Staphylococcus aureus (MRSA), there have been few studies focused on the molecular characterization of methicillin-susceptible Staphylococcus aureus (MSSA). In this cross-sectional study, 85 MSSA isolates were characterized by antimicrobial susceptibility testing, virulence genes analysis, accessory gene regulator (agr) typing, and S. aureus protein A locus (spa) typing.Compared to methicillin-resistant agr types detected in tested strains were mainly type I (76.5%), II (12.9%), and III (10.6%). Of 85 MSSA examined isolates, 48 (56.5%) isolates were toxinogenic with 27 producing pvl (31.8%) and 21 tst (24.7%). The findings of the study show a high genetic diversity in MSSA strains warranting continued surveillance to provide critical insights into control and treatment of MSSA infections.In present study, 9 different clonal complexes namely CC8-MSSA-t037 (22.4%), CC8-MSSA-t008 (11.8%), CC7-MSSA-t091 and CC30-MSSA-t021 (each 9.4%), CC8-MSSA-t037 (8.3%), CC398-MSSA-t034 (7.1%), CC22-MSSA-t005 (5.9%), CC5-MSSA-t002 and CC15-MSSA-t084 (each 4.7%), CC22-MSSA-t790 and CC59-MSSA-t437 (each 3.5%), CC22-MSSA-t1869, CC5-MSSA-t045, and CC45-MSSA-t015 (each 2.3%), CC30-MSSA-t318 and CC15-MSSA-t491 (each 1.2%) were found.  Staphylococcus aureus is a common hospital- and community-acquired pathogen [S. aureus strain diversity appears to differ by geographic region, there has been a dramatic increase in the prevalence of S. aureus strains associated with human infections around the world and this appears to be especially true for methicillin-susceptible Staphylococcus aureus (MSSA) [S. aureus infections [pathogen . It is rpathogen . Althougs (MSSA) –5. MSSA s (MSSA) , 6. Comps (MSSA) . Antimicfections , 8. Knowfections , 10. AltS. aureus isolates phenotypically by using standard microbiological techniques. Polymerase chain reaction (PCR) assay targeting the S. aureus-specific nuc gene was applied to verify the isolates [S. aureus isolates susceptible to cefoxitin disc  in a disc diffusion assay using established methods (CLSI 2018) and negative for the presence of mecA gene by PCR were considered as MSSA strains [Eighty-five MSSA isolates were obtained from hospitalized patients at four hospitals affiliated to Shahid Beheshti University of Medical Sciences during an 9-month collection period from March 2019 to November 2019. This study protocol was approved by the Ethics Committee of the Shahid Beheshti University of Medical Sciences in Tehran, Iran (IR. SBMU. MSP.REC. 1398. 774). Furthermore, we confirmed isolates , 12. TheS. aureus ATCC 25923, ATCC 43300, and ATCC 29213 strains.In present study, Kirby–Bauer disk diffusion method was applied to determine the antimicrobial susceptibility of isolates based on the clinical and laboratory standards institute (CLSI) criteria (CLSI 2018). The antimicrobial agents included penicillin, teicoplanin, gentamicin, kanamycin, amikacin, tobramycin, clindamycin, erythromycin, tetracycline, linezolid, rifampicin, mupirocin, ciprofloxacin, quinupristin–dalfopristin, and trimethoprim–sulfamethoxazole . The minimal inhibitory concentrations (MIC) values of vancomycin was evaluated by broth microdilution method. Susceptibility test was quality controlled by using eta, and etb), Panton-Valentine leukotoxin gene (pvl), and toxic shock syndrome toxin (tsst-1) by PCR assay [S. aureus ATCC49775 and toxin positive S. aureus strains obtained from our previous were used as reference strains [S. aureus strain ATCC 25923 were also used as negative control.Genomic DNA was isolated using the phenol–chloroform extraction method. All of the isolates were screened for virulence encoding genes namely: exfoliative toxin genes (CR assay –14. The agr type detection using primer set comprising a common forward primer (Pan) and reverse primers  specific to each agr group [agr types were identified by comparing the banding patterns of isolates to RN6390 (agr type I), RN6607 (agr type II), RN8465 (agr type III), RN4550 (agr type IV), and RN6911 (negative control), as reference strains. PCR amplification was used for spa typing as described previously [spa gene amplified by PCR with forward (5′-AGACGATCCTTCGGTGAGC-3′) and reverse (5′-GCTTTTGCAATGTCATTTACTG-3′) primers. The purified PCR products were sequenced and then edited. The Ridom SpaServer database (http://www.spaserver.ridom.de) was applied to determine the spa type of each isolate. Each set of PCR reactions include a spa-type t008 isolate from our previous study as positive control sample (14), and a reaction mixture containing no template DNA as a control for possible false positive results.Multiplex PCR was performed for gr group . agr typeviously . In thispvl (31.8%) and 21 tst (24.7%).The sources of isolates included: skin and soft tissue wounds (44.7%), purulent exudates from wounds or abscesses 17.7%), urine (14.1%), blood (11.8%), sputum (8.2%), and other body fluids (3.5%). Out of 85 MSSA isolates, 29 isolates were obtained from hospital H1 (34.1%), 25 isolates from hospital H2 (29.4%), 20 isolates from hospital H3 (23.5%), and 11 isolates from hospital H4 (13%). Among 85 MSSA isolates tested, the highest rate of resistance was detected for penicillin (74.1%), and gentamicin (54.1%). , II (12.9%), and III (10.6%). spa results showed 16 types corresponding to nine clonal complexes (CCs), namely CC8 (42.3%), CC22 (11.8%), CC30 (10.6%), CC7 (9.4%), CC5 (7.1%), CC398 (7.1%), CC15 (5.9%), CC59 (3.5%), and CC45 (2.3%). spa type t037 was the most common spa type identified among 85 MSSA isolates, with a frequency of 22.3%, followed by t008 (11.8%), t021 and t091 (each 9.4%), t030 (8.2%), t034 (7%), t005 (5.9%), t084 and t002 (each 4.7%), t790 and t437 (each 3.5%), t1869, t045, and t015 (each 2.4%), t318 and t491 (each 1.2%) (Table 5%), II 1.9%, and Inducible clindamycin resistance was observed in CC8-MSSA-t037 (n = 5), CC30-MSSA-t021 (n = 4) CC8-MSSA-t008 (n = 3) isolates; while constitutive resistance phenotype was observed in CC8-MSSA-t037 (n = 8), CC8-MSSA-t030 (n = 3), CC-MSSA-t034 (n = 4), CC-MSSA-t091 (n = 5), CC-MSSA-t045 (n = 4), CC-MSSA-t005 (n = 4), and CC-MSSA-t790 (n = 1) isolates. Detailed results of the genotype distribution are presented in Table spa types among MSSA isolates. We detected CC8 in 42.3% of isolates. Although multi-resistant CC8 was previously reported as one of the main international CCs of MRSA, the predominance of the CC8-MSSA clone has previously been reported in Europe [Infection with MSSA as the most common pathogen in hospitalized patients is becoming increasingly problematic globally and requires special attention . As presn Europe  and Afrin Europe , 19. We n Europe , Irelandn Europe , and Nign Europe . We confn Europe , 21. As n Europe .We noted a relatively low prevalence of CC7 (9.4%), CC5 (7.1%), CC398 (7.1%), CC15 (5.9%), CC59 (3.5%), and CC45 (2.3%) in our study. A study conducted in China during the 4-year period indicated that CC22-t309 26.0%), CC188-t189 (5.1%), CC796-t796 (4.8%), CC121-t435 (4.8%), and CC398-t571 (3.6%) were the most dominant clones among MSSA isolates . They de.0%, CC18pvl encoding gene. Prevalence PVL positive MSSA strains has varied among studies from different geographic regions including China (34.4%) [Present results demonstrated that out of the 85 tested MSSA, 27 isolates 31.8%) carried 1.8% carr 34.4%) , Ireland (34.4%) , Colombi.4% , Ire (34.4%) , Russia  (34.4%) . In a re (34.4%) . However (34.4%) , 19. In  (34.4%) , 26, 28, (34.4%) .tst positive. This reported rate was different from the earlier studies in Africa (7%) [tst gene were associated with CC8-MSSA-t037 (11.8%), CC8-MSSA-t0307 (5.9%), CC22-MSSA-t790 (3.5%), and CC59-MSSA-t437 (3.5%) clones. A multicenter study from china reported that 4.0% of MSSA isolates carried tst gene which belonged to CC5 clone [tst-positive MSSA was distributed into four STs and the majority of them belonged to CC30 clone [tst encoding gene among S. aureus clinical isolates (21.3%) [tst were significantly higher in MSSA isolates in comparison to MRSA isolates (18.1% vs. 11.6%) [tst-positive MSSA clones seems geographically different and CC8 may represent a newly emerging clone in Iran.In our strain collection, 24.7% of isolates were ica (7%) , China (ica (7%)  and Turkica (7%) . Contrarica (7%) , 31 whicC5 clone . A recenSSA-t037 .8%, CC8- (21.3%) . In a st. 11.6%) . Overallagr genotypes are strictly associated with the clonal lineages [agr type I, as the most predominant type (76.4%), was associated with CC8, CC22, CC7, CC45, CC398, and CC59 isolates. In line with our results, Croes et al. demonstrated that CC7, CC8, CC22, CC25 and CC45 clonal lineages harbored agr I [agr type I (68.8%), followed by agr III (18.7%), and agr IV (12.5%) among MSSA isolates [According to the evidence, the lineages , 35. In ant type 6.4%, wasOur research had some limitations. Firstly, present work lacks detailed clinical information about the patients, Secondly, our samples were not collected consecutively. Thirdly, whole genome sequencing technique was not applied in the present work due to some of technical limitations.


