation, and can be expressed as;
The consequences of KS scheme revealed that the minimum energy state can be established by limiting energy of the energy functional, and it can be done using an agreeable solution of a set of single-particle equations. In the KS scheme, just one critical difficulty is that Exc (exchange-correlation energy) cannot be found exactly. If Exc is determined accurately, it is a precise solution for a many-body problem. There is currently no such exact solution exists, hence approximations are employed to estimate Exc with LDA and GGA being the most commonly used approximations.
In this part, we will go through some of the major advances that lead to contemporary DFT in order to lay a foundation that will help us to comprehend both the theory’s foundations and limits. Bloch (1929) was the first to write about the exchange contribution, and it has become well-known as a result of quantum Monte-Carlo simulations of uniform gases [8], which are parameterized in simple formulations [9, 10]. The Local Density Approximation (LDA) [11], proposed by Kohn and Sham, asserts that the exchange-correlation functional at any point in space is simply dependent on that location’s spin density. LDA is quite correct for geometries, but it often over-binds atoms/molecules roughly by 1 eV per bond, rendering it ineffective for thermo-chemistry [12]. The Generalized Gradient Approximation (GGA) [13, 14] is an extension to the LDA component that includes terms that are dependent on density derivatives. Perdew was the first to apply real-space cutoffs to make GGAs, which led to the development of the PW86 functional model [13]. The PW91 functional [15] was the pinnacle of this comprehensive development, and it produces useful precision for binding energies, as proven in 1993 of around 6–10 kcal/mol [16]. PBE [17] is the most widely used GGA to investigate materials today, whereas BLYP [18] and Lee-Yang-Parr correlation [19] is the most generally employed GGA in chemistry. A hybrid GGA [20] is one that combines a normal GGA plus a Hartree-Fock component, in which the kinetic energy density is also employed to define the GGA component. The GGA, Hartree-Fock, and kinetic energy density components are all present in a meta-hybrid, while hybrid or meta-hybrid component of a double-hybrid includes an involvement from second-order Moller-Plesset perturbation theory [21]. The Density Functional (DF) consists of a part of GGA, LDA, Hartree-Fock exchange or hybrids, and/or a meta-GGA, commonly known as the exchange-and-correlation (XC) functional (meta-GGA or meta-hybrid). Furthermore, the addition of an orbital-dependent correlation, it may also be reliant on virtual Kohn-Sham orbitals (double-hybrids) [22]. A comparison of simplicity versus accuracy of existing approximations in DFT is shown in Figure 2.
A comparison of simplicity versus accuracy of existing approximations in DFT [
The functionals currently utilized in DFT simulations constitute a natural hierarchy, and no systematic approach to the precise functional can be claimed. The available functional form is clearly improving, resulting in a considerably more accurate representation of ground state properties. The most important recent advancements are those that include the non-local aspect of the exchange potential in some way. Table 1, summarizes the present hierarchy.
Commonly used Exc functionals.
This section focuses on the evolution of new functionals in DFT during the last decades.
The exchange correlation energy (Exc) can be calculated using DFT fluctuation dissipation in the form of coupling constant and frequency [24, 25, 26]. The direct random-phase approximation (RPA) [27, 28] or time dependent TD-Hartree, are the results of ignoring the exchange kernel of TDFT. A fifth-rung approximation is generated as a result of this methodology, and this can be expensive to examine, although the relative burden is always reducing [29, 30]. It only examines bubble diagrams in the many-body expansion of the energy, so direct RPA over-correlates systems by ignoring extra contributions at higher levels that diminish correlation. It also has issues with self-interaction since, even when just one electron is involved, it yields low correlation energies, and the dissociation energies of molecules are erroneous [23].
The meta-GGA [31] is a novel component that extends beyond density and gradient, and is commonly used to indicate the KS orbitals’ kinetic energy density. The objective of a successful meta-GGA is to achieve hybrid accuracy without incurring the computational expense of the exact exchange contribution. The incorporation of atom-centered basis functions, the cost of accurate exchange is reasonable, however, it can be costly while using periodic boundary conditions in addition of basis sets. Perdew and colleagues, and plenty of others, have worked on meta-GGAs for decades, with multiple failed attempts [32]. SCAN (strongly constrained and suitably normed semi-local density functional) [33], the most current effort has undergone a number of conventional tests and looks to have a good chance of becoming part of the pantheon of widely employed functionals. The G3 data-set [34] is a common collection of chemical compounds that LDA over-binds around 3 eV, while PBE reduces it to approximately 1 eV, and SCAN around 1/4 eV. On the S22 data-set [35] of weakly bonded systems, SCAN has 2–3 times less errors than PBE does, while SCAN decreases miscalculations of lattice constant and other parameters on the LC20 data [36] set around 0.05 Å, and to around 0.01 Å in PBE. The PBE [37], on contrary to SCAN, only improves underestimation of chemical barrier height by 30 percent, while hybrids on the other hand are frequently 2–3 times superior with conventional varieties. Therefore, one can conclude that SCAN achieves accuracies comparable to hybrid functionals for several characteristics at a fraction of the computing cost [38].
