Evaluation of Cashew Apple Juice for Surfactin Production by Bacillus subtilis LAMI008

Bacillus subtilis LAMI008 strain isolated from the tank of Chlorination at the Wastewater Treatment Plant on Campus do Pici in Federal University of Ceará, Brazil has been screened for surfactin production in mineral medium containing clarified cashew apple juice (MM-CAJC). Results were compared with the ones obtained using mineral medium with glucose PA as carbon source. The influence on growth and surfactin production of culture medium supplementation with yeast extract was also studied. The substrate concentration analysis indicated that B. subtilis LAMI008 was able to degrade all carbon sources studied and produce biosurfactant. The highest reduction in surface tension was achieved with the fermentation of MM-CAJC, supplemented with yeast extract, which decreased from 58.95?±?0.10 to 38.10?±?0.81 dyn cm^?1. The biosurfactant produced was capable of emulsifying kerosene, achieving an emulsification index of 65%. Surfactin concentration of 3.5 mg L^?1 was obtained when MM-CAJC, supplemented with yeast extract, was used, thus indicating that it is feasible to produce surfactin from clarified cashew apple juice, a renewable and low-cost carbon source.     

Evaluation and Functional Characterization of a Biosurfactant Produced by Lactobacillus plantarum CFR 2194

The study details the investigations on the ability of Lactobacillus plantarum CFR 2194, an isolate from kanjika, a rice-based ayurvedic fermented product, to produce biosurfactant. Surfactant production, as a function of fermentation time, indicates that the maximum production occurred at 72 h under stationary conditions. Isolation, partial purification, and characterization of the biosurfactant produced have been carried out, and Fourier transform infrared spectroscopy (FTIR) spectra demonstrated that biosurfactants were constituted by protein and polysaccharide fractions, i.e., possessed the structure typical of glycoprotein, which is affected by the medium composition and the phase of growth of the biosurfactant-synthesizing strain. Critical micelle concentration (cmc) of the biosurfactant was found to be 6 g l^?1. The emulsification index (EI), emulsification activity (EA), and emulsion stability (ES) values of the biosurfactant have confirmed its emulsification property. Aqueous fractions of the produced biosurfactant exhibited a significant antimicrobial activity against the food-borne pathogenic species: Escherichia coli ATCC 31705, E. coli MTCC 108, Salmonella typhi, Yersinia enterocolitica MTCC 859, and Staphylococcus aureus F 722. More importantly, the biosurfactant from L. plantarum showed antiadhesive property against above food-borne pathogens. The results thus indicate the potential for developing strategies to prevent microbial colonization of food contact surfaces and health-care prosthesis using these biosurfactants.     

Evaluation of waste products in the synthesis of surfactants by yeasts

The highest yields of biosurfactants were obtained by: (i) Pseudozyma antarctica (107.2 g L^?1) cultivated in a medium containing post-refining waste; (ii) Pseudozyma aphidis (77.7 g L^?1); and (iii) Starmerella bombicola (93.8 g L^?1) both cultivated in a medium with soapstock; (iv)Pichia jadinii (67.3 g L^?1) cultivated in a medium supplemented with waste frying oil. It was found that the biosurfactant synthesis yield increased in all strains when the cell surface hydrophobicity reached 70–80 %, enabling the microbial cells to make good contact with hydrophobic substrates. The lowest surface tension of the post-cultivation medium was from 32.0 mN m^?1 to 37.8 mN m^?1. However, this parameter (which was also determined by a drop collapse assay) was of limited use in monitoring biosurfactant synthesis in this study. The crude glycerol was not a good substrate for biosurfactant synthesis although, in the case of P. aphidis, 67.4 g L^?1 of biosurfactants were obtained after cultivation in the medium supplemented with glycerol fraction (GF2). In a low-cost medium containing soapstock and whey permeate or molasses, about 90 g L^?1 of mannosylerythritol lipids were synthesised by P. aphidis and approximately 40 g L^?1 by P. antarctica.     

