Marine Bacterium Derived Lipopeptides: Characterization and Cytotoxic Activity Against Cancer Cell Lines

A marine Bacillus circulans DMS-2 was able to grow and produce biosurfactant on glucose mineral salts medium (GMSM) with a reduction in the surface tension up to 27 mN m⁻¹. The microorganism produced 1.64 ± 0.1 g l⁻¹ of crude biosurfactant. The lipopeptide nature of the produced biosurfactant was confirmed by primulin and ninhydrin assays using High Performance Thin Layer Chromatography (HPTLC). Preparative thin layer chromatography (TLC) was performed to purify the lipopeptides from the crude biosurfactant. The critical micelle concentrations (CMC) of the crude and purified products were found to be 90 and 40 mg l⁻¹ respectively. Fourier transform infrared spectrophotometer (FTIR) and matrix assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectral analysis revealed the identity of the produced lipopeptides as surfactin (m/z 1,023 Da) and fengycin (m/z 1,495 Da) isoforms. The purified marine lipopeptides displayed a significant antiproliferative activity against the human colon cancer cell lines HCT-15 (IC₅₀ 80 μg ml⁻¹) and HT-29 (IC₅₀ 120 μg ml⁻¹).     

Evaluation Antimicrobial and Antiadhesive Properties of the Biosurfactant Lunasan Produced by <Emphasis Type="Italic">Candida sphaerica</Emphasis> UCP 0995

Different groups of biosurfactants exhibit diverse properties and display a variety of physiological functions in producer microorganisms; these include enhancing the solubility of hydrophobic/water-insoluble compound, heave metal binding, bacterial pathogenesis, cell adhesion and aggregation, quorum sensing and biofilm formation. Candida sphaerica was grown in a low cost medium, consisting of distilled water supplemented with 9% refinery residue of soybean oil and 9% corn steep liquor, for 144 h at 28°C and 150 rpm. The cell-free supernatant obtained at the end of the experiments was submitted to extraction, and afterward the biosurfactant was isolated using methanol with a yield of 9 g l⁻¹. The critical micelle concentration of the biosurfactant was found to be 0.25 mg ml⁻¹ with a surface tension of 25 mN m⁻¹. Several concentrations of the biosurfactant (0.625–10 mg ml⁻¹) were used to evaluate its antimicrobial and antiadhesive activities against a variety of microorganisms. The biosurfactant showed antimicrobial activity against Streptococcus oralis (68%), Candida albicans (57%), and Staphylococcus epidermidis(57.6%) for the highest concentration tested. Furthermore, the biosurfactant at a concentration of 10 mg ml⁻¹ inhibited the adhesion between 80 and 92% of Pseudomonas aeruginosa, Streptococcus agalactiae, Streptococcus sanguis12. Inhibition of adhesion with percentages near 100% occurred for the higher concentrations of biosurfactant used. Results gathered in this study point to a potential use of the biosurfactant in biomedical applications.     

Biosurfactant production by a thermophilic Bacillus subtilis strain

A strain of Bacillus subtilis was able to grow and produce a biosurfactant on 2% sucrose at 45°C. As a result of biosurfactant synthesis the surface tension of the medium was reduced from 68 dynes cm⁻¹ to 28 dynes cm⁻¹. The strain had the capacity to produce the biosurfactant at high NaCl concentrations (4%) and a wide range of pH (4.5–10.5). The biosurfactant retained its surface-active properties after heating at 100°C for 2 h and at different pH values (4.5–10.5). A maximum amount of biosurfactant was produced when urea or nitrate ions were supplied as nitrogen source. The use of the biosurfactant at high temperatures, acidic, alkaline and saline environments is discussed. As a result of its action, 62% of oil in a sand pack column could be recovered, indicating its potential application in microbiologically enhanced oil recovery. Received 28 March 1996/ Accepted in revised form 16 September 1996     

Production of biosurfactant at mesophilic and thermophilic conditions by a strain of Bacillus subtilis