Andreas Savin was the first to create the range separation hypothesis, which is quite precise [39, 40], through which coulomb repulsion may be easily expressed by combining a short-ranged input with a long-ranged involvement that do not have coulomb singularity at zero separation, and decays quicker than the inverse of the separating distance. In KS equation generalizations, one contribution is treated as an interaction, while the other is compensated by a redefined XC contribution. The HSE06 functional [41] is a hybrid with a range separation that manages long-ranged exchanges with an approximation, short-ranged exchanges with accuracy in an extended insulator [42], and this combination frequently yields exact gaps for moderate-gap semiconductors and insulators [37].
Over the last two decades, tremendous progress has been made in addressing the challenges associated with weak van der Waals (vdW) interactions. Traditional functionals do a good job at manipulation of covalent, ionic, and metallic interactions due to their semi-local nature, but they fall short when it comes to longer, weaker bonds, and cannot offer weak binding that drops off as a function of R6 (R is the distance among two atoms) [38]. To account these impacts, modifications must be applied to the conventional functionals and this can be accomplished in one of three ways. There is a succession of approximations produced by Langreth and Lundqvist and collaborators [43] for the evolution of explicit non-local functionals of electron density, while these approximations are generated non-empirically, notably beginning with contributions of correlation energy. Additionally, these functionals may be useful for any materials, ranging from solids to molecules, and have been designed by supposing systems that contain a gap [44]. RPA, which incorporates approximations to the vdW forces by default, as well as the Becke and Johnson technique [45], leverages the exchange hole’s dipole moment to approximate C6, as well as higher coefficients.
The inadequacy of traditional approximations to anticipate band gaps of semiconductors and insulators is a critical flaw. The LDA undervalues gap between bulk Si and Ge by a factor of two, making Germanium a metal, whereas GGAs performs a bit good but underestimate as well. The ability to give precise and dependable gaps has always been a strong suit of the GW approach [46]. In the last two decades, precise gap computation utilizing hybrid functionals such as HSE06 [41] has been a huge success, and is accomplished through the use of a generalized KS scheme [47]. In this case, rather than using pure Kohn-Sham theory, the orbital reliant element of the functional is considered as in Hartree-Fork approach to overcome flaws of other Exc functionals [48].
In principle, DFT is exact; however its effectiveness depends on the development as well as advancement in exchange-correlation (Exc) functionals which may be achieved by optimizing against larger data-sets and using improved functional arrangements that are more flexible and contain more elements. Smoothness has also been prioritized in recent enhancements, which helps to alleviate problems like grid-size convergence and self-consistent field iterations. In this section, we will go through some of DFT’s challenges that may differ from those that appears to be “solved” to those that are still being explored. There are numerous more that are less well-known, and yet crucial to DFT’s future growth as well as use.
DFT’s inadequacy for strongly correlated systems utilizing typical approximations has been acknowledged since its inception, and this can be investigated as well as linked to standard approximation localization or delocalization inaccuracies when integer or half-integer electron quantities are found in distinct locations [49]. In quantum computational physics and chemistry, the Kohn-Sham gap among two states becomes too narrow, and the wave function of a many-body system is very nearly equal to mixing of two slater determinants, which is referred to as static correlation. The failure of approximations under these situations cause the challenges, not the KS scheme itself, as demonstrated by the two-site Hubbard model, in which the precise KS system is simple to design, even when one deal with strongly correlated systems [50]. This problem can be addressed by breaking the symmetry of evenly spaced atomic chains into multiple solutions, and one of which will have the least amount of energy [51]. This is such a significant issue; hence, a great deal of research has been done on it, particularly by Weitao Yang’s group [52], but also by Scuseria [53] and Becke [54].
