Molasses as a whole medium for biosurfactants production by Bacillus strains and their application

Two types of biosurfactant (BS)-producing bacteria, Bacillus licheniformis TR7 and Bacillus subtilis SA9, were isolated from mangrove sediment in the south of Thailand. The BS production was done by using only molasses as a whole medium for growth and production. Under optimized conditions, the yields of TR7 and SA9 BS were found to be 3.30 and 3.78 g/l, respectively. It could reduce the surface tension of pure water to 28.5 and 29.5 mN/m, with the critical micelle concentrations of about 10 and 30 mg/l, respectively. Good thermal, pH, and salt stability were exhibited. Both BSs could recover oil more effectively than the two synthetic surfactants. In addition, TR7 and SA9 BS could enhance the solubility of polyaromatic hydrocarbons (PAHs). Thus, these BSs have the potential for the removal of oil and PAHs from the combined contaminated environment and facilitate its bioremediation. These studies indicate that molasses, as a renewable, relatively inexpensive and easily available resource, can be used for important biotechnological processes.     

Interactive optimization of biosurfactant production by Paenibacillus alvei ARN63 isolated from an Iranian oil well

The potential of an indigenous bacterial strain isolated from an Iranian oil field for the production of biosurfactant was investigated in this study. After isolation, the bacterium was characterized to be Paenibacillus alvei by biochemical tests and 16S ribotyping. The biosurfactant, which was produced by this bacterium, was able to lower the surface tension of media to 35 mN/m. Accordingly, thin layer chromatography (TLC) and FT-IR has been carried out to determine compositional analysis of the produced biosurfactant. After all the tests related to characterization of the biosurfactant produced by the isolated bacterium, it was characterized as lipopeptide derivative. The combination of central composite rotatable design (CCRD) and response surface methodology (RSM) was exploited to optimize biosurfactant production. Therefore, variations of four impressive parameters, pH, temperature, glucose and salinity concentrations were selected for optimization of growth conditions. The empirical model developed through RSM in terms of effective operational factors mentioned above was found to be adequate to describe the biosurfactant production. A maximum reduction in surface tension was obtained under the optimal conditions of 13.03 g/l glucose concentration, 34.76 °C, 51.39 g/l total salt concentration and medium pH 6.89.     

Synthesis,Characterization, and Oil Recovery Application of Biosurfactant Produced by Indigenous <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> WJ-1 Using Waste Vegetable Oils

A bacterial strain was isolated and cultured from the oil excavation areas in tropical zone in northern China. The biochemical characteristics and partial sequenced 16S rRNA gene of isolate, WJ-1, was identical to those of cultured representatives of the species Pseudomonas aeruginosa. This bacterium was able to produce a type of biosurfactant. Compositional analysis revealed that the extracted biosurfactant was composed of high percentage lipid (∼74%, w/w) and carbohydrate (∼20%, w/w) in addition to a minor fraction of protein (∼6%, w/w). The best production of 50.2 g/l was obtained when the cells were grown on minimal salt medium containing 6.0% (w/v) glucose and 0.75% (w/v) sodium nitrate supplemented with 0.1% (v/v) element solution at 37 °C and 180 rpm after 96 h. The optimum biosurfactant production pH value was found to be 6.0–8.0. The biosurfactant of WJ-1, with the critical micelle concentration of 0.014 g/L, could reduce surface tension to 24.5 mN/m and emulsified kerosene up to EI₂₄ ≈95. The results obtained from time course study indicated that the surface tension reduction and emulsification potential was increased in the same way to cell growth. However, maximum biosurfactant production occurred and established in the stationary growth phase (after 90 h). Thin layer chromatography, Fourier transform infrared spectrum, and mass spectrum analysis indicate the extracted biosurfactant was affiliated with rhamnolipid. The core holder flooding experiments demonstrated that the oil recovery efficiency of strain and its biosurfactant was 23.02% residual oil.     

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.     