Lipopeptide surfactant production by Bacillus subtilis grown on low-cost raw materials

The production of biosurfactant by Bacillus subtilis ATCC 6633 was investigated using commercial sugar, sugarcane juice and cane molasses, sugarcane juice alcohol stillage, glycerol, mannitol, and soybean oil. Commercial sugar generated the minimum values of surface tension, with the best results (28.7 mN/m, (relative critical micelle concentration [CMC⁻¹] of 78.6) being achieved with 10 g of substrate/L in 48 h. At a pH between 7.0 and 8.0, a higher production of surface-active compounds and a greater emulsifier activity was also observed. Enrichment of the culture medium with trace minerals and EDTA showed maximum yields, whereas supplementation with yeast extract stimulated only cell growth. The kinetic studies revealed that biosurfactant production is a cell growth-associated process; surface tension, CMC, and emulsification index values of 29.6 dyn/cm, 82.3, and 57%, respectively, were achieved, thus indicating that it is feasible to produce biosurfactants from a renewable and low-cost carbon source.     

Comparative study of biosurfactant produced by microorganisms isolated from formation water of petroleum reservoir

Biosurfactant produced by Pseudomonas aeruginosa, Bacillus subtilis and Rhodococcus erythropolis that isolated from the formation water of Chinese petroleum reservoir has been compared in surface abilities and oil recovery. Maximum biosurfactant production reached to about 2.66 g/l and the surface tension of liquid decreased from 71.2 to 22.56 mN/m using P. aeruginosa. Three strains exhibited a good ability to emulsify the crude oil, and biosurfactant of P. aeruginosa attained an emulsion index of 80% for crude oil which was greater than other strains. Stability studies were carried out under the extreme environmental conditions, such as high temperature, pH, salinity and metal ions. Results showed an excellent resistance of all biosurfactants to retain their surface-active properties at extreme conditions. It was found that the biosurfactants from three isolated bacteria showed a good stability above pH of 5, but at lower pH (from 1 to 5) they will harmfully be affected. They were able to support the condition up to 20 g/l salinity. P. aeruginosa biosurfactant was even stable at the higher salinity. Regarding temperature, all produced biosurfactants demonstrated a good stability in the temperature up to 120 °C. But stability of three biosurfactants was affected by monovalent and trivalent ions. Oil recovery experiments in physical simulation showed 7.2-14.3% recovery of residual oil after water flooding when the biosurfactant of three strains was added. These results suggest that biosurfactants of these indigenous isolated strains are appropriate candidates for enhanced oil recovery with a preference to biosurfactant of P. aeruginosa.     

Production of a Bioemulsifier with Potential Application in the Food Industry

Biosurfactants are of considerable interest due to their biodegradability, low degree of toxicity, and diverse applications. However, the high production costs involved in the acquisition of biosurfactants underscore the need for optimization of the production process to enable viable application on an industrial scale. The aims of the present study were to select a species of Candida that produces a biosurfactant with the greatest emulsifying potential and to investigate the influence of components of the production medium and cultivation conditions. Candida utilis achieved the lowest surface tension (35.53 mN/m), best emulsification index (73 %), and highest yield (12.52 g/l) in a medium containing waste canola frying oil as the carbon source and ammonium nitrate as the nitrogen source. The best combination of medium components and cultivation conditions was 6 % (w/v) glucose, 6 % (w/v) waste canola frying oil, 0.2 % (w/v) ammonium nitrate, 0.3 % (w/v) yeast extract, 150 rpm, 1 % inoculum (w/v), and 88 h of fermentation. The greatest biosurfactant production and the lowest surface tension were achieved in the first 24 h of production, and the maximum biomass production was recorded at 72 h. The biosurfactant produced from C. utilis under the conditions investigated in the present study has a potential to be a bioemulsifier for application in the food industry.     

Application of Biosurfactant from Sphingobacterium spiritivorum AS43 in the Biodegradation of Used Lubricating Oil

This study aimed at investigating the application of biosurfactant from Sphingobacterium spiritivorum AS43 using molasses as a substrate and fertilizer to enhance the biodegradation of used lubricating oil (ULO). The cell surface hydrophobicity of bacteria, the emulsification activity, and the biodegradation efficiency of ULO were measured. The bacterial adhesion in the hydrocarbon test was used to denote the cell surface hydrophobicity of the used bacterial species. The results indicate a strong correlation between cell surface hydrophobicity, emulsification activity, and the degree of ULO biodegradation. The maximum degradation of ULO (62 %) was observed when either 1.5 % (w/v) of biosurfactant or fertilizer was added. The results also revealed that biosurfactants alone are capable of promoting biodegradation to a large extent without added fertilizer. The data indicate the potential for biosurfactant production by using low-cost substrate for application in the bioremediation of soils contaminated with petroleum hydrocarbons or oils.     