One of the biggest problems for DFT is to preserve some aspect of simplicity as its foundations. When DFT functionals get as complicated as full configuration interaction, one of the theory’s most significant properties, namely simplicity, is lost, which is particularly true in terms of computational environment. This simplicity, however, must not be at the expense of accuracy, nor should it become an exclusively empirical approach. The precise representation of binding energies and geometries of simple molecules was one of DFT’s first major hurdles in chemistry. Becke, Perdew, Langreth, and Parr presented the density’s first derivative in the form of generalized gradient approximation in the 1980s, which was the first step towards chemists being able to correctly use DFT. In the early 1990s, Becke described the proportion of Hartree-Fock exact exchange (HF) which is included in the functionals, and as a result of this effort, B3LYP [55], the utmost extensively utilized of all the functionals, was developed, and has demonstrated outstanding performance in variety of systems. Despite the introduction of new concepts into more current functionals of varying complication, it remains the prevalent, and DFT will likely benefit from developing functionals that improves on B3LYP [56].
To provide a comprehensive chemistry explanation, it is indispensable to go beyond explaining a molecule in equilibrium geometry to similarly explain weakly interacting atoms or molecules, and chemical reaction transition states. It’s challenging to describe reaction barriers with LDA or GGA functional since they consistently underestimate the difficulty of transitioning from one condition to another. Formerly the functionals may be utilized to represent potential energy surfaces, and this systematic imperfection must be corrected. Transition states, covalent bonding, and van der Waals attraction are all challenging to represent precisely and effectively, though efforts are to be made to address these problems. This is especially true when DFT becomes more widely applied to biologically important regions, where all of these interactions might occur at the same time [57].
The enactment of DFT, as evidenced by significant errors for one-electron systems, is another important issue. In DFT, a single electron system has no exceptional role; in fact, one electron can interrelate with itself, as the self-interaction error has long proved. Of course, there is no self-interaction in the accurate functional; the exchange energy precisely cancels the coulomb energy of single electron. In increasingly complicated systems, they can be linked to systematic flaws like static correlation and delocalization error, and despite most recent advancements, even the simplest systems can contain mistakes in most recent functionals [58]. Hence, these basic systems should not be overlooked since they hold the vital knowledge of functionals that can lead to advancements [57].
The applications of warm dense matter vary from modeling planetary interiors to inertial confinement fusion [59], which is a completely new field for DFT, and has been exploded in the last decade, with considerable temperatures on the electronic scale of roughly 105 K but not to the point that the Thomas- Fermi hypothesis or classical performance takes precedence. This domain is so “new” that temperature-dependent exchange-correlation energy of a uniform gas, which is the input to thermal LDA, is just now being computed with remarkable precision [60]. Figure 3 summarizes some of the potential application areas of DFT.
Potential application areas of DFT [
Density Functional Theory is a powerful and commonly employed quantum mechanical tool for investigating various aspects of matter. This field’s research ranges from the development of novel analytical approaches focused on the design of precise exchange-correlation functionals to the use of this technique to predict the molecular and electronic configuration of atoms, molecules, and solids in both gas and solution phases. Designing and evolution of more efficient density functionals is a continuous endeavor since there are still challenges to be resolved, and getting all of the attributes correct at a reasonable computing cost is a quantum fantasy. The future research will focus on developing even more consistently precise density functionals for specific applications, allowing researchers to take use of DFT’s comparatively high accuracy at cheap processing cost, and the possibility of even more improvements awaits.