Biosurfactant production by Rhodococcus erythropolis grown on glycerol as sole carbon source

The production of biosurfactant by Rhodococcus erythropolis during the growth on glycerol was investigated. The process was carried out at 28°C in a 1.5-L bioreactor using glycerol as carbon source. The bioprocess was monitored through measurements of biosurfactant concentration and glycerol consumption. After 51 h of cultivation, 1.7 g/L of biosurfactant, surface, and interfacial tensions values (with n-hexadecane) of 43 and 15 mN/m, respectively, 67% of Emulsifying Index (E ₂₄), and 94% of oil removal were obtained. The use of glycerol rather than what happens with hydrophobic carbon source allowed the release of the biosurfactant, originally associated to the cell wall.     

A Novel Endoglucanase (Cel9P) From a Marine Bacterium <Emphasis Type="Italic">Paenibacillus</Emphasis> sp. BME-14

By constructing a genomic library, an endoglucanase gene (cel9P) was cloned from Paenibacillus sp. BME-14 which was isolated from the sea. It had an open-reading frame of 1,629 bp, encoding a peptide of 542-amino acid residue with a calculated molecular mass of 60 kDa. The enzyme showed the highest amino acid identity of 52% with other known endoglucanases and had a C-terminal catalytic domain belonging to the glycosyl hydrolases family 9. The optimum pH and temperature for enzymatic activity was pH 6.5 and 35 °C. The metal ions of Ca²⁺, Mg²⁺, and Mn²⁺ had a positive effect on the activity while Hg²⁺, Cu²⁺, and EDTA had a negative effect. Notably, Cel9P had 65% of the maximal activity at 5 °C. Based on the special characteristic of Cel9P, it had a potential significance for study of cold-active mechanism and industry applications.     

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.     

Production and Physico-chemical Characterization of a Biosurfactant Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> OBP1 Isolated from Petroleum Sludge

Pseudomonas aeruginosa strain OBP1, isolated from petroleum sludge, was used to produce biosurfactant from a modified mineral salt medium with 2% n-hexadecane as sole source of carbon. The crude biosurfactant was fractionated using TLC and HPLC. Using FTIR spectroscopy, ¹H NMR, and LC-MS analyses, the chemical structure of the purified fraction of crude biosurfactant was identified as rhamnolipid species. The LC-MS spectra show that monorhamnolipid (l-rhamnopyranosyl-β-hydroxydecanoyl-β- hydroxydecanoate, Rha-C₁₀-C₁₀) was produced in abundance with the predominant congener [M–H]⁻ ions for l-rhamnopyranosyl-l-rhamnopyranosyl-3-hydroxydecanoyl-3-hydroxydecanoate (Rha-Rha-C₁₀-C₁₀). Seven different carbon substrates and five nitrogen sources were examined for their effect on rhamnolipid production. Using n-hexadecane (20 g/l) as carbon substrate and urea along with (NH₄)₂SO₄ (2 g/l each) as nitrogen source was found to be the best, with a maximum yield of 4.8 g/l. The biosurfactant reduced the surface tension of water to 31.1 mNm⁻¹ with a critical micelle concentration of 45 mg/l. The biosurfactant showed a better emulsifying activity against a variety of hydrocarbon and achieved a maximum emulsion index of 82% for diesel. The purified biosurfactant showed a significant antibacterial activity against Staphylococcus aureus at a minimum inhibitory concentration of 8 μg/ml.     

Optimization and production of a biosurfactant from the sponge-associated marine fungus Aspergillus ustus MSF3

Marine endosymbiotic fungi Aspergillus ustus (MSF3) which produce high yield of biosurfactant was isolated from the marine sponge Fasciospongia cavernosa collected from the peninsular coast of India. Maximum production of biosurfactant was obtained in Sabouraud dextrose broth. The optimized bioprocess conditions for the maximum production was pH 7.0, temperature 20 °C, salt concentration 3%, glucose and yeast extract as carbon source and nitrogen sources respectively. The response surface methodology based analysis of carbon and nitrogen ratio revealed that the carbon source can increase the biosurfactant yield. The biosurfactant produced by MSF3 was partially characterized as glycolipoprotein based on the estimation of macromolecules and TLC analysis. The partially purified biosurfactant showed broad spectrum of antimicrobial activity. The strain MSF3 can be used for the microbially enhanced oil recovery process.     