The ability of a Bacillus subtilis strain to grow and produce biosurfactant on different carbon and nitrogen sources under thermophilic conditions (45°C) was studied. The strain was able to reduce surface tension to 34 dynes cm⁻¹ on 2% sucrose, and 32 dynes cm⁻¹ on starch after 96 h of growth. The biosurfactant was stable at 100°C and within a wide pH range (3.0–11.0). Biosurfactant formation at mesophilic conditions (30°C) was also studied. The organism was able to produce the maximum amount of biosurfactant when nitrate ions were supplied as the nitrogen source. The potential application of the biosurfactant in oil recovery from desert oil fields, acidic and alkaline environments is demonstrated. The biosurfactant was identical to surfactin as confirmed by TLC and IR analysis. Received 29 May 1997/ Accepted in revised form 03 October 1997     

Effects of Rhamnolipids from Pseudomonas aeruginosa DS10-129 on Luminescent Bacteria: Toxicity and Modulation of Cadmium Bioavailability

In this study, the mixture of mono- and di-rhamnolipids produced by Pseudomonas aeruginosa DS10-129 was characterized for its toxicity and modulatory effects on Cd availability to different bacteria. Gram-negative naturally bioluminescent Vibrio fischeri and recombinant bioluminescent Pseudomonas fluorescens, P. aeruginosa, Escherichia coli, and Gram-positive Bacillus subtilis were used as model organisms. Rhamnolipids reduced the bioluminescence of these bacteria in less than a second of exposure even in relatively low concentrations (30-min EC₅₀ 45–167 mg l⁻¹). Toxicity of Cd to Gram-negative bacteria (30-min EC₅₀ values 0.16 mg l⁻¹ for E. coli, 0.96 mg l⁻¹ for P. fluorescens, and 4.4 mg l⁻¹ for V. fischeri) was remarkably (up to 10-fold) reduced in the presence of 50 mg l⁻¹ rhamnolipids. Interestingly, the toxicity of Cd to Gram-positive B. subtilis (30-min EC₅₀ value 0.49 mg l⁻¹) was not affected by rhamnolipids. Rhamnolipids had an effect on desorption of Cd from soil: 40 mg l⁻¹ rhamnolipids increased the water-extracted fraction of Cd twice compared with untreated control. However, this additionally desorbed fraction of Cd remained bound with rhamnolipids and was not available to bacteria. Hence, in carefully chosen concentrations (still effectively complexing heavy metals but not yet toxic to soil bacteria), rhamnolipids could be applied in remediation of polluted areas.     

Biosurfactant production by free and alginate entrapped cells of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis>

Production of biosurfactant by free and alginate-entrapped cells of Pseudomonas fluorescens Migula 1895-DSMZ was investigated using olive oil as the sole carbon and energy source. Biosurfactant synthesis was followed by measuring surface tension and emulsifying index E24 over 5 days at ambient temperature and at neutral pH. Diffusional limitations in alginate beads affected the kinetics of biosurfactant production when compared to that obtained with free cells culture. Nevertheless, the emulsion stability was improved and fewer by-products interfered with the biosurfactant activity. A decrease in pH down to 5 in the case of immobilized cells was observed during the first 3 days, after which it returned to its initial value. The minimum values of surface tension were 30 and 35 dynes cm⁻¹ achieved after 40 and 72 h with free and immobilized cells, respectively, while the corresponding maximum E24 values were 67 and 62%, respectively. After separation by acetone precipitation, the biosurfactant showed a rhamnolipid-type in nature, and had a good foaming and emulsifying activities. The critical micellar concentration was found to be 290 mg l⁻¹. The biosurfactant also showed good stability during exposure to high temperatures (up to 120 °C for 15 min), to high salinity (10% NaCl) and to a wide range of pH (4–9).     

Effects of carbon and nitrogen sources on rhamnolipid biosurfactant production by <Emphasis Type="Italic">Pseudomonas nitroreducens</Emphasis> isolated from soil

Rhamnolipid biosurfactant production by Pseudomonas nitroreducens isolated from petroleum-contaminated soil was investigated. The effects of carbon, nitrogen and carbon to nitrogen ratio on biosurfactant production were examined using mineral salts medium as the growth medium. The tenso-active properties (surface activity and critical micelle concentrations of the produced biosurfactant were also evaluated. The best carbon source, nitrogen source were glucose and sodium nitrate giving rhamnolipid yields of 5.28 and 4.38 g l⁻¹, respectively. The maximum rhamnolipid production of 5.46 g l⁻¹ was at C/N (glucose/sodium nitrate) of 22. The rhamnolipid biosurfactant reduced the surface tension of water from 72 to ~37 mN/m. It also has critical micelle concentration of ~28 mg l⁻¹. Thus, the results presented in our reports show that the produced rhamnolipid can find wide applications in various bioremediation activities such as enhanced oil recovery and petroleum degradation.     