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Saxena is a vice dean and professor at King George's Medical University, Lucknow, India. His research interests involve understanding the molecular mechanisms of host defense during human viral infections and developing new predictive, preventive, and therapeutic strategies for them using Japanese encephalitis virus (JEV), HIV, and emerging viruses as a model via stem cell and cell culture technologies. His research work has been published in various high-impact factor journals (Science, PNAS, Nature Medicine) with a high number of citations. He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award, and Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India. 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He is the president of the Travel Medicine Committee of the Pan-American Infectious Diseases Association (API), as well as the president of the Colombian Association of Infectious Diseases (ACIN). He is a member of the Committee on Tropical Medicine, Zoonoses, and Travel Medicine of ACIN. He is a vice-president of the Latin American Society for Travel Medicine (SLAMVI) and a Member of the Council of the International Society for Infectious Diseases (ISID). Since 2014, he has been recognized as a Senior Researcher, at the Ministry of Science of Colombia. He is a professor at the Faculty of Medicine of the Fundacion Universitaria Autonoma de las Americas, in Pereira, Risaralda, Colombia. He is an External Professor, Master in Research on Tropical Medicine and International Health, Universitat de Barcelona, Spain. He is also a professor at the Master in Clinical Epidemiology and Biostatistics, Universidad Científica del Sur, Lima, Peru. In 2021 he has been awarded the “Raul Isturiz Award” Medal of the API. Also, in 2021, he was awarded with the “Jose Felix Patiño” Asclepius Staff Medal of the Colombian Medical College, due to his scientific contributions to COVID-19 during the pandemic. He is currently the Editor in Chief of the journal Travel Medicine and Infectious Diseases. His Scopus H index is 47 (Google Scholar H index, 68).",institutionString:"Institución Universitaria Visión de las Américas, Colombia",institution:null},{id:"332819",title:"Dr.",name:"Chukwudi Michael",middleName:"Michael",surname:"Egbuche",slug:"chukwudi-michael-egbuche",fullName:"Chukwudi Michael Egbuche",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/332819/images/14624_n.jpg",biography:"I an Dr. Chukwudi Michael Egbuche. I am a Senior Lecturer in the Department of Parasitology and Entomology, Nnamdi Azikiwe University, Awka.",institutionString:null,institution:{name:"Nnamdi Azikiwe University",country:{name:"Nigeria"}}},{id:"284232",title:"Mr.",name:"Nikunj",middleName:"U",surname:"Tandel",slug:"nikunj-tandel",fullName:"Nikunj Tandel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/284232/images/8275_n.jpg",biography:'Mr. Nikunj Tandel has completed his Master\'s degree in Biotechnology from VIT University, India in the year of 2012. He is having 8 years of research experience especially in the field of malaria epidemiology, immunology, and nanoparticle-based drug delivery system against the infectious diseases, autoimmune disorders and cancer. He has worked for the NIH funded-International Center of Excellence in Malaria Research project "Center for the study of complex malaria in India (CSCMi)" in collaboration with New York University. The preliminary objectives of the study are to understand and develop the evidence-based tools and interventions for the control and prevention of malaria in different sites of the INDIA. Alongside, with the help of next-generation genomics study, the team has studied the antimalarial drug resistance in India. Further, he has extended his research in the development of Humanized mice for the study of liver-stage malaria and identification of molecular marker(s) for the Artemisinin resistance. At present, his research focuses on understanding the role of B cells in the activation of CD8+ T cells in malaria. Received the CSIR-SRF (Senior Research Fellow) award-2018, FIMSA (Federation of Immunological Societies of Asia-Oceania) Travel Bursary award to attend the IUIS-IIS-FIMSA Immunology course-2019',institutionString:"Nirma University",institution:{name:"Nirma University",country:{name:"India"}}},{id:"334383",title:"Ph.D.",name:"Simone",middleName:"Ulrich",surname:"Ulrich Picoli",slug:"simone-ulrich-picoli",fullName:"Simone Ulrich Picoli",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/334383/images/15919_n.jpg",biography:"Graduated in Pharmacy from Universidade Luterana do Brasil (1999), Master in Agricultural and Environmental Microbiology from Federal University of Rio Grande do Sul (2002), Specialization in Clinical Microbiology from Universidade de São Paulo, USP (2007) and PhD in Sciences in Gastroenterology and Hepatology (2012). She is currently an Adjunct Professor at Feevale University in Medicine and Biomedicine courses and a permanent professor of the Academic Master\\'s Degree in Virology. She