Escherichia coli, Staphylococcus aureus, and Candida albicans showed a reduced proliferation of pathogens when compared to the polyethylene references. In particular, turmeric resulted in being more effective against E. coli when compared to neem, while they had similar performances against S. aureus. Against C. albicans, only neem was able to show a good antifungal behavior, at high concentrations. Tensile testing showed that the addition of reinforcing particles reduced the mechanical properties of polyethylene, and in the case of turmeric, it was further reduced by UV irradiation.With the increased scientific interest in green technologies, many researches have been focused on the production of polymeric composites containing naturally occurring reinforcing particles. Apart from increasing mechanical properties, these additions can have a wide range of interesting effects, such as increasing the resistance to bacterial and fungal colonization. In this work, different amounts of two different natural products, namely neem and turmeric, were added to polyethylene to act as a natural antibacterial and antifungal product for food packaging applications. Microscopic and spectroscopic characterization showed that fractions of up to 5% of these products could be dispersed into low-molecular weight polyethylene, while higher amounts could not be properly dispersed and resulted in an inhomogeneous, fragile composite. In vitro testing conducted with  Salmonella, Norovirus, Listeria, and Escherichia coli each year [Foodborne illnesses are a serious problem worldwide. Statistics from the Centers for Disease Control and Prevention report that one in six Americans suffer from foodborne illnesses, such as ach year , resultiach year  and thouach year .Annual reports of food poisoning cases around the world can be correlated with various factors such as climate , averageA forth key parameter is represented by “food hygiene”, which strongly depends on education. A survey conducted on food handlers in the plantation sector of Sri Lanka in 2016 showed that only about 60% of the 375 interviewed had good knowledge of food practice and hygiene, and these results could be well correlated with the levels of education . A 2018 Other than education, a major role is also played by the perception of risk: producers often tend to underestimate the levels of risk represented by their products, focusing more on quality , while mCodex Alimentarius” distributed by the Food and Agriculture Organization of the United Nations [The problem of food hygiene is not limited to businesses: consumers were demonstrated to be optimistically biased when considering the chances of food poisoning from self-prepared foods . General Nations .One of the most common sources of food poisoning arises from improperly cleaned and dried equipment  such as Polyethylene is the most common synthetic plastic material used in the world, accounting for about 34% of the total plastic market . This is due to the simple nature of its basic chemical structure, composed of ethylene groups (C 115 °C) .Polyethylene has low strength, hardness, and rigidity, but high ductility and impact strength and a low friction coefficient against most materials. Under constant load, it is easily subjected to high creep due to its low melting temperature.Many grades of polyethylene are commonly used in industry, ranging from low- (LDPE) to medium- (MDPE) or even high-density (HDPE), depending on the structure.3. It has more branching (about 2% of the carbon atoms) than HDPE, which results in weaker intermolecular forces, lower tensile strength, and higher resilience. Furthermore, because its molecules are less tightly packed and less crystalline due to the side branches, its density is lower. It is mainly applied in containers, corrosion-resistant surfaces, weldable parts, juice and milk cartons (where it is applied as a film), plastic wraps, and toys.LDPE is defined by a density range of 0.917–0.930 g/cmFor many years, packaging materials have been chosen solely by using three criteria; compatibility with food, stability at various conditions, and manufacturing costs .Recent trends towards greener technologies have pushed plastic companies and researchers to develop more environmentally friendly products, which can be bio-degraded , compostNeem is the common name for the tree Azadirachta indica, in the mahogany family Meliaceae. Neem is typically grown in tropical and sub-tropical regions where it is used in traditional medicine, as well as in clothes to protect against insect attack. It contains various bioactive chemicals, of which only two were reported to have antibacterial and antifungal properties, namely quercetin  and sitoS. aureus) and Gram negative (E. coli) bacteria and C. albicans fungi. Mechanical testing was also conducted to evaluate the effects of the particles on the strength of polyethylene and the material reaction to UV light exposure.In this research, turmeric and neem powders were added to polyethylene to produce bioactive composites with antibacterial and antiviral properties. The bioactive effects were tested against Gram positive . All images were collected at various magnifications ranging from 10× to 150×. Initial roughness values were measured on 10 randomized 100 × 100 2, and the acquisition time was set to 30 s. The instrument was operated using dedicated software . Ten different spectra were acquired for each sample before and after biological testing. The average spectra are presented, unless otherwise specified.Fourier Transformed Infra-Red spectroscopy (FTIR) spectra were collected at room temperature using an FTIR spectrometer  equipped with a Michelson 28 deg interferometer with corner-cube mirrors, covering a range between 250,000 and 5 cmS. aureus (NBRC 13276), E. coli (E1 NBRC 3972), and C. albicans (NBRC 1594) were cultured by the streak plate method. The first plate was prepared using bacteria beads in the agar plate. The second plate was prepared through four sector quadrant streaks using one colony from the first plate. Both plates were incubated under 37 E. coli, and 108 broth was used for C. albicans, then incubated on a shaking incubator  at 37 Two-hundred microliters of the bacterial and fungal solutions were dropped onto the surface of the samples (5 cm × 5 cm) and covered with parafilm (4 cm × 4 cm). Each sample was replicated three times to calculate the average. Then, the samples were arranged in a container and incubated for 24 h at 37 The samples with dimensions of 1 cm × 1 cm were treated in UV Form Cure  equipped with 13 LEDs at 405 nm. The temperature was maintained at 25 The tensile testing of the samples in dumbbell shapes was performed using the MCT 2150 Desktop Tensile-Compression Tester  at an elongation rate of 50 mm/min. The tensile properties were determined for three samples of each concentration.p > 0.05). Similarly, the total height of the maximum roughness amplitude (Rt) is increased with the addition of reinforcing particles, but even if the differences are statistically significant between the reference and the 5% neem samples, the increase is relatively small (about 20%).2 asymmetric stretching), 2850 cm2 symmetric stretching), 1475 cm3 symmetric deformation), 1365 cmBands related to polyethylene appear at about 2920 cmrmation) .The turmeric spectrum is similar to the ones previously reported in the literature for pure curcumin , featuri2.The spectrum of polyethylene is not much influenced by the presence of the turmeric powders as various intense bands, such as the ones located at about 1630 cm2 can be either absorbed by the composite when exposed to air or due to the interaction between curcumin and polyethylene.COThe spectrum of neem powder is quite similar to the reference for turmeric of Due to the complexity of the system, it is impossible to deconvolve the spectrum into its basic constituents. Nevertheless, it can be observed that the three regions, related to aromatic C-O stretching vibrations , the curcumin mixed into the polyethylene matrix is still able to react to the alkaline solution. Moreover, curcumin is released from the composite polymer into the solution, which also becomes red.For both the composite and solution, the intensity of the color is a function of the fraction of curcumin.E. coli for 24 h, as compared to the control culture. It can be observed that the antibacterial effect of turmeric is stronger than neem, even at relatively low concentrations. For neem, only a concentration of about 5.0% wt. was able to show a statistically significant decrease in CFU.S. aureus. Both neem and turmeric show comparable antibacterial properties against this specific strain, and the effects are already statistically significant at low concentrations. Overall, turmeric seems to be more effective than neem, particularly at lower concentrations.C. albicans. Unlike previous results against bacteria, low concentrations of neem and turmeric do not seem to have a statistically relevant anti-fungal effect, and only neem seems to have a strong effect against C. albicans at high concentrations.During this degradation, the breaking of the C-C bonds in the central chain is responsible for the loss in color emission.a contained in the leaves is also photochemically degraded into hydroxy-pheophytin a, pheophytin a, and hydroxy-lactone-pheophytin a [For neem, the original green pigmentation caused by the chlorophyll phytin a , thus le regions . FerulicMechanical testing results, performed in a tensile configuration on the samples with the highest fractions of reinforcing powders, are presented in The addition of turmeric not only reduces both the maximum load (from 5.5 to 4.7 MPa) and the deformation (from 27% to 18%), but it also makes the composite much more sensible to UV exposure. After just one hour of treatment, the load further decreases to about 4.5 MPa, and the elongation to about 15%; however, the properties stabilize afterwards, and no further decrease is observed for longer exposure times.Neem seems to have the greatest influence on the mechanical properties, reducing the elongation from 27% to about 15%, but the values are not much affected by UV exposure.ffsZ gene) in the Z-ring [The bioactive molecule responsible for most of the biological effects in turmeric as reported in the literature is curcumin. It has been postulated that the small curcumin molecule is able to penetrate both Gram positive and Gram negative bacteria, leading to a permanent damage of the membrane, which causes leakage . Moreovee Z-ring .E. coli (Gram negative) and S. aureus (Gram positive), have different membrane structures, curcumin was able to penetrate both, as suggested by the simple test of Figure S2. Five milliliters of PBS were used to wash the samples, and a small amount (100 Even if the two bacteria strains, For neem-reinforced composites, the antibacterial effect was supposed to be the result of the presence of both quercetin and sitosterol . While nC. albicans. The antifungal effect of neem powder is also associated with the presence of quercetin, which has shown effects against various strains of Candida [On the other hand, neem has shown greater efficiency against fungal infection of  Candida .The drawback of using neem or turmeric powders to produce a polymer matrix composite is the decrease of the mechanical properties. Both turmeric and neem cause a decrease of the mechanical properties, particularly elongation, with a loss of strength and elongation comparable to other polymer matrix composites from the literature .The better performances of turmeric-reinforced polyethylene when compared to neem-reinforced can be explained by considering the capability of curcumin to bond to the chains of polyethylene, resulting in a higher interface adhesion between the particles and the matrix.Combining polyethylene with turmeric makes the composite material more sensitive to UV irradiation, leading to a reduction of the mechanical properties over time. Even if the loss of the mechanical properties was somehow limited, short time UV irradiation did not improve the mechanical performances as in the case of pure polyethylene.Mechanical testing proved that the structure of turmeric-polyethylene composites can be degraded with UV exposure, but the degradation was not strong enough to lead to decomposition.In this research, up to 5% of natural products, namely turmeric and neem powders, were successfully added to polyethylene in order to obtain a composite material.Both neem and turmeric powders improve the antibacterial efficiency of polyethylene, with turmeric showing efficiency against both Gram positive and negative strains.C. albicans fungal colonization.Neem-based composites, on the other hand, were more effective against The mechanical strength of polyethylene was compromised by the presence of reinforcing particles, decreasing the maximum load and, more specifically, the elongation.