Biosorption of cadmium by a Cd2+-hyperresistant Bacillus cereus strain HQ-1 newly isolated from a lead and zinc mine

A Cd²⁺-hyperresistant bacterial strain HQ-1 was isolated from a lead–zinc mine. The strain was characterized and identified as Bacillus cereus based on morphology, physiological tests and 16S rRNA gene analysis. The minimal inhibitory concentration of Cd²⁺ for the bacterium was 0.012 mol/l. Isotherms for cadmium (Cd) biosorption by cells of B. cereus strain HQ-1 were investigated. The equilibrium data could be fitted by a Langmuir isotherm equation. The possible functional sites that might be influenced by the sorption were determined. The results indicate that this B. cereus strain has excellent potential for biosorption of Cd. Physiological characterization of the isolate also indicates possible application of this strain for bioremediation of sites with Cd contamination.     

Heme oxygenase 2 of the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 is induced under a microaerobic atmosphere and is required for microaerobic growth at high light intensity

Cyanobacteria, red algae, and cryptomonad algae utilize phycobilin chromophores that are attached to phycobiliproteins to harvest solar energy. Heme oxygenase (HO) in these organisms catalyzes the first step in phycobilin formation through the conversion of heme to biliverdin IXα, CO, and iron. The Synechocystis sp. PCC 6803 genome contains two open reading frames, ho1 (sll1184) and ho2 (sll1875), whose products have in vitro HO activity. We report that HO2, the protein encoded by ho2, was induced in the cells growing under a microaerobic atmosphere [0.2% (v/v) O₂], whereas HO1 was constitutively expressed under both aerobic and microaerobic atmospheres. Light intensity did not have an effect on the expression of both the HOs. Cells, in which ho2 was disrupted, were unable to grow microaerobically at a light intensity of 40 μmol m⁻² s⁻¹, but did grow microaerobically at 10 μmol m⁻² s⁻¹ light intensity. These cells grew normally aerobically at both light intensities. Comparative analysis of complete cyanobacterial genomes revealed that possession of two HOs is common in cyanobacteria. In phylogenetic analysis of their amino acid sequences, cyanobacterial HO1 and HO2 homologs formed distinct clades. HO sequences of cyanobacteria that have only one isoform were most similar to HO1 sequences. We propose that HO2 might be the more ancient HO homolog that functioned under low O₂ tension, whereas the derived HO1 can better accommodate increased O₂ tension in the environment.     

Desulfurization of dibenzothiophene by Bacillus subtilis recombinants carrying dszABC and dszD genes

The desulfurization (dsz) genes from Rhodococcus erythropolis DS-3 were successfully integrated into the chromosomes of Bacillus subtilis ATCC 21332 and UV1 using an integration vector pDGSDN, yielding two recombinant strains, B. subtilis M29 and M28 in which the integrated dsz genes were expressed efficiently under the promoter, Pspac. The dibenzothiophene (DBT) desulfurization efficiency of M29 was 16.2 mg DBT l⁻¹ h⁻¹ at 36 h, significantly higher than that of R. erythropolis DS−3 and B. subtilis M28 and also showed no product inhibition. The interfacial tension of the supernatant fermented by M29 varied from 48 mN m⁻¹ to 4.2 mN m⁻¹, lower than that of the recombinant strain, M28, reveals that the biosurfactant secreted from M29 may have an important function in the DBT desulfurization process.     