Optimizing carbon/nitrogen ratio for biosurfactant production by a Bacillus subtilis strain

A Bacillus subtilis strain isolated from contaminated soil from a refinery has been screened for biosurfactant production in crystal sugar (sucrose) with different nitrogen sources (NaNO(3), (NH(4))(2)SO(4), urea, and residual brewery yeast). The highest reduction in surface tension was achieved with a 48-h fermentation of crystal sugar and ammonium nitrate. Optimization of carbon/nitrogen ratio (3, 9, and 15) and agitation rate (50, 150, and 250 rpm) for biosurfactant production was carried out using complete factorial design and response surface analysis. The condition of C/N 3 and 250 rpm allowed the maximum increase in surface activity of biosurfactant. A suitable model has been developed, having presented great accordance experimental data. Preliminary characterization of the bioproduct suggested it to be a lipopeptide with some isomers differing from those of a commercial surfactin.     

Purification and Characterization of Chitinase from <Emphasis Type="BoldItalic">Paenibacillus</Emphasis> sp. D1

A 56.56-kDa extracellular chitinase from Paenibacillus sp. D1 was purified to 52.3-fold by ion exchange chromatography using SP Sepharose. Maximum enzyme activity was recorded at pH 5.0 and 50 °C. MALDI-LC-MS/MS analysis identified the purified enzyme as chitinase with 60% similarity to chitinase Chi55 of Paenibacillus ehimensis. The activation energy (E _a) for chitin hydrolysis and temperature quotient (Q ₁₀) at optimum temperature was found to be 19.14 kJ/mol and 1.25, respectively. Determination of kinetic constants k _m, V _max, k _cat, and k _cat/k _m and thermodynamic parameters ΔH*, ΔS*, ΔG*, ΔG*_E–S, and ΔG*_E–T revealed high affinity of the enzyme for chitin. The enzyme exhibited higher stability in presence of commonly used protectant fungicides Captan, Carbendazim, and Mancozeb compared to control as reflected from the t _1/2 values suggesting its applicability in integrated pest management for control of soil-borne fungal phytopathogens. The order of stability of chitinase in presence of fungicides at 80 °C as revealed from t _1/2 values and thermodynamic parameters E _a(d) (activation energy for irreversible deactivation), ΔH*, ΔG*, and ΔS* was: Captan > Carbendazim > Mancozeb > control. The present study is the first report on thermodynamic and kinetic characterization of chitinase from Paenibacillus sp. D1.     

Antiadhesive action of a marine microbial surfactant

The antiadhesive action of a lipopeptide biosurfactant from a marine bacterium was investigated. The effect of cultivation conditions on the adhesion property of few bacterial strains was studied. It was observed that the static cultures showed greater adhesion due to scarcity of oxygen. The biosurfactant upon surface conditioning was found to be effective in removal of the microbial adhesion at a concentration as low as 0.1 g L⁻¹. The percentages of inhibition of adhesion against different test bacterial strains ranged from 15 to 89% using 0.1–10 g L⁻¹ of purified biosurfactant. These percentages of adhesion inhibition were found to be significantly higher than the previously reported values. The antiadhesive efficacy of the biosurfactant was also evident from confocal laser scanning microscopy studies.     

Production of Microbial Surfactants from Oily Sludge-Contaminated Soil by Bacillus subtilis DSVP23