HOT graphene containing carbon hexagons, octagons, and tetragons. A corresponding series of nanotubes are also constructed by rolling up the HOT graphene sheet. Ab initio calculations are performed on geometric and electronic structures of the HOT graphene and the HOT graphene nanotubes. Dirac cone and high Fermi velocity are achieved in a non-hexagonal structure of HOT graphene, implying that the honeycomb structure is not an indispensable condition for Dirac fermions to exist. HOT graphene nanotubes show distinctive electronic structures depending on their topology. The  n (n ≥ 3) HOT graphene nanotubes reveal the characteristics of semimetals, while the other set of nanotubes  n shows continuously adjustable band gaps (0~ 0.51 eV) with tube size. A competition between the curvature effect and the zone-folding approximation determines the band gaps of the  n nanotubes. Novel conversion between semimetallicity and semiconductivity arises in ultra-small tubes .We report a new graphene allotrope named  In the lower dimension, the carbon nanotube is a honeycomb structure rolled into a hollow cylinder with nanometric diameter and μm length [kF shifting [Because of its bonding flexibility, carbon-based systems show an unlimited number of different structures with an equally large variety of physical properties. These physical properties are, in great part, the result of the dimensionality of these structures . Graphenm length –10. As tm length –13. The m length –18. Grapm length  or semicm length –21, withm length , 16. Furm length , and Dirm length . Such rem length . Comparishifting , 25, σ-πshifting  are intrshifting , 28, theshifting , 30, catshifting , sensorsshifting –35, transhifting , 36, etcshifting –39. Hencshifting . Howevershifting –42.HOT graphene. HOT graphene nanotubes were also constructed by rolling up HOT graphene sheet along with different directions. The electronic property, curvature effect, kF shifting effect, etc. of HOT graphene and nanotubes were calculated using ab initio calculations based on density function theory (DFT).In this study, we designed a new allotrope of graphene that has two-dimensional Dirac fermions without an exclusively hexagonal structure. The new allotrope was constructed with interlaced carbon hexagons, octagons, and tetragons, and was named HOT graphene and HOT graphene nanotubes were performed by using a first-principles method based on the density-functional theory (DFT) with the generalized gradient approximation (GGA) in the form of Perdew-Burke-Ernzerh (PBE) exchange-correlation functional [k-point mesh resolution of 2π × 0.03 Å− 1. The unit cell basis vectors (representing unit cell shape and size) and atomic coordinates were fully relaxed in each system until the forces on all the atoms were smaller than 0.01 eV/Å.The present calculations on nctional , as implnctional , 45. Thenctional  samplingHOT graphene  adjacent to Γ. The 3D band structure  of the Dirac fermions, evaluated from the gradient of the linear dispersions of the band structures, is 6.27 × 105 m/s, which is a little lower than 8.1 × 105 m/s [5 m/s [F implies high mobility of carriers in the HOT graphene.The geometric structure of ene Fig. a shows aure Fig.  presents 105 m/s  for grapHOT graphene tubes are rolled up from the HOT graphene sheet in various directions symbolized by an index on the 2D HOT graphene lattice  n basing on two unit vectors a and b. Symbol  [HOT graphene sheet, n stands for the number of perimeter units , are selected in our study. This is because the helical arrangement of atoms in other directions is barely periodic and possesses a nearly infinite length of unit cell along the tube axis. Such a large unit cell is unrealistic in our calculation. Different rolling directions between  n and  n tubes result in the differences in geometry and bonding situation. Two tubes, 6 and 4, are depicted in Fig. 4–C6–C8 (orange) and C8–C6–C4 (blue) which is exactly opposite to each other. These two opposite patterns alternate along the circumferential direction of the tube. In direction , polygons arranging along the tube axis also have two patterns: C4–C8 (blue) and C6–C6 (orange). Two C4–C8 patterns alternate with one C6–C6 pattern along the circumferential direction.The ice Fig. a. This iol  , 20, 21 its Fig. b and c uEcur as:n tubes exhibit a lower energy cost than the graphene nanotubes while the  n tubes are nearly the same as the armchair graphene nanotubes except for several ultra-small tubes. Such results also suggest that it is possible to synthesize the HOT graphene nanotubes in experiments. It should be noted that tube 1 is too small because its diameter is even shorter than the bond length of carbon.To reveal the energy cost in rolling up a sheet into tubes, we define the curvature energy Fig.  Ecur as:HOT graphene sheet is rolled up into a nanotube, its 2D BZ reduces to 1D BZ as a result of the new periodic boundary conditions in nanotubes. The periodic boundary conditions along the circumferential direction of the tube only allow wave vectors “around” the nanotube circumference and these vectors are quantized [k lines [HOT graphene always cross the Dirac point  n nanotubes. Tube 6 is calculated to evaluate the semimetallicity of this set of  n nanotubes in Fig. HOT graphene nanotubes shows a crossing point of energy bands at the Fermi level and the corresponding DOS shows no states at the Fermi level which verifies the semimetallicity of the system. As the Dirac cone in  n tubes is originated from the HOT graphene sheet, the calculated coordinate of the Dirac point in the 6 nanotube is the same as that in HOT graphene sheet which is . The calculated fermi velocity at the Dirac point in 6 nanotube is 6.76 × 105 m/s, close to 6.27 × 105 m/s in the HOT graphene sheet.The calculated electronic band structure and DOS of nanotube 6 Fig. b indicatuantized . The per[k lines . As the n nanotubes with different tube radii  n are semimetallic (n ≥ 3) and transform to metal (n = 2) and then turn back to semimetal (n = 1). Such a change under a small radius resulted from the so-called curvature effect [k point at which occupied and unoccupied bands do cross (at a point we label kF) and shift the kF away from the original Dirac points, which is called the kF shifting effect [n HOT graphene nanotubes, the kF shifts away from its original position (kHOT) in the HOT graphene sheet ). According to the calculated coordination of Dirac points, the kF shifting effect becomes non-negligible at 5 with a tube radius of 5.988 Å, whose Dirac point (kF) shifts to  from original point (kHOT) at  in HOT graphene sheet. As the tube radius gets smaller, the kF keeps shifting and reaches point  in tube 3. In tube 2, the kF shifts to  where the Dirac point moves down below the Fermi level, resulting in a metallic system. The vanishing of the semimetallicity in 2 indicates a deviation from the kF shifting effect in the  n nanotubes (n ≥ 3). Moreover, the 1 tube becomes semimetallic again in its band structure and the DOS 1 shows a σ-π hybridization that a low-lying σ* band intersects the Fermi level and joins the Dirac cone  n nanotubes (~6.76 × 105 m/s). An obviously different shape of its band structure is also shown in Fig. HOT graphene nanotube 1 is originated from the σ-π hybridization. In summary, with the increasing curvature, the kF shifting effect emerges at n = 5, becomes more effective at 4 ≥ n ≥ 2, and is finally replaced by the σ-π hybridization effect at n = 1.The band structure evolution of  dii Fig. a revealse effect . In the g effect , 25. As  to 0,0.026,0 fromanotubes , 24. HowHOT graphene . The valence band maximum (VBM) and conduction band minimum (CBM) get closer from tube 6 to tube 4 and then crosses with each other in tube 3 with a radius of 2.17 Å. This crossing point is exactly on the Fermi level located at . The calculated DOS  indicates that the band gap is adjustable with tube sizes. Also, instead of being monotonous, the dependence of the band gap as the tube size has a zigzag shape , a band gap will appear in nanotube. Furthermore, this distance between kF and the k lines is proportional to the band gaps, as the band dispersion near the Dirac cone is linear [Δkm measures the shortest distance between kF and km lines of quantum number m. We calculated this distance Δkm between the Dirac points (kHOT) and the allowed k lines in the HOT graphene and plot it (red line) together with the band gaps (black line) in Fig. k lines have a quantum number m. When n ≤ 7  is between the kHOT and the first k line, k1, which sits at the Γ point constantly (insert graph (a) in Fig. Δkm is constant as both k1 and kHOT are constant independents of the tube size. However, as the allowed k lines become denser in larger nanotubes (7 ≤ n ≤ 17), the k2 becomes the nearest one to kHOT . n = 17 is a turning point where k2 almost intersects the Dirac point resulting in a local minimum of the distance Δkm . And it is revealed that the band gaps change in cycles: the km gets closer to the Dirac point (kHOT) causing a decline of band gap, then traverses the Dirac point resulting in a local minimum, then gets farther from the Dirac point causing a rising of band gap, and is finally replaced by the next line km + 1 entering the next cycle. In summary, the reason why the band gaps are changing with tube size (n ≥ 7) is that the k lines are moving with different tube sizes, thereby the change the distance Δkm between kHOT and the allowed k lines which is proportional to the band gaps.In another rolling direction, the calculated band structure of 6 Fig. b shows sene Fig. a, and opene Fig. b. In thiDOS Fig. b presentd at 0.071,0,0. Thd at 0.071,0,0. Thd at 0.071,0,0. Th k lines . Thus, ts linear . The Δkmd at 0.071,0,0. Thd at 0.071,0,0. Thd at 0.071,0,0. Thd at 0.071,0,0. Thd at 0.071,0,0. Thd at 0.071,0,0. Thd at 0.071,0,0. Thance Δkm , the banΔkm curve and the band gap curve have similarities in shape, the differences between them are also obvious, which is that the Δkm plot shows a “delay” in change at n ≥ 7 and becomes completely different from the band gap plot at 3 ≤ n ≤ 7. The reason is that the Fermi point in HOT graphene nanotubes (kF) is assumed to have the same coordinate as the original Fermi point in HOT graphene (kHOT) in the preceding section where the band gap changing was explained. However, the Fermi point (kF) in nanotubes shifts away from the origin Fermi point (kHOT) in HOT graphene sheet under the curvature effect. Therefore, the kF shifting (ΔkF = kF–kHOT) effect contributes to the mismatches between the distance Δkm and the band gaps. As calculated in the  n tubes  n tubes also shifts to the outer BZ towards the symmetric point X under the curvature. Therefore, when the nearest km sits between the kHOT and the Γ point, the distance Δkm underestimates the band gap , the kF shifting effect is enhanced under the large growth rate of the curvature; consequently, it causes a drastic kF shifting and changes the band gap. When the radius is getting smaller than n = 8, the k1 becomes the closest to the kHOT indicating a constant Δkm. However, the kF shifting effect is so strong to move the kF farther from k1 but closer to the k2 . The kF keeps shifting to the k2 at a high velocity so that the distance between kF and k2 gets smaller and smaller. Therefore, from 7 to 3, the band gap decreases 3 that gives rise to the semimetallicity 2 , while the k line moving leads in big tubes (n ≥ 8) where the kF shifting effect is faded. The amount of the kF shifting is estimated to be 0.0015 2π/Å at n = 15 and 0.0238 2π/Å at n = 3 as the band gaps are 0 eV where the shifted kF point is on the allowed k lines. It can be seen that the kF shifting is 15.86 times bigger in a small tube 3) than in a big tube 15).Though the bes Fig. , the kF  k2 Fig. e. The kFses Fig. . At last = 3Fig. f. This cHOT graphene, carbon atoms are