Laboratory production and characterization of a new biosurfactant from <Emphasis Type="Italic">Candida glabrata</Emphasis> UCP1002 cultivated in vegetable fat waste applied to the removal of hydrophobic contaminant

Biosurfactant production by Candida glabrata was studied using vegetable fat waste as substrate. A factorial design was initially carried out to investigate the effects and interactions of waste, yeast extract and glucose on the surface tension after 144 h cultivation. Maximum surface tension reduction was achieved with vegetable fat waste at 5% and yeast extract at 0.2%. The biosurfactant containing cell-free broth retained its surface-active properties after incubation at high temperatures, at a wide range of pH values and salt concentrations. Comparison between three solvent systems for surfactant recovery showed that ethyl acetate extracted both crude extracellular and intracellular biosurfactant with high product recovery. The isolated extracellular biosurfactant showed a CMC of 1% and the surface tension at that point was 24 mN m⁻¹. Preliminary chemical composition revealed the presence of carbohydrates, proteins and lipids. The application of the crude biosurfactant to a soil–water-hydrophobic contaminant system was investigated and the apparent critical micelle concentration was determined at 7% of the broth, although the best oil removal (92.6%) had been obtained with 10% of the cell-free broth. The cost of application of the biosurfactant in soils was estimated based on the cost of a commercial biosurfactant.     

Chitinolytic enzymes from the newly isolated <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> SBK1: study of the mosquitocidal activity

Aeromonas hydrophila SBK1 (GenBank accession no. HM802878.1), a potent chitinolytic bacterium, was isolated from a pool of 30 chitinolytic isolates. The isolate showed higher chitinolytic activity in respect to clear zone to colony size ratio of 2.15. Maximum production of chitinolytic enzymes, viz., β-N-acetyl-glucosaminidase and chitinase (specific activity 655.3 and 71.6 U mg⁻¹, respectively) by A. hydrophila SBK1 was observed in the synthetic media, containing (w/v)-colloidal chitin, 4.0%; peptone, 0.3%; phosphate, 0.3% (0.15% of each KH₂PO₄ and K₂HPO₄); NaCl, 0.25%; MgSO₄, 0.05%; KCl, 0.05%; pH 7.0 and at 35°C after 72 h of incubation. Both carbon-to-nitrogen (C/N) and carbon-to-phosphate (C/P) ratio of 13.33 were found optimum for chitinase production. Enzyme productivity increased about twofold in optimized culture condition in respect to its un-optimized state. The crude enzyme showed optimum activity against Culex quinquefasciatus larvae in native water at pH 7.0 and 35°C (LD₅₀ 0.60 U ml⁻¹ at 48 h). Therefore, the studied chitinases can be used as an effective mosquitocidal agent.     

Production of tetramethylpyrazine by batch culture of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> with optimal pH control strategy

The effects of initial culture pH ranging from 5.0 to 7.5 on biomass content, precursor 3-hydroxy-2-butanone (HB) accumulation, and 2,3,5,6-tetramethylpyrazine (TTMP) formation by Bacillus subtilis CCTCC M 208157 were investigated in shake flask fermentation. Weak acidic conditions were found to favor cell growth and precursor HB accumulation, while TTMP could be synthesized more efficiently in conditions with initial pH towards neutrality. Batch bioprocess of TTMP fermentation by Bacillus subtilis CCTCC M 208157 at various controlled pH values ranging from 5.5 to 7.0 was then examined in 7.5-l fermentor. The results suggested that optimum pH for cell growth and precursor HB accumulation was 5.5 with maximum cell growth rate (Q _x) and precursor HB accumulation rate (Q _HB) of 0.833 g l⁻¹ h⁻¹ and 1.118 g l⁻¹ h⁻¹, respectively, while optimum pH for TTMP formation was 7.0 with maximum TTMP formation rate (Q _TTMP) of 0.095 g l⁻¹ h⁻¹. A pH-shifted strategy was accordingly developed to improve TTMP production in bioreactor fermentation by shifting the culture pH from 5.5 to 7.0 after 48 h of cultivation. By applying the strategy, final TTMP concentration of 7.43 g l⁻¹ was obtained, being 22.2% greater than that of constant-pH fermentation.     