Exopolysaccharides and Antimicrobial Biosurfactants Produced by Paenibacillus macerans TKU029

Paenibacillus macerans TKU029 can produce exopolysaccharides (EPSs; 3.46 g/L) and a biosurfactant (1.78 g/L) in a medium with 2 % (w/v) squid pen powder as the sole carbon/nitrogen source. The biosurfactant can reduce the surface tension of water from 72.30 to 35.34 mN/m at a concentration of 2.76 g/L and reach an emulsification index of 56 % after a 24-h reaction with machine oil. This biosurfactant is stable at 121 °C for 20 min, over a pH range from 3 to 11, and in <5 % salt solutions. It also shows significant antimicrobial activity, which remains active after treatment at 121 °C and at pH values from 4 to 10, against Escherichia coli BCRC13086, Staphylococcus aureus BCRC10780, Fusarium oxysporum BCRC32121 and Aspergillus fumigatus BCRC30099. Furthermore, human skin shows from 37.3 to 44.3 % hydration after being treated with TKU029 EPSs for 180 min. These results imply that EPSs and the biosurfactant from this strain have potential in cosmetics, for removal of oil contamination, and as antimicrobial agents.     

Synthesis and properties of the cationic fluorocarbon emulsifier-free latex in a new micellar system

Cationic fluorocarbon emulsifier-free latex (CFEL) based on hexafluorobutyl methacrylate (FA), styrene, butyl acrylate, and methacrylatoethyl trimethyl ammonium chloride is successfully prepared in a new micellar system in which the fluorinated surface active monomer (FSM) based on isophorone diisocyanate, dodecafluoroheptanol, and allyl polyethylene glycol is used. The chemical structure of FSM is characterized by Fourier transform infrared spectroscopy, ¹H-NMR, and its surface-active properties have been investigated by surface tension determinator. Besides, effect of FSM, FA, and also the curing temperature on the latex and film properties has been investigated by the coagulation ratio (W _c ), precipitation ratio (W _p ), Nano-ZS particle sizer, contact angle, and water absorption ratio, respectively. The results show that the FSM is successfully prepared. The CMC of FSM is 2.37 g L^?1 and the γ _CMC is 26.31mN m^?1 accordingly. The more FSM content makes more stable emulsion and have only little adverse effect on its film properties. When the FSM content increases from 1.05 to 13.11 %, the W _c and W _p decrease by 83.5 and 32.1 %, respectively, and the surface free energy (γ) of CFEL film only increases by 8.3 %. The more FA content makes less stable emulsion but have favorable effect on its film properties. When the FA content increases from 0 to 25.11 %, the γ is decreased by 55.1 %. The curing temperature has much impact on film property. For example, the γ from 27.47 to 20.36 mJ?·?m^?2 when the curing temperature rises from 30 to 110 °C.     

Antifungal Lipopeptides Produced by <Emphasis Type="Italic">Bacillus</Emphasis> sp. FJAT-14262 Isolated from Rhizosphere Soil of the Medicinal Plant <Emphasis Type="Italic">Anoectochilus roxburghii</Emphasis>

This study aimed to develop biocontrol Bacillus and explore bacterial biocontrol substances. According to the blood agar test, strain FJAT-14262 was screened as a biosurfactant-producer. The biosurfactant-producing ability of FJAT-14262 was further confirmed by the oil spreading tests because of its amphipathic character. Furthermore, its fermentation supernatant could decrease the surface tension from 74.1 to 32.7 mN m^?1. Fourier transform infrared spectroscopy (FT-IR) analysis indicated that the biosurfactant produced by the strain FJAT-14262 was a kind of lipopeptides. Reverse-phase high-performance liquid chromatography (RP-HPLC) and liquid chromatography-mass spectrometry (LC-MS) analysis demonstrated that this lipopeptide contained surfactin with polar amino acids and hydrophobic fatty acid chains. Moreover, bioinformatic analysis revealed that the nonribosomal peptide synthetases genes srfAA, srfAB, and srfAC were structurally conserved in the FJAT-14262 genome. Importantly, the crude surfactant exhibited strong inhibitory activities against Fusarium oxysporum, suggesting that strain FJAT-14262 could be a potential biological control agent against Fusarium wilt.     