all three-fold coordinated, thus the fourth valence electron plays a key role in its conductivity. The calculated band decomposed charge density ± 0.15 eV around the Fermi level . In another set of HOT graphene nanotubes, the band gaps show adjustability with different tube radius 15 nanotube show the different distribution of electrons from the semiconductive tubes. More importantly, it exhibits a similar distribution to the semimetallic HOT graphene sheet and  n tubes. The localized π bond of 15 only locates on the 8–8 bonds. This redistribution of electron in  n tubes causes the conductivity change.In vel Fig.  shows thvel Fig. a have anvel Fig. b shows ting Fig. e in nanoing Fig. e. Stronging Fig. . Therefoius Fig. . The chaius Fig. a. The CBius Fig. b. The 4–HOT graphene is constructed by carbon hexagons, octagons, and tetragons showing Dirac cone and high Fermi velocity, which implies that the honeycomb structure is not an indispensable condition for Dirac fermions to exist. The semiconductivity of HOT graphene is dependent on the localized π bonding. A corresponding series of nanotubes is rolled up from the HOT graphene sheet and shows distinctive electronic structures depending on the topology. The set of  n (n ≥ 3) HOT graphene nanotubes reveals a character of semimetallicity and Dirac cones that are composed by π states. A non-negligible kF shifting along the allowed k line arises under the curvature effect when the tube radius gets smaller (3 ≤ n ≤ 5). However, the ultra-small nanotube 2 begins to deviate from the π state-based kF shifting effect showing a transformation to metallicity. Finally, an σ-π hybridization takes the place of the π states at Fermi level in nanotube 1, where a low-lying σ* band intersection appears at the Fermi level and forms a semimetallicity again. Another set of tubes  n shows various band gaps (0~ 0.51 eV), which is continuously adjustable with the tube size. The band gaps of  n (n ≥ 3) nanotubes turn out to be determined by a competition mechanism between the k line moving and the kF shifting effect. The zone-folding approximation indicates a k line moving and results in the zigzag and periodical band gap changing curve in big tubes (n ≥ 8), while the kF shifting effect gets stronger and causes a dramatic decrease of band gaps in small tubes (7 ≥ n ≥ 3). Zero-gap semimetallic tubes appear periodically under the competition.A new graphene allotrope named


Experimental reproducibility in aquatic microbial ecology is critical to predict the dynamics of microbial communities. However, controlling the initial composition of naturally occurring microbial communities that will be used as the inoculum in experimental setups is challenging, because a proper method for the preservation of those communities is lacking. To provide a feasible method for preservation and resuscitation of natural aquatic prokaryote assemblages, we developed a cryopreservation procedure applied to natural aquatic prokaryotic communities. We studied the impact of inoculum size, processing time, and storage time on the success of resuscitation. We further assessed the effect of different growth media supplemented with dissolved organic matter (DOM) prepared from naturally occurring microorganisms on the recovery of the initially cryopreserved communities obtained from two sites that have contrasting trophic status and environmental heterogeneity. Our results demonstrated that the variability of the resuscitation process among replicates decreased with increasing inoculum size. The degree of similarity between initial and resuscitated communities was influenced by both the growth medium and origin of the community. We further demonstrated that depending on the inoculum source, 45–72% of the abundant species in the initially natural microbial communities could be detected as viable cells after cryopreservation. Processing time and long-term storage up to 12 months did not significantly influence the community composition after resuscitation. However, based on our results, we recommend keeping handling time to a minimum and ensure identical incubation conditions for repeated resuscitations from cryo-preserved aliquots at different time points. Given our results, we recommend cryopreservation as a promising tool to advance experimental research in the field of microbial ecology. Experimental studies have played a central role in the field of microbial ecology over the past 55 years  and haveHowever, their confidence and reproducibility rely on maintaining comparable initial conditions, which include the identity and density of organisms in the starting community. Differences in community initial composition and inherent environmental variability may be propagated and amplified during the course of an experiment , effectiThe assembly of bacterial strains into artificial communities to be used as starting communities in experimental setups is one option to circumvent these caveats and it has been applied in multiple studies to test ecological theory, such as the biodiversity-ecosystem functioning (BEF) relationships e.g., . ExperimTo better link the findings from experiments performed on a limited number of strains with processes taking place in natural environments, experimental manipulation of naturally co-occurring microbes is a valuable tool e.g., . HoweverThe composition of natural microbial communities is highly dynamic and time-space dependent, with concomitant short- and long-time phenomena affecting their succession at synoptical and seasonal scales or due to regime shifts . We suggCryopreservation is the preservation of biological material at cryogenic ultralow temperatures (−80 or −196°C) for its optimal long-term preservation . It is uThe effectivity of cryopreservation has been verified for both axenic and non-axenic cultures during the last decades . More reEven if cells remain viable after cryopreservation, appropriate thawing and culturing conditions are required to recover the activity of individual community members and effectively resuscitate the preserved communities . HoweverCulture media based on the original environmental conditions, such as filtered seawater and artificial fresh- or sea-water amended with low concentration of nutrients, have been shown to increase the culturing success . It has In this study, we propose a method to improve the control of initial microbial communities of experiments through the cryopreservation and resuscitation of natural aquatic microbial assemblages in either sterile filtered seawater or artificial seawater (ASW) with DOM supplements that were prepared from the particulate organic fraction of seawater . The met–1 with rather stable salinity concentrations (33.20–39.99 PSU) and Canet is a shallow coastal Lagoon characterized by high productivity and large-scale salinity changes  to retain bacteria and all particles in larger size classes. These filters were then stored frozen (−20°C) until further processing.Either sterile filtered water from the SOLA station, hereafter F-SEA, filtered through 0.22 μm, 47 mm cellulose filter  or inorganic ASW basic medium  supplemeTM, Fisher Scientific, Strasbourg, France) while immersed in liquid nitrogen. Next, the filter pieces were resuspended in a known volume of ultrapure water (60 mL) and autoclaved at 120°C for 20 min. Subsequently, the sterile suspensions containing the filter pieces were filtered through pre-combusted  GF/F 47 mm filters  to remove particles. Finally, the obtained DOM supplements were stored frozen at −20°C until use.To prepare the DOM used as supplement, the filters were ground in a stainless-steel mortar and pestle  and the second containing mixed DOM from SOLA and the Warnow River (hereafter SW-DOM). S-DOM obtained from the equivalent of 0.1 L of sample water was added to 1 L of ASW medium, while DOM supplements from the equivalent of 0.05 L from the sample water obtained from SOLA and 0.17 L from the Warnow River were added to 1 L ASW medium in the SW-DOM medium.3–, NO2–, and PO43–) were determined. Aliquots of the water samples and DOM concentrates were filtered through two pre-combusted  25 mm GF/F filters . Aliquots for DOC measurements (10 mL) were stored in combusted glass ampoules and acidified . The ampoules were closed and maintained at room temperature until analyzed on a Shimadzu TOC-V following a protocol published elsewhere (3–), nitrite (NO2–), and phosphate (PO43–) were determined by standard colorimetric technique  , dissolved organic nitrogen (DON), dissolved organic phosphorous (DOP), and nutrients  .8 cells/mL, 5 to 106 cells/mL  using a vacuum pump (∼600 mg Hg) in order to concentrate prokaryotic cells. Drying of the filters was carefully avoided during the filtration, except at its end, when the pressure was set to a minimum, to prevent cell damage, and the final water volume covering the filter was slowly filtered. Immediately after removal of the sample water, the pump was turned off and 0.5 mL of inorganic ASW medium was added to the top of the filter, which was then used to resuspend the cells from the filter by pipetting up and down. This volume was subsequently transferred into previously prepared 2 mL tubes with 0.5 mL cryoprotectant containing 10% (v/v) sterile-filtered DMSO  in inorganic ASW, equilibrated at 4°C . Next, the filters were completely immersed into the medium with cryoprotectant and resuspend cells and the tubes were kept at 4°C for 15 min for equilibration . The tubWe additionally prepared 10 mL aliquots of 0.8 μm pre-filtered water samples without prior cell concentration via filtration. Sterile-filtered DMSO was added as cryoprotectant . Then, the aliquots were kept for 15 min at 4°C before flash-freezing in liquid nitrogen and transferred to −80°C for long-term storage .The abundance of prokaryotic cells in the 0.8 μm pre-filtered inoculum water of SOLA and Canet was estimated via flow cytometry as detailed elsewhere . In shorAdditionally to the preparation of filters with cryopreserved communities, 1.5 L of the 0.8 μm  pre-filtered water from both locations was filtered onto 0.22 μm filters  for later DNA extraction and 16s rRNA gene sequencing to characterize the initial communities.in situ temperature  and then added (together with the filter where applicable) to the culture medium. The cells were incubated for 5–6 days close to the 15–18°C,  in glass15–18°C, . In all 6 cells/mL). For experiment 1b, cryopreserved water aliquots  from the 0.8 μm prefiltered SOLA seawater were diluted 10-fold and resuscitated in triplicates in S-DOM medium . The starting inocula in this experiment contained 1.32, 3.30, 6.60, and 9.90 million cells, respectively. The resuscitated cells in both experiments were incubated for 6 days , and cryopreserved water aliquots from 0.8 μm pre-filtered SOLA seawater (experiment 1b). For experiment 1a, filters containing concentrated cryopreserved communities were prepared from 100, 200, 500, 750, and 1500 mL 0.8 μm pre-filtered SOLA seawater. These inocula contained, according to the results from flow cytometry analyses, 66, 132, 330, 495, and 990 million cells in total. Triplicate cryopreserved communities of each volume were resuscitated in specific volumes of S-DOM medium  in order to ensure constant initial cell abundance in the incubations (1.1 × 10r 6 days . Since l8 cells) and Canet (8.04 × 108 cells) were incubated in 900 mL medium in 1 L Duran Schott bottles. Triplicate SOLA inocula were incubated in F-SEA, ASW supplemented with S-DOM extract, and ASW supplemented with SW-DOM extract. Triplicate Canet inocula were incubated only in ASW supplemented with S-DOM or SW-DOM. The water of the F-SEA treatment was prepared at the same day as the cryopreserved SOLA community and inoculated on the evening of that same day after the communities had been cryopreserved for a few hours. This was done to avoid or minimize chemical changes