Degradation of hydrocarbons and biosurfactant production by Pseudomonas sp. strain LP1

Pseudomonas sp. strain LP1, an organism isolated on the basis of its ability to grow on pyrene, was assayed for its degradative and biosurfactant production potentials when growing on crude, diesel and engine oils. The isolate exhibited specific growth rate and doubling time of 0.304 days⁻¹ and 2.28 days, respectively on crude oil (Escravos Light). The corresponding values on diesel were 0.233 days⁻¹ and 2.97 days, while on engine oil, were 0.122 days⁻¹ and 5.71 days. The organism did not show significant biosurfactant production towards crude oil and diesel, but readily produced biosurfactant on engine oil. The highest Emulsification index (E₂₄) value for the biosurfactant produced by LP1 on engine oil was 80.33 ± 1.20, on day 8 of incubation. Biosurfactant production was growth-associated. The surface-active compound which exhibited zero saline tolerance had its optimal activity at 50°C and pH 2.0.     

Crude biosurfactant from thermophilic Alcaligenes faecalis: Feasibility in petro-spill bioremediation

The thermophilic bacterium Alcaligenes faecalis isolated from the crude oil contaminated soil of Upper Assam, India. The isolated bacterium was first screened for the ability to produce biosurfactant. The strain growing at 42 °C could produce higher amount of biosurfactant in medium supplemented with 2% (v/v) diesel as sole source of carbon and energy. Biochemical characterizations including FT-IR and MS studies suggested the biosurfactant to be glycolipid. Tensiometric studies revealed that the biosurfactant produced by the bacterial strain could decrease the surface tension (??) at air-water interface from 71.6 to 32.3 mNm⁻¹ after 96 h of growth on hydrocarbon and possessed a low critical micelle concentration (CMC) value of approximately 38 mgl⁻¹, indicating high surface activity. The culture supernatant containing the biosurfactant was found to be functionally stable at varying pH (2-12), temperature (100 and 121 °C) and salinity (1-6% NaCl, w/v) conditions. Both the culture broth and the cell free supernatant exhibited high emulsifying activity against the different hydrocarbons and the crude oil components. The increase in cell surface hydrophobicity and glycolipid production by the strain suggested the existence of biosurfactant enhanced interfacial uptake of the hydrocarbons. Moreover, the partially purified biosurfactant exhibited antimicrobial activity by inhibiting the growth of several bacterial and fungal species. The strain represented a new class of biosurfactant producers and could be a potential candidate for the production of glycolipid biosurfactant which could be useful in a variety of biotechnological and industrial processes, particularly in the oil industry.     

A halotolerant,thermotolerant, and facultative biosurfactant producer: Identification and molecular characterization of a bacterium and evolution of emulsifier stability of a lipopeptide biosurfactant

A halotolerant, thermotolerant, and facultative biosurfactant producing bacterium was identified as a strain of Bacillus mojavensis based on the phenotypic data, a phylogenetic analysis, and DNA-DNA relatedness with closely-related species. This strain grew at temperatures and salinities up to 55°C and 0 ∼ 10% (w/v) NaCl, respectively, and under anaerobic conditions. A batch fermentation showed that this strain secreted a lipopeptide biosurfactant that can reduce surface tension to 27 mN/m while growing on mineral medium. The emulsifying activity of the cell-free supernatant and stability of the formed emulsions were studied at various temperatures and salinities. The results showed that the ability to significantly reduce surface tension was not sufficient to form stable emulsions. The ability of this strain to grow and reduce surface tension under a wide range of salinities and temperatures gives it an advantage for many applications.     

Bacillus manliponensis sp. nov., a new member of the Bacillus cereus group isolated from foreshore tidal flat sediment

A Gram-positive, endospore-forming, new Bacillus species, strain BL4-6^T, was isolated from tidal flat sediment of the Yellow Sea. Strain BL4-6^T is a straight rod, with motility by peritrichate flagella. The cell wall contains meso-diaminopimelic acid, and the major respiratory quinone is menaquinone-7. The major fatty acids are iso-C_15:0 and summed feature 3 (containing C_16:1 ω7c/iso-C_15:0 2OH, and/or iso-C_15:0 2OH/C_16:1 ω7c). Cells are catalase-positive and oxidase-negative. The G+C content of the genomic DNA is 38.0 mol%. Based on a comparative 16S rRNA gene sequence analysis, the isolate belongs to the genus Bacillus, forms a clade with the Bacillus cereus group, and is closely related to Bacillus mycoides (98.5%), Bacillus cereus (98.5%), Bacillus anthracis (98.4%), Bacillus thuringiensis (98.4%), Bacillus weihenstephanensis (98.1%), and Bacillus pseudomycoides (97.5%). The isolate showed less than 85% similarity of the gyrA gene sequence and below 95% similarity of the rpoB gene sequence to the members of this group. DNA-DNA relatedness between strain BL4-6^T and B. cereus group was found to be in a range of 22.8–42.3%, and thus BL4-6^T represents a unique species. On the basis of these studies, strain BL4-6^T (=KCTC 13319^T =JCM 15802^T) is proposed to represent the type strain of a novel species, Bacillus manliponensis sp. nov.     