The indigenous microbial community utilizing aliphatic, aromatic, and polar components from the oily sludge as sole source of carbon and energy was selected from the soil samples of Ankleshwar, India for biosurfactant production. Evaluation of biosurfactant production was done using screening assays such as surface tension reduction, hemolytic activity, emulsification activity, drop-collapse assay, and cell surface hydrophobicity studies. Maximum biosurfactant (6.9?g/l) production was achieved after 5?days of growth from Bacillus subtilis DSVP23 which was identified by 16S RNA technique (NCBI GenBank accession no. EU679368). Composition of biosurfactant showed it to be lipopeptide in nature with 15.2% protein content and 18.0% lipid content. Functional group analysis was also done by using Fourier transform infrared spectroscopy which showed it to be a protein-bound lipid thereby imparting them special properties. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric and nuclear magnetic resonance revealed that the major constituents of lipopeptide are leucine and isoleucine. Gas chromatographic analysis data indicated that oily sludge components of chain length C₁₂?CC₃₀ and aromatic hydrocarbons were degraded effectively by B. subtilis DSVP23 after 5?days of incubation. These results collectively points toward the importance of B. subtilis DSVP23 as a potential candidate for bioremediation studies.     

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.     

Solubilization of organics I: 1H NMR chemical shift perturbations,diffusometry, and NOESY indicate biphenyls internalize in micelles formed by cetyltrimethylammonium bromide

Polychlorinated biphenyls are a class of persistent environmental contaminants, and micellar solubilization can be applied to remediate them. The intermolecular aggregates of biphenyl (BP) analogs and cetyltrimethyl ammonium bromide (CTAB) were studied by chemical shift perturbation, nuclear magnetic resonance (NMR) diffusometry, quantitative proton NMR, and nuclear Overhauser effect (NOE) spectroscopy to understand the structural determinants of their solubilization. The micelles of CTAB solubilized BPs readily, but its capacity depended strongly on the nature of the functional group (BPCH₂OH > > BPCHO > BPCOOH ≈ BPCl ≈ BP). Upon internalization, the BPs diffused much slower, introduced significant low-frequency ¹H chemical shift changes for CTAB, and displayed strong intermolecular NOEs. The semiquantitative analysis of NOEs revealed further that the BPs are located in the palisade layer closer to the N⁺(CH₃)₃ head group, away from the hydrophobic core. ¹H NMR offers a simple high-throughput screening assay for evaluating and quantitating the solubilization of organics in micelles. The intermolecular NOEs and site-specific perturbation of chemical shifts add further insights on the location of solubilizates in micelles, which may be important for designing surfactants specific for environmental pollutants.     

Scale up and Application of Biosurfactant from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in Enhanced Oil Recovery

There is a lack of fundamental knowledge about the scale up of biosurfactant production. In order to develop suitable technology of commercialization, carrying out tests in shake flasks and bioreactors was essential. A reactor with integrated foam collector was designed for biosurfactant production using Bacillus subtilis isolated from agricultural soil. The yield of biosurfactant on biomass (Y _p/x), biosurfactant on sucrose (Y _p/s), and the volumetric production rate (Y) for shake flask were obtained about 0.45 g g⁻¹, 0.18 g g⁻¹, and 0.03 g l⁻¹ h⁻¹, respectively. The best condition for bioreactor was 300 rpm and 1.5 vvm, giving Y _x/s, Y _p/x, Y _p/s, and Y of 0.42 g g⁻¹, 0.595 g g⁻¹, 0.25 g g⁻¹, and 0.057 g l⁻¹ h⁻¹, respectively. The biosurfactant maximum production, 2.5 g l⁻¹, was reached in 44 h of growth, which was 28% better than the shake flask. The obtained volumetric oxygen transfer coefficient (K _L a) values at optimum conditions in the shake flask and the bioreactor were found to be around 0.01 and 0.0117 s⁻¹, respectively. Comparison of K _L a values at optimum conditions shows that biosurfactant production scaling up from shake flask to bioreactor can be done with K _L a as scale up criterion very accurately. Nearly 8% of original oil in place was recovered using this biosurfactant after water flooding in the sand pack.     