in the F-SEA medium that could occur during prolonged storage time. The incubation of SOLA communities in F-SEA was performed to test the resuscitation procedure in an environment as close as possible to the natural environment of the initial SOLA community. Incubations in the S-DOM and SW-DOM media differed in their C:N:P ratio  were resuscitated in 900 mL of S-DOM medium. In experiment 4, three sets of cryopreserved filter-concentrated communities from SOLA (4.12 × 108 cells) were resuscitated after cryopreservation for 1.5, 6, and 12 months. The resuscitation was performed in 900 mL S-DOM medium in 1 L Duran Schott bottles. In both experiments, the communities were incubated for 5–6 days . Concentration and quality of the eluted DNA were tested by electrophoresis (1% agarose gel) and Quantus fluorometer using the PicoGreen assay .Filters for DNA extractions were stored at −30°C. DNA extraction was performed after cutting the filters in small pieces and using a modified protocol of the QIAmp DNA Mini Kit  with an initial bead-beating step in ATL buffer using a FastPrep-241, with some modifications (R-code is available in https://github.com/angelrainf/gesifus.cryo.dada2). A total of 700 amplicon sequence variants (ASVs) were identified across all sequenced samples and the obtained ASVs were taxonomically assigned using the Genome Taxonomy Database (GTDB)  using the primers pair 515yf-926r . For thee (GTDB) . Raw seqDifferences in the community composition of incubations in experiments 3 and 4 were estimated using ARISA analysis. Extracted DNA from the communities was amplified with KAPA2G Fast Ready Mix  in 20 μL reactions using 10 μM of the primers 1406F and 23SRY  and 1 μL®, Foster City, CA, United States).PCR products were subsequently purified through Sephadex G-50 fine  and quantified with a Quantus fluorometer with the Picogreen assay . Then, 2.5 μL of purified PCR products was mixed with 0.2 μL of MapMarker1000  and 15 μL of formamide  and, denatured at 95°C for 3 min. Samples were then sequenced in a Sequencer 3130XL  and the areas of the identified T-RF peaks were imported into the T-Rex  online software for noise filtering, alignment, and binning by default parameters  were identified using the GeneMapper software  to statistically compare the Shannon diversity between initial and resuscitated communities. For this purpose, we used the mean Shannon diversity of the technical replicates from the initial community as a reference value and we subtracted Shannon diversities from the triplicate incubations from this mean. The one-sample t-test was then applied to test the null hypothesis that the difference in diversity between the initial sample used as inoculum and the incubations did not differ from zero.In experiment 2, we compared initial communities of SOLA and Canet with resuscitated communities after several days of incubation. However, while triplicate values in the initial communities represent technical replicates, triplicates after resuscitation represent biological replicates with presumably larger variance. Differences in variance among compared datasets violate assumptions for two-sample To assess and subsequently visualize the similarity of resuscitated communities to the initial starting communities in dependence of the growth medium and inoculum (experiment 2), pairwise Bray–Curtis distance indices between all communities were calculated. This was done based on the relative abundance of ASVs in each community obtained after rarefication to the minimum number of processed reads obtained for the individual libraries  that exhibited cell growth and that were thus viable during the incubations. In this analysis, we compared absolute abundance estimates of individual ASVs in the initial communities with estimates obtained from the resuscitated communities. Absolute abundances of individual ASVs were calculated by multiplying the relative abundances of each ASV by the total cell counts of the communities from which they were identified. Because of the biases during DNA extraction and/or amplification, this is not a proper estimation of the absolute cell numbers of individual ASVs within a community . HoweverLog2-fold-change (L2FC) of the absolute abundances of each ASV exceeding 0.5% relative abundance in the initial sample between the incubation start (day 0) and the final incubation day was used to estimate their growth in each incubation. Positive L2FC values indicate that the tested ASV exhibited more cells in the incubation medium after 5 days than in the starting community, implying that cells affiliating with this ASV were actively replicating.t-test (two-tailed) to the replicate L2FC values of each AVS per treatment . In the F-SEA incubations, we omitted one of the three replicates from the t-test, where the community was different from the other two replicates due to the overgrow of members of the Enterobacterales order. ASVs with (significantly) positive L2FC in at least one medium were considered as viable. P-values were adjusted with the Benjamini–Hochberg correction to account for false discovery rates during multiple comparisons , we furthermore applied a Mantel test between the Bray–Curtis distance matrix and the Euclidean distance matrix of storage time to evaluate the effect of storage time on the dissimilarity among samples.To visualize the effect of handling and storage time, Hierarchical Clustering (Ward.D2) was performed on the normalized ARISA community fingerprint data based on Bray–Curtis distances from the incubations obtained in experiments 3 and 4. Analyses of similarities  were performed to evaluate the effect of the handling and storage time. In the case of the storage time analysis, an additional 6 cells/mL), and the difference was generally greater in incubations with smaller inoculum, except for the smallest inoculum in experiment 1a (The resuscitation and subsequent incubation of cryopreserved inocula in S-DOM media succeeded independently from the inoculum type (concentrated by filtration or sample water) and inoculum size . Both iniment 1a .We observed for both inoculum types that the variance among replicates tended to decrease with increasing inoculum size . Moreove6 cells/mL) compared to the S-DOM (0.80 ± 0.26 × 106 cells/mL) and SW-DOM (0.43 ± 0.13 × 106 cells/mL), a difference caused by one of the F-SEA triplicate incubations reaching much higher abundances than the other two replicates . On the other hand, the resuscitation of SOLA inocula in SW-DOM resulted in a significant decrease in Shannon diversity compared to the initial SOLA community  were viable  were viable . However, the composition of communities originating from cryopreserved inocula processed with delay was less similar (Bray–Curtis distances 0.741 ± 0.246). Two replicates clustered closely together while the community composition of the third replicate seemed to be more similar to communities from the other incubations . Further6 and 0.91 ± 0.23 × 106 cells/mL. However, because of a slightly longer lag phase of communities originating from inocula stored for 1.5 months, the communities were, in this case, possibly sampled just before reaching the plateau phase. Incubations from aliquots with a storage time of 6 or 12 months were in contrast sampled shortly after reaching the maximal cell densities (6 cells/mL).Growth curves of cryopreserved communities that were resuscitated after a storage time of 1.5 and 12 months were similar, with lag phases of 3–4 days and carrying capacities between 0.65 ± 0.14 × 10ensities . Differep = 0.07, p-value cutoff of p < 0.05 and could point to an effect of the storage time on community composition. Post hoc pairwise comparisons indicated that possible differences in the community composition occurred primarily between the samples with a storage time of 1.5 months vs the samples with 6 and 12 months of storage. Pairwise comparison between samples with 6 and 12 months storage time did not indicate any influence of storage time on community composition in this case. Furthermore, a mantel test did not support a significant increase in community dissimilarity with increasing storage time of the cryopreserved inocula  without a preceding filtration step to concentrate cells resulted in higher CVs and similar variances. The concentration of cells on a filter further allows the complete removal of the initial water sample, avoiding the carry-over of substrates into the incubation medium and, therefore, providing additional control over the initial substrate conditions during the resuscitation process. Additionally, since the concentrated cells were cryopreserved in a liquid volume of only 2 mL, the amount of DMSO needed as a cryoprotectant is reduced to a minimum. This could be important since the ability to metabolize DMSO to dimethyl sulfide is widespread among microorganisms , and mayOverall, the CV observed among triplicates for larger inocula in our experiments . This was evident in the analyses by individual ASVs , ASVs grConversely, Canet resuscitated communities, which originated from a highly variable source environment, attained in all incubations a comparably high degree of similarity of the community composition as well as diversity levels relative to the initial community. This result is further striking because Canet inocula were not incubated in sterile-filtered Canet water, but exclusively in media with DOM supplements that did not even originate from the Canet Lagoon . We specThe overrepresentation of taxa in the incubations such as ASVs affiliating with the genera Alteromonas  and Arcobacter  likely reflect their niche adaptation as opportunists within the SOLA and Canet communities, respectively. Members of the genus Alteromonas are known for their ability to exploit complex organic substrates  and theipadj < 0.1). This observation is in concordance with the cultivability of the oligotrophic SAR11 clade in low nutrient sterile-seawater  were critically assessed to offer new opportunities for future research in microbial ecology based on the manipulation of natural communities. Resuscitation was achieved for contrasting communities harvested from the coastal waters of the Northwest Mediterranean Sea. Based on the findings of this study, we recommend using sufficiently large community aliquots for cryopreservation to improve reproducibility, while on the other hand avoiding extended handling time. Finally, also long-term storage times from 6 up to 12 months did not seem to cause major changes in the community composition of the resuscitated communities.The cryopreservation protocol presented in this study can be applied to current challenges in microbial ecology since it enables to control the starting community in various experimental setups based on complex natural communities. Our method will, for instance, allow all kinds of experimental setups, where communities are mixed after a time delay and possibly in different ratios to address the priority effect after community coalescence events. Experiments could also be repeated retrospectively after the evaluation of a first experimental series, with the option to change a specific parameter in the setup while controlling for the starting community. This, for instance, could be applied to consecutively scrutinize culturing settings that cause initially rare opportunistic species to overgrow other members of their communities, with the aim to develop protocols for improved culturing of natural communities under laboratory conditions. The opportunity to completely remove the original sample medium makes the presented method furthermore highly feasible for transplant experiments, where the carry-over of substances in the original sample water should be avoided. The implementation of cryopreservation to natural communities in combination with high-throughput sequencing may furthermore increase the success for the cultivation of target organisms: preceding analyses of meta-“omic” data can be used to identify samples, where a certain target taxon is abundant and may, after the application of genome binning approaches, additionally reveal genes of the target taxon that indicate specific growth requirements. Cryopreserved communities from selected samples can then in retrospect be resuscitated under optimized conditions to enrich or even isolate the target taxon.Overall, we believe that cryopreservation techniques applied to natural microbial communities have a high potential to advance the research field of microbial ecology. The presented tests and analyses can guide researches on the methodological aspects that should be considered for the cryopreservation of natural aquatic communities in dependency of the research question to be addressed.https://www.ebi.ac.uk/ena, PRJEB38947.