Isolation and characterization of halophilic Archaea able to produce biosurfactants

Halotolerant microorganisms able to live in saline environments offer a multitude of actual or potential applications in various fields of biotechnology. This is why some strains of Halobacteria from an Algerian culture collection were screened for biosurfactant production in a standard medium using the qualitative drop-collapse test and emulsification activity assay. Five of the Halobacteria strains reduced the growth medium surface tension below 40 mN m⁻¹, and two of them exhibited high emulsion-stabilizing capacity. Diesel oil-in-water emulsions were stabilized over a broad range of conditions, from pH 2 to 11, with up to 35% sodium chloride or up to 25% ethanol in the aqueous phase. Emulsions were stable to three cycles of freezing and thawing. The components of the biosurfactant were determined; it contained sugar, protein and lipid. The two Halobacteria strains with enhanced biosurfactant producers, designated strain A21 and strain D21, were selected to identify by phenotypic, biochemical characteristics and by partial 16S rRNA gene sequencing. The strains have Mg²⁺, and salt growth requirements are always above 15% (w/v) salts with an optimal concentration of 15–25%. Analyses of partial 16S rRNA gene sequences of the two strains suggested that they were halophiles belonging to genera of the family Halobacteriaceae, Halovivax (strain A21) and Haloarcula (strain D21). To our knowledge, this is the first report of biosurfactant production at such a high salt concentration.     

An efficient biosurfactant-producing and crude-oil emulsifying bacterium Bacillus methylotrophicus USTBa isolated from petroleum reservoir

An efficient biosurfactant-producing bacterium was isolated and cultured from petroleum reservoir in northeast China. Isolate was screened for biosurfactant production using haemolytic assay, Cetyl Trimethyl Ammonium Bromide agar plate assay (CTAB) and the qualitative oil-displacement test. Based on partial sequenced 16S rDNA analysis of isolate, USTBa, identified as Bacillus methylotrophicus with 100% identity. This bacterium was able to produce a type of biosurfactant with excessive foam-forming properties. The maximum biosurfactant production was obtained when the cells were grown on minimal salt medium containing 2% (v/v) crude-oil as the sole source of carbon at 35 °C and 180 rpm after 192 h. This strain had a high emulsification activity and biosurfactant production of 78% and 1.8 g/L respectively. The cell free broth containing biosurfactant could reduce the surface tension to 28 mN/m. Fourier transform infrared (FT-IR) spectrum of extracted biosurfactant indicates the presence of carboxyl, hydroxyl and methoxyl functional groups. Elemental analysis of the biosurfactant by Energy dispersive X-ray spectroscopy (EDS) reveals that the biosurfactant was anionic in nature. The strain USTBa represented as a potent biosurfactant-producer and could be useful in variety of biotechnological and industrial processes, particularly oil industry.     

Interfacial Tension of the Lipid Membrane Formed from Phosphatidylcholine–Decanoic Acid and Phosphatidylcholine–Decylamine Systems

Interfacial tension has been determined for phosphatidylcholine (PC)–decanoic acid (DA) and PC–decylamine (DE) membranes. PC (lecithin), DA and DE were used in the experiments; the interfacial tension values of the pure components are 1.62 × 10⁻³, −2.38 × 10⁻² and −3.88 × 10⁻² N/m (hypothetical values for DA and DE), respectively. The 1:1 complexes were formed during formation of PC–DA and PC–DE membranes. The following parameters describing the complexes were determined: the surface concentrations of the lipid membranes formed from these complexes, A₃ ^{- 1} A_{3}^{ - 1} ; the interfacial tensions of such membranes, γ ₃; and the stability constants of these complexes, K.