Production of Microbial Rhamnolipid by Pseudomonas Aeruginosa MM1011 for Ex Situ Enhanced Oil Recovery

Recently, several investigations have been carried out on the in situ bacteria flooding, but the ex situ biosurfactant production and addition to the sand pack as agents for microbial enhanced oil recovery (MEOR) has little been studied. In order to develop suitable technology for ex situ MEOR processes, it is essential to carry out tests about it. Therefore, this work tries to fill the gap. The intention of this study was to investigate whether the rhamnolipid mix could be produced in high enough quantities for enhanced oil recovery in the laboratory scale and prove its potential use as an effective material for field application. In this work, the ability of Pseudomonas aeruginosa MM1011 to grow and produce rhamnolipid on sunflower as sole carbon source under nitrogen limitation was shown. The production of Rha-C₁₀-C₁₀ and Rha₂-C₁₀-C₁₀ was confirmed by thin-layer chromatography and high-performance liquid chromatography analysis. The rhamnolipid mixture obtained was able to reduce the surface and interfacial tension of water to 26 and 2 mN/m, respectively. The critical micelle concentration was 120 mg/L. Maximum rhamnolipid production reached to about 0.7 g/L in a shake flask. The yield of rhamnolipid per biomass (Y _RL/x ), rhamnolipid per sunflower oil (Y _RL/s ), and the biomass per sunflower oil (Y _x/s ) for shake flask were obtained about 0.01, 0.0035, and 0.035 g g^?1, respectively. The stability of the rhamnolipid at different salinities, pH and temperature, and also, its emulsifying activity has been investigated. It is an effective surfactant at very low concentrations over a wide range of temperatures, pHs, and salt concentrations, and it also has the ability to emulsify oil, which is essential for enhanced oil recovery. With 120 mg/L rhamnolipid, 27 % of original oil in place was recovered after water flooding from a sand pack. This result not only suggests rhamnolipids as appropriate model biosurfactants for MEOR, but it even shows the potential as a biosurfactant of choice for actual MEOR applications.     

Enhanced Biodegradation of Alkane Hydrocarbons and Crude Oil by Mixed Strains and Bacterial Community Analysis

In this study, two strains, Acinetobacter sp. XM-02 and Pseudomonas sp. XM-01, were isolated from soil samples polluted by crude oil at Bohai offshore. The former one could degrade alkane hydrocarbons (crude oil and diesel, 1:4 (v/v)) and crude oil efficiently; the latter one failed to grow on alkane hydrocarbons but could produce rhamnolipid (a biosurfactant) with glycerol as sole carbon source. Compared with pure culture, mixed culture of the two strains showed higher capability in degrading alkane hydrocarbons and crude oil of which degradation rate were increased from 89.35 and 74.32?±?4.09 to 97.41 and 87.29?±?2.41 %, respectively. In the mixed culture, Acinetobacter sp. XM-02 grew fast with sufficient carbon source and produced intermediates which were subsequently utilized for the growth of Pseudomonas sp. XM-01 and then, rhamnolipid was produced by Pseudomonas sp. XM-01. Till the end of the process, Acinetobacter sp. XM-02 was inhibited by the rapid growth of Pseudomonas sp. XM-01. In addition, alkane hydrocarbon degradation rate of the mixed culture increased by 8.06 to 97.41 % compared with 87.29 % of the pure culture. The surface tension of medium dropping from 73.2?×?10^?3 to 28.6?×?10^?3 N/m. Based on newly found cooperation between the degrader and the coworking strain, rational investigations and optimal strategies to alkane hydrocarbons biodegradation were utilized for enhancing crude oil biodegradation.     