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: SB and GPM designed the study. SB, GPM, and AR-F collected samples, performed the experiments, and analyzed the data. AR-F and SB mainly wrote the manuscript, while GPM also contributed. All authors commented on the manuscript, and read and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Exceptional advancement has been made in the development of graphene optical nanoantennas. They are incorporated with optoelectronic devices for plasmonics application and have been an active research area across the globe. The interest in graphene plasmonic devices is driven by the different applications they have empowered, such as ultrafast nanodevices, photodetection, energy harvesting, biosensing, biomedical imaging and high-speed terahertz communications. In this article, the aim is to provide a detailed review of the essential explanation behind graphene nanoantennas experimental proofs for the developments of graphene-based plasmonics antennas, achieving enhanced light–matter interaction by exploiting graphene material conductivity and optical properties. First, the fundamental graphene nanoantennas and their tunable resonant behavior over THz frequencies are summarized. Furthermore, incorporating graphene–metal hybrid antennas with optoelectronic devices can prompt the acknowledgment of multi-platforms for photonics. More interestingly, various technical methods are critically studied for frequency tuning and active modulation of optical characteristics, through in situ modulations by applying an external electric field. Second, the various methods for radiation beam scanning and beam reconfigurability are discussed through reflectarray and leaky-wave graphene antennas. In particular, numerous graphene antenna photodetectors and graphene rectennas for energy harvesting are studied by giving a critical evaluation of antenna performances, enhanced photodetection, energy conversion efficiency and the significant problems that remain to be addressed. Finally, the potential developments in the synthesis of graphene material and technological methods involved in the fabrication of graphene–metal nanoantennas are discussed. Innovative electromagnetic optical nanoantennas (NA) functioning at terahertz band, infrared and optical frequencies play a vital role in the emerging field of photonics and plasmonics since these antennas have been considered as the best tools for controlling, manipulation and propagation of light the interaction of light with electrons present in materials . The funMicrowave standards are not valid for terahertz region, as the size of the antennas is miniaturized to micrometer scale ; thus, tThe design of optical nanoantenna is analogous to conventional microwave- and millimeter-wave antennas, as it controls the energy conversion of light into localized energy and optical radiation at the subwavelength scale . VariousThe wavelength of photons is much larger than the wavelength of the electron . Hence, Graphene, the wonder material, is an allotrope of carbon having a monoatomic layer of carbon atoms. The carbon atoms in a two-dimensional graphene structure are arranged in a honeycomb lattice due to the bonding of atoms in a hexagon manner. The discovery of graphene has attracted marvelous research curiosity in numerous arenas credited to its distinguishing characteristics . GraphenGraphene displays high wave localization and can underpin the propagation of SPPs, with acceptable loss. It also portrays a remarkable tunability through doping of certain chemicals and by electric/magnetic biasing . SignifiAll essential components of optical transceivers can utilize graphene plasmonics: optical switches, optical waveguides and modulators phase shifters. Furthermore, biasing of graphene through magneto-static fields has been demonstrated to result in giant Faraday rotation and non-reciprocal propagation ,39. MoreIt is worth noting that, to date, graphene-based photonics devices  have only been proposed and scrutinized hypothetically, and the electromagnetic response evaluated through simulators by using numerical solvers ,43,44,45In this article, state-of-the-art graphene plasmonic nanoantennas and hybrid metal–graphene nanoantennas are discussed in detail. The latest techniques in the development of graphene nanoantennas for photonics applications are identified. In Graphene is a single atomic thick sheet of carbon atoms arranged in a hexagon manner , as illua”, is also called lattice constant. The hopping energy between the most adjacent atoms is t ≈ 2.8 eV. The Hamiltonian can be written as a series of summations in space as follows:The vectors lated as :(3). The result obtained from Equation (5) is often expressed in a different form by using  components for F is the Fermi velocity roughly equal to ≈106 m/s.At first Brillouin zone, six Dirac points occur due to contact and the short distance between conduction and valance bands. By employing a Taylor expansion series at these Dirac points, the graphene dispersion relation is estimated as linear:The mobility of electrons in graphene at the conduction band is also referred to as intraband transition. The intraband transition according to Boltzmann equation can be calculated asE and p, respectively, and the electron probability density function is denoted by f in k space. Here, τ is used for relaxation time and time approximation. Equation (8) can be rewritten as Equation (9) by applying a Fourier transform.The kinetic energy and momentum of the electrons are represented by v and the electric field vector is denoted by E. E = hv and v = dw/dk. Finally, integrating Equation (9) by v and then again at k-space, the following equation is obtained:In Equation (9), group velocity is represented by g” are equivalent to four for the graphene layer considered in Equation (10).In a unit cell, two carbon atoms and spin degeneracy “Manipulating In the following, Hall impedance, which occurs due to a magnetic field is ignored and electrical impedance is considered, that is,e represents the charge on the electron, angular frequency is denoted by ω, relaxation time is τ, ℏ is the reduced plank constant, and d and Equation (12) agrees with Kubo formalism for the intraband transition part. The unique electronic band structure of graphene directly affects the conductivity of graphene, as well as the other parameters, for instance, chemical potential, chemical doping, relaxation time, electrons mobility and the surrounding temperature operating frequency. In addition, these characteristics parameters are correlated with each other ,60,61. Aquencies . In contittivity . In-planrding to . The othrding to ,66,67. NCompared to noble metals, graphene supports the generation and travel of SPPs at the frequencies mentioned above. In particular, a free space standing graphene sheet supporting transverse magnetic (TM) plasmon mode can be calculated as:sppk is surface plasmons wave vector and Zc is the characteristic impedance of the graphene sheet Undeniably, a standout among the most exciting properties of graphene is the ability to control the complex conductivity, which results in frequency tunability. This should be effortless by applying an external gate (electrostatic) bias to manipulate the graphene field-effect [Equations (14) and (15) can be used as an approximation for calculating the loss and leading wavelength of plasmons in innovative designs, such as graphene patches and graphene nanoribbons. d-effect , when thd-effect . The extTypically, the gating of the graphene sheet controls the chemical potential of graphene by varying the bias voltage across the graphene sheet. The change in chemical potential by gate voltage tunes the graphene conductivity for the desired requirements. In addition, the variation of chemical potential enables the frequency tunability feature because of carrier injection, which increases the graphene conductivity; however, the chemical potential is electrically controlled by varying bias voltage on the graphene sheet. The relation between chemical potential and the bias voltage is derived through Equation (16). Ongoing examinations have affirmed that this tunability is exceptional in a broad range and quick up to some hundreds of gigahertz, thus empowering reconfiguration abilities in all graphene antennas. In a related context, magneto-statically biasing the graphene can produce a discontinuous band structure and become discretized in Landau levels. This behavior of conductivity can be exploited as a gyro tropic tensor, which leads to non-reciprocal responses ,71,72. IVg is the gate potential applied at gate pads of the graphene-based device and cμ is the chemical potential. The increase in the gate voltage actively tunes the graphene conductivity, which provides frequency reconfigurability of the device without changing the geometric dimensions.t, m and b indicate top, middle, and bottom layers, respectively), It is worth mentioning that carbon-based nanomaterial composites have to overcome certain shortcomings, for instance low conductivity and toughness . Additione stack . Remembene stack (20)ns=2µc) are recognized for each of the graphene sheets, the complex conductivity of the graphene stack–metal hybrid structure can be extracted asOnce the Fermi energies  is a computational technique employed for solving electromagnetic sources as surface or volume integral calculations in the frequency domain. The advantage of MoM is that it only considers the EM sources where the surface or volume current on the surface of geometry needs to be discretized. Moreover, MoM is exceptionally effective for electrically large electronic devices and is widely utilized for calculating radiation and scattering properties by deriving surface currents through Green’s function. The analytical equations based on dyadic Green’s function are derived for graphene in . Araneo An effective way to validate the computational results of graphene-based devices is by designing the equivalent resonant circuits and conventional transmission line (TL) models for graphene-based antenna simulations. Lovat demonstrated the EM interaction of graphene by deriving equivalent circuits as the EM wave propagates through the graphene sheet . The effNanoantennas operating in THz band are of great importance in everyday life due to their numerous utilizations in the various photonics fields. Initially, graphene-based antennas were hypothetically studied, while recent studies by Sergai and colleagues  demonstrHossein et al. proposed a graphene–metal hybrid antenna by employing waveguide feed , as depiThe use of graphene nanoribbons (GNRs) in a dipole antenna is presented in . The autMore work on the graphene patch antenna has been done by the same researchers. To increase the directivity of a graphene patch antenna, the double-layer graphene stack and the dielectric lens are utilized. These methods can be directly employed in applications that require high directivity due to high tunability