has experience in the field of Microbiology, with an emphasis on Bacteriology, working mainly on the following topics: bacteriophages, bacterial resistance, clinical microbiology and food microbiology.",institutionString:null,institution:{name:"Universidade Feevale",country:{name:"Brazil"}}},{id:"229220",title:"Dr.",name:"Amjad",middleName:"Islam",surname:"Aqib",slug:"amjad-aqib",fullName:"Amjad Aqib",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229220/images/system/229220.png",biography:"Dr. Amjad Islam Aqib obtained a DVM and MSc (Hons) from University of Agriculture Faisalabad (UAF), Pakistan, and a PhD from the University of Veterinary and Animal Sciences Lahore, Pakistan. Dr. Aqib joined the Department of Clinical Medicine and Surgery at UAF for one year as an assistant professor where he developed a research laboratory designated for pathogenic bacteria. Since 2018, he has been Assistant Professor/Officer in-charge, Department of Medicine, Manager Research Operations and Development-ORIC, and President One Health Club at Cholistan University of Veterinary and Animal Sciences, Bahawalpur, Pakistan. He has nearly 100 publications to his credit. His research interests include epidemiological patterns and molecular analysis of antimicrobial resistance and modulation and vaccine development against animal pathogens of public health concern.",institutionString:"Cholistan University of Veterinary and Animal Sciences",institution:null},{id:"62900",title:"Prof.",name:"Fethi",middleName:null,surname:"Derbel",slug:"fethi-derbel",fullName:"Fethi Derbel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/62900/images/system/62900.jpeg",biography:"Professor Fethi Derbel was born in 1960 in Tunisia. He received his medical degree from the Sousse Faculty of Medicine at Sousse, University of Sousse, Tunisia. He completed his surgical residency in General Surgery at the University Hospital Farhat Hached of Sousse and was a member of the Unit of Liver Transplantation in the University of Rennes, France. He then worked in the Department of Surgery at the Sahloul University Hospital in Sousse. Professor Derbel is presently working at the Clinique les Oliviers, Sousse, Tunisia. His hospital activities are mostly concerned with laparoscopic, colorectal, pancreatic, hepatobiliary, and gastric surgery. He is also very interested in hernia surgery and performs ventral hernia repairs and inguinal hernia repairs. He has been a member of the GREPA and Tunisian Hernia Society (THS). During his residency, he managed patients suffering from diabetic foot, and he was very interested in this pathology. For this reason, he decided to coordinate a book project dealing with the diabetic foot. Professor Derbel has published many articles in journals and collaborates intensively with IntechOpen Access Publisher as an editor.",institutionString:"Clinique les Oliviers",institution:null},{id:"300144",title:"Dr.",name:"Meriem",middleName:null,surname:"Braiki",slug:"meriem-braiki",fullName:"Meriem Braiki",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/300144/images/system/300144.jpg",biography:"Dr. Meriem Braiki is a specialist in pediatric surgeon from Tunisia. She was born in 1985. She received her medical degree from the University of Medicine at Sousse, Tunisia. She achieved her surgical residency training periods in Pediatric Surgery departments at University Hospitals in Monastir, Tunis and France.\r\nShe is currently working at the Pediatric surgery department, Sidi Bouzid Hospital, Tunisia. Her hospital activities are mostly concerned with laparoscopic, parietal, urological and digestive surgery. She has published several articles in diffrent journals.",institutionString:"Sidi Bouzid Regional Hospital",institution:null},{id:"229481",title:"Dr.",name:"Erika M.",middleName:"Martins",surname:"de Carvalho",slug:"erika-m.-de-carvalho",fullName:"Erika M. de Carvalho",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/229481/images/6397_n.jpg",biography:null,institutionString:null,institution:{name:"Oswaldo Cruz Foundation",country:{name:"Brazil"}}},{id:"186537",title:"Prof.",name:"Tonay",middleName:null,surname:"Inceboz",slug:"tonay-inceboz",fullName:"Tonay Inceboz",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/186537/images/system/186537.jfif",biography:"I was graduated from Ege University of Medical Faculty (Turkey) in 1988 and completed his Med. PhD degree in Medical Parasitology at the same university. I became an Associate Professor in 2008 and Professor in 2014. I am currently working as a Professor at the Department of Medical Parasitology at Dokuz Eylul University, Izmir, Turkey.\n\nI have given many lectures, presentations in different academic meetings. I have more than 60 articles in peer-reviewed journals, 18 book chapters, 1 book editorship.