Candida Biofilm Disrupting Ability of Di-rhamnolipid (RL-2) Produced from Pseudomonas aeruginosa DSVP20

Biosurfactant produced from Pseudomonas aeruginosa DSVP20 was evaluated for its potential to disrupt Candida albicans biofilm formed on polystyrene (PS) surfaces in this investigation. P. aeruginosa DSVP20 exhibited optimum production of biosurfactant (5.8 g?L^?1) after 96 h of growth with an ability to reduce surface tension of the aqueous solution from 72 to 28 mN?m^?1. Analysis of purified biosurfactant with FT-IR, ¹H and ¹³C NMR and MALDI-TOF MS revealed it to be di-rhamnolipid (RL-2) in nature. Biofilm disrupting ability of RL-2 (0.16 mg?mL^?1) on Candida cells when checked using XTT reduction assay revealed that about 50 % of the cells remain adhered to 96-well plate after 2 h of treatment, while up to 90 % reduction in pre-formed C. albicans biofilm on PS surface was observed with RL-2 (5.0 mg?mL^?1) in a dose-dependent manner. Microscopic analyses (SEM and CLSM) further confirm the influence of RL-2 on disruption of Candida biofilm extracellular matrix on PS surface which can be exploited as a potential alternative to the available conventional therapies.     

First Report of a Lipopeptide Biosurfactant from Thermophilic Bacterium Aneurinibacillus thermoaerophilus MK01 Newly Isolated from Municipal Landfill Site

A biosurfactant-producing thermophile was isolated from the Kahrizak landfill of Tehran and identified as a bacterium belonging to the genus Aneurinibacillus. A thermostable lipopeptide-type biosurfactant was purified from the culture medium of this bacterium and showed stability in the temperature range of 20–90 °C and pH range of 5–10. The produced biosurfactant could reduce the surface tension of water from 72 to 43 mN/m with a CMC of 1.21 mg/mL. The strain growing at a temperature of 45 °C produces a substantial amount of 5 g/L of biosurfactant in the medium supplemented with sunflower oil as the sole carbon source. Response surface methodology was employed to optimize the biosurfactant production using sunflower oil, sodium nitrate, and yeast extract as variables. The optimization resulted in 6.75 g/L biosurfactant production, i.e., 35 % improved as compared to the unoptimized condition. Thin-layer chromatography, FTIR spectroscopy, ¹H-NMR spectroscopy, and biochemical composition analysis confirmed the lipopeptide structure of the biosurfactant.     

Utilization of Banana Peel as a Novel Substrate for Biosurfactant Production by Halobacteriaceae archaeon AS65

In this study, biosurfactant-producing bacteria was evaluated for biosurfactant production by using banana peel as a sole carbon source. From the 71 strains screened, Halobacteriaceae archaeon AS65 produced the highest biosurfactant activity. The highest biosurfactant production (5.30 g/l) was obtained when the cells were grown on a minimal salt medium containing 35 % (w/v) banana peel and 1 g/l commercial monosodium glutamate at 30 °C and 200 rpm after 54 h of cultivation. The biosurfactant obtained by extraction with ethyl acetate showed high surface tension reduction (25.5 mN/m), a small critical micelle concentration value (10 mg/l), thermal and pH stability with respect to surface tension reduction and emulsification activity, and a high level of salt tolerance. The biosurfactant obtained was confirmed as a lipopeptide by using a biochemical test FT-IR, NMR, and mass spectrometry. The crude biosurfactant showed a broad spectrum of antimicrobial activity and had the ability to emulsify oil, enhance PAHs solubility, and oil bioremediation.     

Application of Lipopeptide Biosurfactant Isolated from a Halophile: Bacillus tequilensis CH for Inhibition of Biofilm

Biosurfactants are amphiphilic molecules having hydrophobic and hydrophilic moieties produced by various microorganisms. These molecules trigger the reduction of surface tension or interfacial tension in liquids. A biosurfactant-producing halophile was isolated from Lake Chilika, a brackish water lake of Odisha, India (19°41′39″N, 85°18′24″E). The halophile was identified as Bacillus tequilensis CH by biochemical tests and 16S rRNA gene sequencing and assigned accession no. KC851857 by GenBank. The biosurfactant produced by B. tequilensis CH was partially characterized as a lipopeptide using thin-layer chromatography, Fourier transform infrared spectroscopy, and nuclear magnetic resonance techniques. The minimum effective concentration of a biosurfactant for inhibition of pathogenic biofilm (Escherichia coli and Streptococcus mutans) on hydrophilic and hydrophobic surfaces was found to be 50 μg ml^?1. This finding has potential for a variety of applications.     