over a wide range. Furthermore, resonant tuning of frequency is established by having impedance reliability, and it is noticed that the efficiency alters the frequency reconfigurability and is not stable, similar to input impedance, which can be explained through the electrical length of the antenna. The plasmon wavelength is increased by varying the chemical potential of graphene layers through biasing, which produces upshift in the resonance frequency, thus increasing the electrical length of the antenna at the same physical size. This standard method is employed for miniaturizing graphene antenna devices, which exploit high inductance of graphene for low chemical potential with the cost of reduced radiation efficiency. Using high chemical potential, the SPP properties of graphene are rarely affected by the bias voltage; thus, the reconfigurability of the graphene antenna is hardly affected.2/Si stack substrate by sandwiching the graphene layer between the metal and the substrate [1 = 260 µm and the thickness of silica is t2 = 300 nm, while gold metal is employed as a radiator for the dipole, as illustrated in Qin et al. worked on the graphene–metal hybrid antenna designed on a SiOubstrate . This teThe idea of graphene-coated wires (GCWs) was recently presented by Diego and colleagues  to furthAdditionally, all power propagates outside the wire material; hence, these type of waveguide cylinders are designed as a resistant for the dielectric losses in the material ,116,117.Furthermore, the authors proposed an equivalent circuit model, which assumes the capacitance between the gap of GCW arms and reactance at the edges of a dipole. The geometry presented in Chen et al. demonstrated the concept of terahertz frequency generation by using a graphene-based antenna along with photomixer to achieve wide-band and tunable performance over the terahertz region , as presConsequently, field outflow in nanodevices with rapid switching time and high current densities are generally exploited in miniaturized nanoelectronics . Mario aMoving toward the infrared (IR) and visible frequencies, researchers widely investigate the effect of graphene antenna responses on photodetection and light enhancement by the manipulation of localized surface plasmons. Chitraleema and coworkers investigated the performance of graphene-metal antennas for enhanced photodetection . A four Brar et al. experimentally demonstrated blackbody emission from graphene resonator and the control of emission with electronic tunability of a graphene antenna placed on a silicon nitride substrate . It is s2 substrate thickness on the propagation properties of SPP on the graphene surface and control of SPP through SiO2 thickness, when it is less than 100 nm. This study again exemplifies the tradeoff between compact size and wavelength response. Graphene has been used as a parasitic element in various research works, for example reconfigurable substrates [The optical properties of graphene are reliant on the exclusive gate biasing, making graphene electrically tunable at optical frequencies. In recent work, Yuwei and coworkers investigated the polarization control of graphene–metal nanoantenna with electrostatic biasing of monolayer graphene . The desbstrates  or high bstrates ,137. TheThe graphene layers can be N-type or P-type doped by applying positive or negative voltage bias. Tunable surface conductivity in a graphene sheet can be achieved by the displacement of Fermi energy level  from the Dirac points, which results from the infusion of hot carriers in the graphene sheet. Graphene sheets sustain metallic responses and can maintain strong plasmonic behaviors from the terahertz region to mid-infrared and even some portion of visible light frequency region, due to the forbidden interband transition by the Pauli blocking. Significantly, single graphene layer with tunable characteristic  empowers the design and manipulation of its surface conductivity and, afterwards, controls the graphene plasmons to tune the plasmonic responses of graphene-loaded nanoantennas. In this section, the reputed antennas with graphene layers as the fundamental element identified for tuning the radiation characteristics and acquiring tunability are discussed. In the following subsections, the features of graphene–metal antennas  are discussed with the aid of graphene tunable conductivity behavior.Remarkable work was demonstrated by Chen et al., who designed a graphene–metal nanoantenna comprised of two symmetric gold strips deposited on two different graphene layers at mid-infrared frequencies . This inThe geometric model is depicted in Naresh and colleagues investigated and developed a bowtie array antenna on the top of graphene sheets with the active swapping of a plasmon resonance at the optical region using a large graphene sheet . The intThe optical antennas are tailored to launch and control the graphene plasmon which arises in the infrared region, and the refraction of these plasmons is analyzed when they pass through prism-shaped 2D bilayer graphene. This unique effect can be utilized for the manipulation of light in nanoscale optoelectronics and plasmonic circuits. The in-situ control of polarization and the phase of propagating far-field light by employing metasurfaces is demonstrated in . Gold re2 layer [2O3 layer between the graphene sheets depends on the graphene backing cavity [Yasir and coworkers experimentally demonstrated the tuning of frequency using multi-layer graphene in a nanoantenna . The geo2 layer . Raman s2 layer . The str2 layer . The cong cavity . Dual-bag cavity . The fieThe performance of the graphene bowtie antenna are enhanced through the optimization of the substrate material and gradual tuning of the gate voltage in . The tunAgreeing to the reciprocity statement, graphene-based tunable antennas are capable of transmitting and receiving electromagnetic waves from terahertz to optical wavelengths. Important antenna characteristic parameters  are the key elements for the antenna to work as a receiver. However, apart from these parameters, optical absorption performs a significant function, aimed at the conversion of free space propagating waves into surface waves with high responsivity and better enhancement in the near field . The varPure single-layer graphene has an optical absorption ability of 2.3%, which is quite unsatisfactory to apprehend intense interactions of light and matter. Different methods have been employed to enhance the optical absorption of graphene antennas and likewise couplings effects, photonic modes and graphene plasmons. For example, varying graphene chemical potential through the electrostatic bias of a tooth-shaped log-periodic graphene antenna achieved tunable absorption at infrared wavelengths as well as high reconfigurable intensity . Electro−1 with a modulated reflection efficiency of 95.9%, as depicted in Yu et al. demonstrated a model for complete absorption using a graphene layer. Complete absorption takes place due to destructive interference between the reflection of a radiating surface and a back reflector . HoweverElectrically changing the chemical potential of graphene with the gate bias progressively transforms its dielectric constant, hence playing a significant role in the continual modulation of antenna parameters ,167,168.2) metal–insulator phase transition is studied experimentally for the broadband modulation of light in the terahertz region [2 surface, and then modulation of terahertz light is further improved by adding a bowtie antenna between the wire and the VO2 layers. Yu et al. demonstrated the realization of metasurfaces for tuning optical ranges and modulation depths [Dong and coworkers demonstrated the enhancement of graphene light interaction using coupled resonance with higher modulation by exploiting as cross-shaped slot metamaterial absorber . Blueshiz region . The plan depths . The metn depths  nanorod n depths  and optin depths  have been depths ,179.Surface plasmon polaritons (SPPs) propagation maintained by single-layer graphene can be vigorously tuned by changing the Fermi level by electrical doping, hence enabling the design of resonant spectral location and their interface with the exciting metallic plasmons. Earlier reports exposed the good coupling behavior of graphene plasmons with metal plasmons arising from the thin metal layer, with an enhanced performance . The autp, modulation amplitude M, and the average reactance sX. The reactance should be inductive in order to sustain highly confined TM waves that powerfully interact with the modulated surface. The exact values of modulation amplitude, radiation rate, and angle regulate the field confinement and wave propagation constant. Equation (13) represents the expression of modulated surface reactance:Fascinating beam steering properties of leaky-wave antennas (LWAs) have been an exceptionally active area of antenna investigation in previous decades ,190,191.M for the effective aperture will bring improved efficiency, and vice versa. However, this configuration simultaneously has 11% radiation efficiency and beam reconfigurability. In a subsequent work by the same authors [Graphene is the ideal stage to actualize this idea at THz and infrared frequencies. It provides an inductive behavior, given as ipulated . Moreoveipulated  based on authors , a sinus authors . AlthougWang and co-authors demonstrated a graphene-based 2D leaky-wave antenna with simultaneous beam steering and frequency reconfigurability features in the terahertz frequency region . GraphenThe novel concept of mantle cloaking is adopted for the beam steering of a leaky-wave antenna in . The autDifferent ways to actualize leaky-wave antennas in the optical region require borrowing basic principles from microwave theory, such as circularly polarized antennas and transmission lines. In particular, the authors of  considerIn another setting, an ongoing and extremely encouraging trend in both the EM and antenna arenas is the interest in non-reciprocal response without magneto-optic properties which require bulky magnets to function, thereby restricting the miniaturization of plasmonic devices and their applicability in other electronics . This isReflectarrays have been a hot topic among multiple domains  ,218. Mor2). In a traditional reflectarray unit cell patches are designed using a metallic material and noble metals. The metallic patches resonate when their physical size is equivalent to a half wavelength of the wave in an actual medium, thereby leading to large geometric structures. This situation becomes diverse when graphene patches are considered, as the graphene patches resonate at much lower frequencies because of the slow propagating plasmonic modes of graphene when compared to their metallic counterparts. However, graphene plasmonic responses prompt a sharp decrease in the electrical size of the array elements, providing overall better performance. For instance, the reflectarray in [The groundbreaking investigation on the graphene-based reflectarray has been firstly presented in the year 2013, by Carrasco and Perruisseau-Carrier . The refarray in  has grapThe total reflectarray is formed from 25,000 graphene elements and is fed through a terahertz horn antenna. Moreover, applying DC bias voltage at the polysilicon gate layer effectively tunes the graphene sheet reactance and accordingly the phases of the array elements. Importantly, the miniaturized size and tunability of beams concerning the phase response of graphene patches is the most significant advancement in reflectarray antennas . HoweverThe generation of vortex beams is the most common application of reflectarrays. Vortex radiation beams are described by orbital angular momentum (OAM), which allows the spectral efficiency to be increased by utilizing the originality of a vast number of phase states. Vortex beams have