\n\nMy research interests are Echinococcus granulosus, Echinococcus multilocularis (diagnosis, life cycle, in vitro and in vivo cultivation), and Trichomonas vaginalis (diagnosis, PCR, and in vitro cultivation).",institutionString:"Dokuz Eylül University",institution:{name:"Dokuz Eylül University",country:{name:"Turkey"}}},{id:"71812",title:"Prof.",name:"Hanem Fathy",middleName:"Fathy",surname:"Khater",slug:"hanem-fathy-khater",fullName:"Hanem Fathy Khater",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/71812/images/1167_n.jpg",biography:"Prof. Khater is a Professor of Parasitology at Benha University, Egypt. She studied for her doctoral degree, at the Department of Entomology, College of Agriculture, Food and Natural Resources, University of Missouri, Columbia, USA. She has completed her Ph.D. degrees in Parasitology in Egypt, from where she got the award for “the best scientific Ph.D. dissertation”. She worked at the School of Biological Sciences, Bristol, England, the UK in controlling insects of medical and veterinary importance as a grant from Newton Mosharafa, the British Council. Her research is focused on searching of pesticides against mosquitoes, house flies, lice, green bottle fly, camel nasal botfly, soft and hard ticks, mites, and the diamondback moth as well as control of several parasites using safe and natural materials to avoid drug resistances and environmental contamination.",institutionString:null,institution:{name:"Banha University",country:{name:"Egypt"}}},{id:"99780",title:"Prof.",name:"Omolade",middleName:"Olayinka",surname:"Okwa",slug:"omolade-okwa",fullName:"Omolade Okwa",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/99780/images/system/99780.jpg",biography:"Omolade Olayinka Okwa is presently a Professor of Parasitology at Lagos State University, Nigeria. She has a PhD in Parasitology (1997), an MSc in Cellular Parasitology (1992), and a BSc (Hons) Zoology (1990) all from the University of Ibadan, Nigeria. She teaches parasitology at the undergraduate and postgraduate levels. She was a recipient of a Commonwealth fellowship supported by British Council tenable at the Centre for Entomology and Parasitology (CAEP), Keele University, United Kingdom between 2004 and 2005. She was awarded an Honorary Visiting Research Fellow at the same university from 2005 to 2007. \nShe has been an external examiner to the Department of Veterinary Microbiology and Parasitology, University of Ibadan, MSc programme between 2010 and 2012. She is a member of the Nigerian Society of Experimental Biology (NISEB), Parasitology and Public Health Society of Nigeria (PPSN), Science Association of Nigeria (SAN), Zoological Society of Nigeria (ZSN), and is Vice Chairperson of the Organisation of Women in Science (OWSG), LASU chapter. She served as Head of Department of Zoology and Environmental Biology, Lagos State University from 2007 to 2010 and 2014 to 2016. She is a reviewer for several local and international journals such as Unilag Journal of Science, Libyan Journal of Medicine, Journal of Medicine and Medical Sciences, and Annual Research and Review in Science. \nShe has authored 45 scientific research publications in local and international journals, 8 scientific reviews, 4 books, and 3 book chapters, which includes the books “Malaria Parasites” and “Malaria” which are IntechOpen access publications.",institutionString:"Lagos State University",institution:{name:"Lagos State University",country:{name:"Nigeria"}}},{id:"273100",title:"Dr.",name:"Vijay",middleName:null,surname:"Gayam",slug:"vijay-gayam",fullName:"Vijay Gayam",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/273100/images/system/273100.jpeg",biography:"Dr. Vijay Bhaskar Reddy Gayam is currently practicing as an internist at Interfaith Medical Center in Brooklyn, New York, USA. He is also a Clinical Assistant Professor at the SUNY Downstate University Hospital and Adjunct Professor of Medicine at the American University of Antigua. He is a holder of an M.B.B.S. degree bestowed to him by Osmania Medical College and received his M.D. at Interfaith Medical Center. His career goals thus far have heavily focused on direct patient care, medical education, and clinical research. He currently serves in two leadership capacities; Assistant Program Director of Medicine at Interfaith Medical Center and as a Councilor for the American\r\nFederation for Medical Research. As a true academician and researcher, he has more than 50 papers indexed in international peer-reviewed journals. He has also presented numerous papers in multiple national and international scientific conferences. His areas of research interest include general internal medicine, gastroenterology and hepatology. He serves as an editor, editorial board member and reviewer for multiple international journals. His research on Hepatitis C has been very successful and has led to multiple research awards, including the 'Equity in Prevention and Treatment Award” from the New York Department of Health Viral Hepatitis Symposium (2018) and the 'Presidential Poster Award” awarded to him by the American College of Gastroenterology (2018). He was also awarded 'Outstanding Clinician in General Medicine” by Venus International Foundation for his extensive research expertise and services, perform over and above the