Supplementary Materials
Table S1. Techniques traditionally used for the detection and conformation of LP/BS or BS-producing microorganisms.
Method type / Method name / Short description / Simplicity / Application in HTS / Precision / Quantitative results / Sensitivity / Comments / ReferencesIndirect (surface/interfacial activity) / Drop collapse / Shape of a drop of cell-free culture supernatant on hydrophobic surface (e.g., on plastic Petri dish or parafilm) is analyzed. A significantly larger dimension of a drop of tested supernatant in comparison to water drop indicates presence of surfactants. / + + + / + / + / + / + / Sensitive to environment conditions. One of the most utilized methods for detection of BS. / (Burch et al., 2010; Chen et al., 2007;Jain et al., 1991; Youssef et al., 2004)
Oil spreading/ oil displacement / A drop of clarified culture is put on a water surface coated with hydrophobic liquid (e.g., hexadecane). If supernatant contains surface active compounds, oil will be displaced. / + + + / + / + / + / + / Sensitive to environment conditions. Dimension of a cleared area can be measured for relative or even semi-quantitative results on biosurfactants yield (but this tend to be an overestimation). / (Satpute et al., 2008; Youssef et al., 2004)
Surface tension (ST) / Surface tension of clarified culture supernatant is measured. Different techniques are utilized and de Nouy platinum ring coupled with automatic tensiometer seems to be the most popular one. / + + / - / + + / + / + / Commonly used for confirming the production of BS. BS-producing microorganisms reduce surface tension (air/water) to values o 35-40 mNm-1. Measurements can be affected by environmental conditions. Semi-quantitative results can be obtained with the use of CMD (critical micelle dilution). / (Joshi et al., 2013; Youssef et al., 2004)
Emulsification index (EI) / Few ml of cell-free culture supernatant is poured into a glass tube together with hydrophobic liquid (e.g., oil) and mixed. Height of emulsion phase is measured after given period of time. / + + + / - / + / + / + / Widely used in BS research. Analysis is time-consuming and sensitive to environment conditions. / (Chen et al., 2007; Lawrance et al., 2014; Satpute et al., 2008)
Indirect / Hemolytic activity (HA) / Microorganisms are cultivated on agar plates with 5% blood. Observation of hemolysis zone around colonies can indicate presence of BS. / + + + / + / + / + / + / Traditionally used for screening for BS-producers, now being replaced by more appropriate techniques. Analysis is time-consuming. HA is not common for BS. / (Morán et al., 2002; Satpute et al., 2008; Youssef et al., 2004)
Antimicrobial activity / Potential BS-producing isolates are cultivated on agar plates simultaneously with e.g. pathogenic bacteria or fungi species. After given period of time size of the clear inhibitory zone around BS-producing colonies is measured. / + + + / + / + / + / + / Antimicrobial activity is not common for BS. Can be helpful during preliminary studies on the biological activity of BS. / (Hsieh et al., 2008)
Direct / Thin layer chromatography (TLC) / Hydrophobic/amphiphilic compounds are extracted from microbial culture. Extract is evaporated, dissolved in methanol and applied to a TLC silica plate. Also direct-colony TLC can be used. Different mobile phases are used for plate developing. Then, compounds are visualized (e.g., UV light, iodine, ninhydrin). / + + / - / + + / - / + / Different reagents can be used to visualize different moieties (lipids, sugars, peptides) and help with preliminary studies on BS structure. Samples should be analyzed simultaneously with standards for comparison. / (Huang et al., 2009; Matsuyama et al., 1992; Peng et al., 2008)
Simplicity is an outcome of availability and price of laboratory equipment and materials needed in a certain test as well as demand for skilled staff: very easy (+ + +), easy (+ +), difficult (+); application in HTS: very good (+ + +), good (+ +), poor (+), not applicable (-); precision is an outcome of repeatability of a certain test and the probability of receiving false-positive or false-negative results: very precise (+ + +), precise (+ +), imprecise (+); quantitative results: very good (+ + +), good (+ +), poor (+), not applicable (-); sensitivity is the relative concentration of surfactant that can be detected: very good (+ + +), good (+ +), poor (+), not applicable (-).
Table S2. Various solvent systems and developers employed in TLC methods. Solvent mixtures compositions are shown in volume ratios.
Microorganisms / BS type / Solvent system(s) / Developer(s) / ReferencesB. subtilis / Surfactin / Chloroform/methanol/ammonium hydroxide (65:35:5) / Warming/heating in distilled water at 100°C / (de Faria et al., 2011)
B. subtilis / Surfactin / Chloroform/methanol/propanol/0.25% KCl/ethyl acetate (25:13:25:9:25) / Warming/heating in distilled water at 100°C / (Vedaraman and Venkatesh, 2011)
Alcaligenes sp. / Lipopeptides / Chloroform/ethanol (95:5)
Chloroform/ethanol/water(85:15:2)
Chloroform/ethanol/water (65:25:4)
Hexane/ethanol/acetic acid (85:15:2)
n-butanol/formic acid/water (16:4:8) / Ninhydrin, bromothymol blue and phenol-sulfuric acid / (Huang et al., 2009)
B. subtilis / Bacillomycin D / n-butanol/methanol/water (39:10:20) / Ninhydrin or 4,4’-
Bis(dimethylamino)diphenylmethane; Pauly reagent / (Tabbene et al., 2011)
B. amyloliquefaciens / Lipopeptides / Butanol/acetic acid/water (6:1:2) / UV-active compounds were detected at 254 and 360nm / (Caldeira et al., 2011)
P. syringae / Lipopeptides / Chloroform/methanol/NH4OH (80:25:4) / Bromothymol blue (0.1% in 10% ethanol) / (Berti et al., 2007)
P. fluorescence / Viscosin / Chloroform/methanol/water (65:25:4) / Ninhydrin 4,4’-tetra methyldiamino-diphenylmethane / (Neu et al., 1990)
P. fluorescens / Pseudofactin / Chloroform/methanol/water (65:25:4) / 0.25% ninhydrin in acetone;
0.1% bromothymol blue in 10% aqueous ethanol; iodine / (Janek et al., 2010)
Rhodococcus sp. / Lipopeptides / Chloroform/methanol/water(85:15:2) / Ninhydrin and iodine / (Peng et al., 2008)
Table S3. Typical vibrational frequencies (cm-1) of lipopeptides’ functional groups (Socrates, 2004).
Approximate frequency (cm−1) / Molecular vibrations of functional groups3200 / N-H stretching of amide A
2955 / C-H asymmetric stretching of -CH3
2930 / C-H asymmetric stretching of >CH2
2898 / C-H stretching of ≥C-H of amino acids
2870 / C-H symmetric stretching of -CH3
2850 / C-H symmetric stretching of >CH2
1740 / >C=O stretching of lipid esters
1715 / >C=O stretching of ester,
1695-1675 / C=O stretching of amide I band
1655 / Amide I of α-helical structures
1637 / Amide I of β-pleated sheet structures
1550-1520 / Amide II band
1468 / C-H deformation of >CH2
1415 / C-O-H in-plane bending
1400 / C=O symmetric stretching of COO- group in amino acids, fatty acids
1310-1240 / Amide III band
1200-900 / C-O-C, C-O dominated by ring vibrations
720 / C-H rocking of >CH2
Supplementary References
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