attract a great deal of interest in previous years on account of promising examinations, guaranteeing that OAM may give improved transmission characteristics compared to some other communication technologies ,236. Alt2 to adjust and tune the chemical potential of graphene. Increasing the chemical potential of graphene layers results in an improved reflection magnitude of −2.5 dB with 360° phase reflection range, as illustrated in The recent investigation by Yanshi et al. developed a graphene-based reflectarray with metamaterial structure for the production of (OAM) vortex beams at terahertz frequency , as depiIn a related study, Jian et al. demonstrated a new technique for the generation of vortex plasmons through cross-shaped metal–graphene nanoantennas . To exciLi Deng and coworkers theoretically inspected a circularly polarized graphene-based reflectarray to achieve wide bandwidth and better gain in the terahertz region . The invThe functionality of common reflectarray antennas is limited to the static beam profile, and once the antenna is fabricated, the beam of the array can hardly be tuned. Thus, there is an increased demand for beam steering array antennas where the beam can be steered electrically through tuning the phases of each array unit cell . GraphenRecently the concept of the graphene-based phase shifter was employed in THz antenna arrays for steering functions . A low-lGraphene is an alluring material that is mostly used in sensing and terahertz imaging devices for wideband photodetection ,257. As −1.Additionally, employing graphene in an antenna integrated photodetector enhances the responsivity and other characteristics of the photodetector, which have received intensive consideration. Moreover, its possible application has been deterred by compromises in the responsivity, functioning speed and bandwidth range of current graphene-based photodetectors. Semih and colleagues demonstrated the design of a photodetector comprised of gold–graphene patch nanostrips, which empower immediate wideband and highspeed photodetection by having high responsivity . The nanIn a recent study, a novel concept for fast and efficient terahertz detection is demonstrated by using antenna integrated graphene p–n junctions . Graphen2, and the maximum photoresponse is achieved at 100 GHz in the simulation, further validated through the experimental measurements depicted in The responsivity of graphene photodetectors is normally less due to the weak optical absorption of single-layer graphene. This problem is overcome by Pong Ma and coworkers through experimental investigations . The autThe concept of solar energy harvesting through rectennas, converting solar energy to DC, has existed for nearly 40 years ,279. Rec2 laser at room temperature [−9 WHz−1/2 with the IR detection of 108 cm Hz1/2W−1. Furthermore, the authors stress the utilization of the above-discussed rectenna IR detector in night-vision devices , and the performance for the night-vision systems is calculated through the noise equivalent temperature difference (NETD). The NETD is the ratio of incident signal temperature needed to match with the detector noise, with the goal of the signal-to-noise ratio approaching one. For sensible car night vision it is necessary to have an IR identifier with an NETD value of 0.3 K [The presentation of graphene has escalated consideration for the enhancement of rectenna responsivity. Dragoman and Aldrigo recently worked on a graphene-based rectenna for efficient energy harvesting and obtained a higher conversion efficiency of 58.43% at terahertz frequency . The autperature . The maxof 0.3 K . In anotof 0.3 K . The rec2/Si substrate, and metal contacts for external terminals are placed on top of the graphene. A change in responsivity occurs by varying the geometric parameters of the graphene diode, as shown in Additionally, the enhancement of the energy harvesting of a coupled nanoantenna with a geometric diode is demonstrated by employing a transmitarray to harvest infrared energy at 19 THz. Two key methods are proposed for the improvement of the obtained voltage through a graphene nanoantenna by Araby et al. . The fir2S, which decreases the sensitivity. A Fano metamaterial with dynamic reconfiguration abilities is employed for switching and sensing [The localized surface plasmon resonance of optical antennas enables different sensing applications  and label-free sensing) due to the high enhancement of electric field and sharp resonant excitation of the metallic structures. However, in visible and infrared regimes, these metals have higher losses due to the lack of interband absorption. Advancements have been made to achieve excellent sensing properties by using graphene–metal hybrid resonators for such applications. Graphene-enabled silver nanoantenna sensors are designed to improve the sensitivity of sliver nanoantenna sensors . An over sensing . The rec sensing . Meanwhi sensing . Nejad e sensing . The sen sensing , a stakiThe mid-infrared region is especially appropriate for biosensing, as it includes the atomic vibrations that interestingly recognize the biochemical nature of molecules such as proteins, lipids and DNA. Among various techniques, IR absorption spectroscopy is a ground-breaking method that gives precise biochemical information in a non-destructive label-free style by getting vibrational patterns. Nevertheless, the vibrational absorption signals obtained from IR spectroscopy are restrictively weak on account of an enormous mismatch between the mid-IR wavelengths and biomolecular sizes. To reduce these hurdles, high sensitivity can be accomplished by manipulating strong optical near-fields in the vicinity of the graphene–metallic resonant structures. In particular, biosensing is a research area where graphene tunability and strong IR light confinement offer excellent prospects. Additionally, the spatial control of graphene plasmons in the IR region makes them very appealing for enhanced light–matter interactions and IR nanophotonics.Rodrigo et al. demonstrated mid-infrared plasmonic biosensing with an integrated graphene resonator using infrared spectroscopy . The weaThe enhanced mid-infrared sensing using the concept of total optical absorption is investigated in  by analy6 cm2.V−1.s−1 [4 cm2.V−1.s−1 to 3 × 104 cm2.V−1.s−1 [4 cm2.V−1.s−1 to 2.5 × 104 cm2.V−1.s−1 were achieved after transferring the monolayer graphene from the metallic copper surface to the insulated wafer [Enormous efforts have been made in recent decades towards the synthesis and fabrication of graphene devices and graphene integrated optoelectronics. There are generally three main methods to synthesize graphene layers, which are arranged into diverse groups because of their different synthesis procedures—that is, mechanical cleavage (MC), epitaxial growth (EG) and chemical vapor deposition (CVD) are the well-known methods for the fabrication of graphene. Graphene is a single layer of carbon atoms and can be extracted from bulk graphite through mechanical cleavage with the help of adhesive tape. In 2005, Novoselov et al. synthesized the first in-lab graphene monoatomic layer through the MC method and experimentally achieved maximum carrier mobilities of up to 10.V−1.s−1 ,311. Alt.V−1.s−1 ,313. The.V−1.s−1 ,317,318..V−1.s−1 ,320. How.V−1.s−1 . To acco.V−1.s−1 ,323,324.ed wafer . Additioed wafer ,326. In ed wafer ,328,329.ed wafer .The fabrication of graphene–metal hybrid antennas and antenna integrated optoelectronic devices require various steps, that is, graphene synthesis, transfer to the proposed substrate, patterning of nanostructures through lithography techniques and lastly metal etching and liftoff. Firstly, a graphene layer is grown using CVD on catalytic metal, with the aid of poly(methyl methacrylate) (PMMA). The most critical part is the transfer of graphene to the target substrate and ensuring that the graphene is not damaged, as depicted in ® IGSC02002) is used for printing a multilayer graphene patch antenna through direct-write dispensing. In the end, the graphene antenna is baked at 60 °C for half an hour, which provides better adhesion with the substrate material [Although the huge advances in the patterning of leading materials have provoked the improvement of microstructures , the litmaterial . The fabGraphene signifies an emergent outpost that unites the various research fields of physics, terahertz plasmonics, optoelectronics, nanophotonics and material science. The remarkable properties of graphene have exposed stimulating prospects in these photonic fields, where novel innovation and finding new solutions to current problems are still evolving. From the perspective of the electronic devices and photonics systems, graphene is a 2D monoatomic layer of carbon atoms with an inherently passivated surface. As the production of this material drives the present market for graphene utilizations, there is a clear hierarchy in how soon the graphene applications will influence the consumer. In the near future, the manufacturing methods for this material will evolve and provide the possibility of accessing graphene materials with low costs for applications utilizing a low grade of graphene. Most likely, in future years, the devices requiring the highest grade of pure graphene of electronic quality will take time to develop and improve the graphene synthesis method. The high mechanical properties of graphene will bring a drastic change in current flexible device technologies, such as solar cells and various other plasmonic devices for imaging and sensing applications.To make an impact on industry-standard device utilizations, the current demanding challenges of the graphene must be overcome. Amongst many others, the huge-scale synthesis of high-quality graphene is of foremost significance, which will have a huge impact on this field. Precisely, developments in graphene can considerably extend the life of graphene plasmons, which is highly associated with the device operation characteristics like absorbance, modulation efficiency, strong light interaction, sensitivity and more. Additionally, quantum and nonlinear effects present in profound subwavelength graphene nanostructures will be extensively explored in the future, which will provide a new stage for quantum information and communication. The unique optical properties of graphene-based optical antennas and graphene antenna integrated optoelectronics will shift the indicative paradigm of nano-optoelectronics. On a more extensive scale, the impact of the incorporated graphene optical antennas on numerous innovations, including ultra-fast photodetectors, nano biosensors, and ultra-fast optical chip interconnects is forthcoming.In this article, a review is presented on the development of graphene-based plasmonic antennas, from their origin to the current situation. Their deficiencies and operating abilities have discussed in detail. Three basic themes stand out in this survey: frequency tunability, graphene integration with noble metal antennas and miniaturization. These are the most remarkable and alluring highlights of graphene, and they are the foundation that enabled the research community to realize graphene tunable antennas, graphene antenna photodetectors and modulators, graphene antenna biosensors, and energy harvesters. Even though the realization of these devices is so difficult, the recent improvements in the fabrication process of graphene promises a bright future. From the theoretical point of view, we believe that research in graphene nanoantennas will pursue a few directions in the coming years, such as the merging of graphene antennas with metamaterials. These directions will lead to a proliferation in both theoretical investigations and practical industrial products for high-speed terahertz communication, terahertz imaging, bio-sensing, photodetectors and energy harvesters.