standard expected in the advancement of healthcare, patient safety and quality of care.",institutionString:"Interfaith Medical Center",institution:{name:"Interfaith Medical Center",country:{name:"United States of America"}}},{id:"93517",title:"Dr.",name:"Clement",middleName:"Adebajo",surname:"Meseko",slug:"clement-meseko",fullName:"Clement Meseko",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/93517/images/system/93517.jpg",biography:"Dr. Clement Meseko obtained DVM and PhD degree in Veterinary Medicine and Virology respectively. He has worked for over 20 years in both private and public sectors including the academia, contributing to knowledge and control of infectious disease. Through the application of epidemiological skill, classical and molecular virological skills, he investigates viruses of economic and public health importance for the mitigation of the negative impact on people, animal and the environment in the context of Onehealth. \r\nDr. Meseko’s field experience on animal and zoonotic diseases and pathogen dynamics at the human-animal interface over the years shaped his carrier in research and scientific inquiries. He has been part of the investigation of Highly Pathogenic Avian Influenza incursions in sub Saharan Africa and monitors swine Influenza (Pandemic influenza Virus) agro-ecology and potential for interspecies transmission. He has authored and reviewed a number of journal articles and book chapters.",institutionString:"National Veterinary Research Institute",institution:{name:"National Veterinary Research Institute",country:{name:"Nigeria"}}},{id:"158026",title:"Prof.",name:"Shailendra K.",middleName:null,surname:"Saxena",slug:"shailendra-k.-saxena",fullName:"Shailendra K. Saxena",position:null,profilePictureURL:"https://s3.us-east-1.amazonaws.com/intech-files/0030O00002bRET3QAO/Profile_Picture_2022-05-10T10:10:26.jpeg",biography:"Professor Dr. Shailendra K. Saxena is a vice dean and professor at King George's Medical University, Lucknow, India. His research interests involve understanding the molecular mechanisms of host defense during human viral infections and developing new predictive, preventive, and therapeutic strategies for them using Japanese encephalitis virus (JEV), HIV, and emerging viruses as a model via stem cell and cell culture technologies. His research work has been published in various high-impact factor journals (Science, PNAS, Nature Medicine) with a high number of citations. He has received many awards and honors in India and abroad including various Young Scientist Awards, BBSRC India Partnering Award, and Dr. JC Bose National Award of Department of Biotechnology, Min. of Science and Technology, Govt. of India. Dr. Saxena is a fellow of various international societies/academies including the Royal College of Pathologists, United Kingdom; Royal Society of Medicine, London; Royal Society of Biology, United Kingdom; Royal Society of Chemistry, London; and Academy of Translational Medicine Professionals, Austria. He was named a Global Leader in Science by The Scientist. He is also an international opinion leader/expert in vaccination for Japanese encephalitis by IPIC (UK).",institutionString:"King George's Medical University",institution:{name:"King George's Medical University",country:{name:"India"}}},{id:"94928",title:"Dr.",name:"Takuo",middleName:null,surname:"Mizukami",slug:"takuo-mizukami",fullName:"Takuo Mizukami",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/94928/images/6402_n.jpg",biography:null,institutionString:null,institution:{name:"National Institute of Infectious Diseases",country:{name:"Japan"}}},{id:"233433",title:"Dr.",name:"Yulia",middleName:null,surname:"Desheva",slug:"yulia-desheva",fullName:"Yulia Desheva",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/233433/images/system/233433.png",biography:"Dr. Yulia Desheva is a leading researcher at the Institute of Experimental Medicine, St. Petersburg, Russia. She is a professor in the Stomatology Faculty, St. Petersburg State University. She has expertise in the development and evaluation of a wide range of live mucosal vaccines against influenza and bacterial complications. Her research interests include immunity against influenza and COVID-19 and the development of immunization schemes for high-risk individuals.",institutionString:'Federal State Budgetary Scientific Institution "Institute of Experimental Medicine"',institution:null},{id:"238958",title:"Mr.",name:"Atamjit",middleName:null,surname:"Singh",slug:"atamjit-singh",fullName:"Atamjit Singh",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/238958/images/6575_n.jpg",biography:null,institutionString:null,institution:null},{id:"333753",title:"Dr.",name:"Rais",middleName:null,surname:"Ahmed",slug:"rais-ahmed",fullName:"Rais Ahmed",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/333753/images/20168_n.jpg",biography:null,institutionString:null,institution:null},{id:"252058",title:"M.Sc.",name:"Juan",middleName:null,surname:"Sulca",slug:"juan-sulca",fullName:"Juan