Diego Coraglia and Paula Jauregi, School of Food Biosciences, The University of Reading
Surfactin is a non-ribosomally synthesized lipopeptide produced by Bacillus subtilis, with biosurfactant properties. Surfactin has a high surface activity, is highly biodegradable, edible, has low toxicity and has anti-microbial and anti-viral properties. There is increasing pressure on industry to substitute toxic chemical surfactants for biodegradable, non-toxic alternatives. However in order to reduce the cost of production to become competitive with chemically synthesized surfactants, a cost effective purification process must be developed.
The aim of this work was to develop a two step ultra filtration process for the separation and purification of surfactin from a clarified fermentation broth. In a first ultra filtration step surfactin is retained by the membrane, (MWCO= 10 and 30 KDa). This is due to the surfactin being produced at concentrations higher than its critical micellar concentration and thus will form micelles with a subsequent increase in size. In a second step, the solvent composition of the retentate is modified by adding methanol 50% (v/v). This results in disruption of the micelles and enables the filtration of surfactin in a second ultra filtration step using the same membranes as in the previous step. This results in the removal of the main protein contaminants in the retentate and hence purification of the surfactin.
Dynamic light scattering (DLS) is a technique that measures the time-dependent fluctuations in the intensity of scattered light from a suspension of particles undergoing random, Brownian motion. Analysis of these intensity fluctuations allows for the determination of the diffusion coefficients, which in turn yields the particle size.
Molecules such as surfactin have low refractive index differences with the continuous phase and as a result they scatter very little light. For such weakly scattering samples, the intensity of scattering observed using conventional DLS instruments (i.e. 90° detection) may not be sufficient for successful sizing measurements to be performed.
The Zetasizer Nano range of instruments incorporates non-invasive back scatter (NIBS) optics. The scattered light is detected at an angle of 173°. The novel optics arrangement maximizes the detection of scattered light while maintaining signal quality. This provides the exceptional sensitivity that is required for measuring the size of weakly scattering molecules and particles.
This application note discusses particle size measurements using dynamic light scattering of the clarified broth and retentates of both ultra filtration steps, This was in order to verify the formation and disruption of micelles at the different experimental conditions which was key to the effective separation of surfactin.
All measurements reported in this application note were performed on a Zetasizer Nano S at 25°C. The Nano S contains a 4mW He-Ne laser, operating at a wavelength of 633nm, and an avalanche photodiode (APD) detector.
Dynamic light scattering measurements were made of surfactin micelles in clarified fermentation broth and separated fractions and the results obtained are summarized in table 1. The z-average diameters reported are the mean hydrodynamic diameters calculated from the intensity of scattered light as defined in the International Standard on dynamic light scattering, ISO22412.
| Sample | Fraction | z-Average Diameter (nm) |
|---|---|---|
| 1 | Clarified fermentation broth | 8 |
| 2 | Standard Surfactin solution | 6.5 |
| 3 | Filtrate UF-1 | No particles |
| 4 | Retentate UF-1 | 8.5 |
| 5 | Retentate UF-1 + methanol 50% | 100 |
| 6 | Filtrate UF-2 | No particles |
The sensitivity of the NIBS optics incorporated in the Zetasizer Nano series enables the accurate and reproducible size determination of the surfactin micelles to be made.
These results led to very important findings as they confirm the presence of surfactin micelles in the fermentation broth (sample 1) with a size comparable to that of the commercial standard surfactin solution (sample 2). Micelles of the same size were also measured in the retentate of the first ultrafiltration step (sample 4). No micelles or particles were found in the filtrate of the broth. (Sample 3)
In addition, the DLS results prove that total disruption of these micelles occurs upon addition of 50% methanol to this retentate and moreover, it shows the appearance of larger particles of around 100 nm diameter which could be due to protein aggregation (sample 5).
For the filtrate UF-1 and UF-2 fractions, the intensity of scattered light detected was similar to that obtained from the dispersant on its own. In addition, the autocorrelation functions obtained showed very poor signal to noise ratios so no size information could be obtained from this data. These results again confirm that under suitable conditions, surfactin is present in the form of free molecules which cannot be detected at these concentrations.
Therefore, the effective separation of surfactin from contaminant proteins is not only due to micelle formation and disruption but also due to protein aggregation.