Investigating the Structural and Functional Effects of Mutating Asn Glycosylation Sites of Horseradish Peroxidase to Asp

Horseradish peroxidase (HRP) has long attracted intense research interest and is used in many biotechnological fields, including diagnostics, biosensors, and biocatalysis. Enhancement of HRP catalytic activity and/or stability would further increase its applications. One of the problems with heterologus expression of HRP especially in prokaryotic host is lack of glycosylation that affects it's stability toward H₂O₂ and thermal inactivation. In this study, two asparagine residues which constitute two of the eight glycosylation sites in native HRP (Asn 13 and 268) with respectively 83% and 65% surface accessibility were substituted with aspartic acid in recombinant HRP. Both mutant proteins expressed in Escherichia coli showed increased stabilities against heat (increase in t _1/2 from 20 min in native rHRP to 32 and 67 min in N13D and N268D) and H₂O₂ (up to threefold). Unexpectedly, despite the distance of the mutated positions from the active site, notable alterations in steady-state k _cat and K _m values occurred with phenol/4-aminoantipyrine as reducing substrate which might be due to conformational changes. No significant alteration in flexibility was detected by acrylamide quenching analyses, but ANS binding experiments purposed lesser binding of ANS to hydrophobic patches in mutated HRPs. Double mutation was non-additive and non-synergistic.     

Cassava flour wastewater as a substrate for biosurfactant production

Five cassava flour wastewater (manipueira) preparations were tested as culture media for biosurfactant production by a wild-type Bacillus sp. isolate. No-solids (F), no-solids diluted (F/2), natural (I), natural diluted (I/2), and decanted (IPS) were the tested manipueira media. The microorganism was able to grow and to produce biosurfactant on all manipueira preparations. The media whose solids were removed (F and F/2) showed better results than preparations with the presence of solids (I, I/2, and IPS). No-solids medium (F) showed a surface tension of 26,59 mN/m and reciprocal of critical micelle concentration of over 100 and was selected as a potential substrate for biosurfactant production.     

Tuning commercial diesel to microemulsified and blended form: phase behavior and implications

Abstract

Microemulsification and blending of commercial diesel is under constant research for possible fuel application. Microemulsions (ME) were prepared using diesel (D), kerosene (K), diesel and kerosene mixtures at various proportions (D?+?K) (oil phase: O), Triton X-100 surfactant (S), n-butanol, isobutanol (i-butanol), n-pentanol and n-octanol cosurfactants (C), and aqueous phase (W) containing water or brine for the study. Electrical conductance studies and temperature-induced separation of phase have been adopted for recognizing the o/w, w/o and bicontinuous zones. Dye probing has been done to explain the mass transfer among these phases. Percent of solubilization of oil in water has been enumerated in some of the ME. The possible fuel applications of the microemulsions are predicted from their density and flame brightness.     

Biofilm inhibition and antimicrobial action of lipopeptide biosurfactant produced by heavy metal tolerant strain Bacillus cereus NK1

Biosurfactants are worthful microbial amphiphilic molecules with efficient surface-active and biological properties applicable to several industries and processes. Among them lipopeptides represent a class of microbial surfactants with increasing scientific, therapeutic and biotechnological interests. A heavy metal tolerant Bacillus strain has been isolated and the biofilm inhibition and antimicrobial activity of biosurfactant produced by the strain have been studied. Biosurfactant production was confirmed by the conventional screening methods including hemolytic activity, drop collapsing test, oil displacement test, emulsification and lipase production assays. The biosurfactant produced by this strain was a lipopeptide and exhibited strong surface activity. The biosurfactant has been characterized using FTIR, TLC and HPLC. The minimum active dose of this biosurfactant when compared with the other chemical surfactants was found as 0.150±0.06 μg. The critical micelle concentration was found to be 45 mg/l. The biosurfactant was found to be stable and active over a wide range of pH, temperature and NaCl concentration. It was also able to emulsify a wide range of hydrocarbons and oils thereby extending its application for the bioremediation of oil contaminated sites. The biosurfactant exhibited significant reduction in biofilm formation by pathogens and showed potent antimicrobial activity against various gram positive, gram negative bacteria and fungi. Agar diffusion assay for heavy metal resistance showed that the isolate was resistant to ferrous, lead and zinc. Considering the biofilm inhibition and antimicrobial property of biosurfactant, it can be utilized as a potential therapeutic molecule for numerous microbial infections. The heavy metal resistance of the strain can also be harnessed as an invaluable biological tool for in situ bioremediation.