Sulca",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/252058/images/12834_n.jpg",biography:null,institutionString:null,institution:null},{id:"191392",title:"Dr.",name:"Marimuthu",middleName:null,surname:"Govindarajan",slug:"marimuthu-govindarajan",fullName:"Marimuthu Govindarajan",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/191392/images/5828_n.jpg",biography:"Dr. M. Govindarajan completed his BSc degree in Zoology at Government Arts College (Autonomous), Kumbakonam, and MSc, MPhil, and PhD degrees at Annamalai University, Annamalai Nagar, Tamil Nadu, India. He is serving as an assistant professor at the Department of Zoology, Annamalai University. His research interests include isolation, identification, and characterization of biologically active molecules from plants and microbes. He has identified more than 20 pure compounds with high mosquitocidal activity and also conducted high-quality research on photochemistry and nanosynthesis. He has published more than 150 studies in journals with impact factor and 2 books in Lambert Academic Publishing, Germany. He serves as an editorial board member in various national and international scientific journals.",institutionString:null,institution:null},{id:"274660",title:"Dr.",name:"Damodar",middleName:null,surname:"Paudel",slug:"damodar-paudel",fullName:"Damodar Paudel",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/274660/images/8176_n.jpg",biography:"I am DrDamodar Paudel,currently working as consultant Physician in Nepal police Hospital.",institutionString:null,institution:null},{id:"241562",title:"Dr.",name:"Melvin",middleName:null,surname:"Sanicas",slug:"melvin-sanicas",fullName:"Melvin Sanicas",position:null,profilePictureURL:"https://mts.intechopen.com/storage/users/241562/images/6699_n.jpg",biography:null,institutionString:null,institution:null},{id:"337446",title:"Dr.",name:"Maria",middleName:null,surname:"Zavala-Colon",slug:"maria-zavala-colon",fullName:"Maria Zavala-Colon",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"University of Puerto Rico, Medical Sciences Campus",country:{name:"United States of America"}}},{id:"338856",title:"Mrs.",name:"Nur Alvira",middleName:null,surname:"Pascawati",slug:"nur-alvira-pascawati",fullName:"Nur Alvira Pascawati",position:null,profilePictureURL:"//cdnintech.com/web/frontend/www/assets/author.svg",biography:null,institutionString:null,institution:{name:"Universitas Respati Yogyakarta",country:{name:"Indonesia"}}},{id:"441116",title:"Dr.",name:"Jovanka M.",middleName:null,surname:"Voyich",slug:"jovanka-m.-voyich",fullName:"Jovanka M. 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In recent years, emerging technologies such as multi-omics, high-throughput technologies, and genome editing tools could assist plant physiologists in unraveling molecular mechanisms in specific critical pathways. The global picture of physiological processes in plants needs to be investigated continually to increase our knowledge, and the resulting technologies will benefit sustainable agriculture.",coverUrl:"https://cdn.intechopen.com/series_topics/covers/13.jpg",hasOnlineFirst:!0,hasPublishedBooks:!0,annualVolume:11409,editor:{id:"332229",title:"Prof.",name:"Jen-Tsung",middleName:null,surname:"Chen",slug:"jen-tsung-chen",fullName:"Jen-Tsung Chen",profilePictureURL:"https://mts.intechopen.com/storage/users/332229/images/system/332229.png",biography:"Dr. Jen-Tsung Chen is currently a professor at the National University of Kaohsiung, Taiwan. He teaches cell biology, genomics, proteomics, medicinal plant biotechnology, and plant tissue culture. Dr. Chen\\'s research interests include bioactive compounds, chromatography techniques, in vitro culture, medicinal plants, phytochemicals, and plant biotechnology. He has published more than ninety scientific papers and serves as an editorial board member for Plant Methods, Biomolecules, and International Journal of Molecular Sciences.",institutionString:"National University of Kaohsiung",institution:{name:"National University of Kaohsiung",institutionURL:null,country:{name:"Taiwan"}}},editorTwo:null,editorThree:null,series:{id:"10",title:"Physiology",doi:"10.5772/intechopen.72796",issn:"2631-8261"},editorialBoard:[{id:"313856",title:"Dr.",name:"Christophe",middleName:"F.E.",surname:"Hano",slug:"christophe-hano",fullName:"Christophe Hano",profilePictureURL:"https://mts.intechopen.com/storage/users/313856/images/system/313856.png",institutionString:"University of Orléans",institution:{name:"University of Orléans",institutionURL:null,country:{name:"France"}}},{id:"33993",title:"Dr.",name:"Jose Carlos",middleName:null,surname:"Jimenez-Lopez",slug:"jose-carlos-jimenez-lopez",fullName:"Jose Carlos Jimenez-Lopez",profilePictureURL:"https://mts.intechopen.com/storage/users/33993/images/system/33993.jpg",institutionString:null,institution:{name:"Spanish National Research Council",institutionURL:null,country:{name:"Spain"}}},{id:"191770",title:"Dr.",name:"Mohamed A.",middleName:null,surname:"El-Esawi",slug:"mohamed-a.-el-esawi",fullName:"Mohamed A. 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