Emulsification potential of a newly isolated biosurfactant-producing bacterium, Rhodococcus sp. strain TA6

An indigenous biosurfactant producing bacterium, Rhodococcus sp. strain TA6 was isolated from Iranian oil contaminated soil using an efficient enrichment and screening method. During growth on sucrose and several hydrocarbon substrates as sole carbon source, the bacterium could produce biosurfactants. As a result of biosurfactant synthesis, the surface tension of the growth medium was reduced from 68mNm(-1) to values below 30mNm(-1). The biosurfactant was capable of forming stable emulsions with various hydrocarbons ranging from pentane to light motor oil. Preliminary chemical characterization revealed that the TA6 biosurfactant consisted of extracellular lipids and glycolipids. The biosurfactant was stable during exposure to high salinity (10% NaCl), elevated temperatures (120°C for 15min) and within a wide pH range (4.0-10.0). The culture broth was effective in recovering up to 70% of the residual oil from oil-saturated sand packs which indicates the potential value of the biosurfactant in enhanced oil recovery.     

Physicochemical and Transmission Electron Micrograph (TEM) Studies of Few Carbohydrate Molecules with Aqueous and Aqueous Geraniol Oil Mixtures

Densities (ρ/10³ kg m^?3), viscosities (η/0.1 N s m^?2) and surface tension (γ/mN m^?1) of aqueous 0.005 to 0.02 mol L^?1 mannitol, glucose and fructose at an interval of 0.005 mol L^?1 and the 0.02, 0.025, and 0.03 v% aqueous geraniol oil were studied. The similar carbohydrates were also studied with 0.025 v% aqueous geraniol. TEM were recorded for sizes of molecules for analysis of probable energy consumption in flow through a capillary of Survismeter, like blood capillaries. The ρ values were used for viscosities (η) and surface tensions (γ). The η were fitted with extended Jones-Dole equation for B/L mol^?1 and D/(L mol^?1)² coefficients to explain carbohydrate-geraniol interactions. The B and γ values illustrate structural behavior of carbohydrates irrespective of the compositions. These data analyze hydrophilic and hydrophobic interactions of geraniol and carbohydrates where electron micrographs depict shortening of sizes and intermolecular distances with geraniol. The size with and without geraniol remained from 0.019–0.035 nm and 0.016–0.038 nm, respectively. The geraniol increased the densities and decreased the surface tension to almost one half of the binary systems with an effective intermixing.     

Cytotoxic Effect of Microbial Biosurfactants Against Human Embryonic Kidney Cancerous Cell: HEK-293 and Their Possible Role in Apoptosis

Two different microbial biosurfactants S9BS and CHBS were isolated from Lysinibacillus fusiformis S9 and Bacillus tequilensis CH. Cytotoxicity effect of these biosurfactants on human embryonic kidney cancerous cell (HEK-293) were studied with the help of 3-(4,5-dimethylthiazol-2yl-)-2, 5-diphenyl tetrazolium bromide (MTT) assay and morphological changes were observed under inverted microscope. The biosurfactants exhibited positive cytotoxic effect on HEK-293 cell line. It was found that LC₅₀ of S9BS and CHBS were 75 and 100 μg ml^?1, respectively. Further cell cycle and apoptosis analysis of biosurfactant-treated HEK-293 cell line were done by FACS. In this study, cytotoxic effect of glycolipid biosurfactant against HEK-293 cell lines is reported for the first time. Mechanism towards increased membrane permeability of biosurfactant-treated cancer cell may be the incorporation of its lipid moiety into the plasma membrane leading to formation of pores and membrane disruption. Hence, these microbial biosurfactants can prove to be significant biomolecule for cancer treatment.     

Synthesis and Properties of Gluconamide-Based Trisiloxane Surfactant

A gluconamide-based trisiloxane surfactant (Si3N2-GA) was prepared via a two-step method. Structure characterization of the Si3N2-GA was performed by fourier transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (¹H NMR). Surface activity and aggregation behavior in aqueous solution of the Si3N2-GA were investigated by surface tension measurements, dynamic light scattering (DLS), and negative-stained transmission electron microscopy (TEM). The results show that the critical micelle concentration (CAC) and surface tension (γ_CAC) at the cmc in aqueous solution of the Si3N2-GA are 3.77 × 10^?4 M and 20.5 mN · m^?1, respectively, and the surfactants can self-assemble into spherical vesicle with diameters in the range from 50 to 150 nm.