Strategy for improving extracellular lipolytic activities by a novel thermotolerant Staphylococcus sp. strain
© Cherif et al; licensee BioMed Central Ltd. 2011
Received: 27 October 2011
Accepted: 11 November 2011
Published: 11 November 2011
Extracellular bacterial lipases received much attention for their substrate specificity and their ability to function under extreme environments (pH, temperature...). Many staphylococci produced lipases which were released into the culture medium. Reports of extracellular thermostable lipases from Staphylococcus sp. and active in alkaline conditions are not previously described.
This study focused on novel strategies to increase extracellular lipolytic enzyme production by a novel Staphylococcus sp. strain ESW. The microorganism needed neutral or alkaline pH values between 7.0 and 12.0 for growth. For pH values outside this range, cell growth seemed to be significantly inhibited. Staphylococcus sp. culture was able to grow within a wide temperature range (from 30 to 55°C). The presence of oils in the culture medium leaded to improvements in cells growth and lipolytic enzyme activity. On the other hand, although chemical surfactants leaded to an almost complete inhibition of growth and lipolytic enzyme production, their addition along the culture could affect the location of the enzyme. In addition, our results showed that this novel Staphylococcus sp. strain produced biosurfactants simultaneously with lipolytic activity, when soapstock (The main co-product of the vegetable oil refining industry), was used as the sole carbon source.
A simultaneous biosurfactant and extracellular lipolytic enzymes produced bacterial strain with potential application in soap stock treatment
Lipolytic enzymes catalyse hydrolysis and synthesis reactions, either in long chain triacylglycerols (lipases) or in short chain fatty acids (esterases) , most of them of industrial interest in areas such as food, detergent, paper or oleochemical industries. Nowadays, there has been an increasing interest in the study of enzymes from extremophiles, since they are not only more thermostable but often more resistant to chemical agents and extreme pH values than their mesophilic homologues [2–4]. The production of microbial lipases has been shown to be influenced by several factors, namely the carbon source, temperature, pH, dissolved oxygen concentration and presence of inducers. These compounds, such as oils and some surfactants, have been described as agents that increase the production of enzymes with lipolytic activity. Also, in some cases they are essential for lipolytic activity to be detected . Last, the engineering of culture conditions has also been shown to be an effective mode to achieve enzyme preparations enriched in selected isoenzymes which are effective for particular biotechnological applications . In previous paper [6–8], many authors show that enzyme production was not fully associated to growth rate, although absolute values of total lipolytic activity and biomass were positively correlated. However, cell growth was relatively low, and lipolytic activity appeared to be largely retained within the biomass. Therefore, it would be interesting to find culture conditions (i.e. medium composition, pH, temperature, aeration), allowing to improve growth and/or favour enzyme secretion. In this work, optimisation of lipolytic enzyme production by a newly isolated Staphylococcus sp. strain has been attempted. The influence of incubation temperature, the effect of pH on the growth and enzyme production, and the influence of some other parameters in the culture medium have been investigated. Finally, Staphylococcus sp. culture was found to be able to grow on soapstocks (one of the major by-products from vegetable oil refining). This rich bacterial substrate was found to be soluble, when biosurfactants and lipolytic enzymes were produced in the culture medium.
The identification of the bacterial strain ESW has been previously determined in our laboratory. The methods used for 16S rRNA gene amplification and sequencing have been previously reported [9, 10]. Sequence data were imported into the sequence editor BioEdit version 5.0.9. The full sequence was aligned using the RDP Sequence Aligner program . The consensus sequence was manually adjusted to conform to the 16S rRNA secondary structure model. Sequences used in the phylogenetic analysis were obtained from the RDP and GenBank databases [11, 12]. Positions of sequence and alignment ambiguity were omitted andpairwise evolutionary distances were calculated using the method of Jukes and Cantor . Strain ESW was affiliated to Staphylococcus genus and designed as Staphylococcus sp. strain ESW.
The microorganism was grown in a liquid medium containing per liter: 5 g yeast extract, 10 g NaCl, 10 g peptone. The medium was autoclaved at 121°C for 20 min. Cultures were carried out in 250 mL Erlenmeyer flasks with 50 mL of medium. Moreover, some experiments were realized in culture media composed only by soapstocks (by-products of vegetable oil industry).
Several experiments were carried out to determine the inducing effect by adding 1% of several oils and surfactants, namely olive oil, soybean oil, trioctanoin, tributyrin, Triton X-100 or Tween 80, to the flasks at the beginning of the cultures. Moreover, the best inducer was tested by adding this compound at 0, 5 or 10 h of growth in order to study the effect of addition time. Samples were taken during the stationary phase after 30 h of growth. Furthermore, the effect of surfactants (Triton X-100, Tween 80 and 20, CHAPS and PEG 200) was also tested by adding each of them at the beginning of the stationary phase after 24 h of growth. Samples were taken immediately before and after the addition of the surfactant and at the 30 h of growth.
Cells were harvested by centrifugation (10 min, 5000 g) and suspended in 4 ml Tris/HCl buffer 50 mM pH 7.5, containing 25 mM EDTA and 25 mM NaCl. The supernatant was reserved for extracellular enzyme analysis. The cell suspension was sonicated in two cycles of 2 min at 50% of the maximum power (Branson Sonifier, model 250). The procedure was carried out in an ice bath, and a 2 min cooling time was allowed between cycles. Then, the mixture was centrifuged for 10 min at 5°C and 5000 g. The supernatant and the pellet were kept for the measurement of intracellular lipolytic activity and of membrane lipolytic activity, respectively.
Cell growth determination
Biomass concentration was measured via turbidimetry at 600 nm and the obtained values were converted to concentration by using a previously determined calibration curve.
Lipolytic activity assay
The lipase activity was assayed by measuring the free fatty acids released from mechanically stirred emulsions of triacylglycerols, using 0.1 N NaOH with a pH-Stat (Metrohm, Switzerland). The kinetic assay was performed, in optimal conditions (pH 12.0 and 60°C) using 0.25 ml TC4 (Sigma) in (30 ml 2.5 mM Tris-HCl, 150 mM NaCl and 0.5 mM Sodium deoxycholate (NaDC)) or in olive oil emulsion obtained by mixing (3×30 s in a Waring blender), 10 ml of olive oil (Sfax-huile, Tunisia) in 90 ml of 10% GA (Gum Arabic). One lipase unit corresponds to 1 μmol of fatty acid released per minute .
Biosurfactant production determination
Surface tension measurement was used to evaluate biosurfactant production when soapstosks was used as culture medium. Samples of the culture media of the selected strains were centrifuged at 8000 xg for 20 min. Surface tension (ST) of the supernatant fluid of the culture was measured by the ring method using a DuNouy ring tensiometer (Kruss T 10, Hamburg, Germany).
Results and discussion
In order to improve growth and/or favour the enzyme secretion, the influence of some key variables such as medium composition (i.e., ion concentrations of mineral water, lipid compounds, surfactants) and culture conditions (i.e., pH, temperature, visible radiation), were assessed. From the preliminary results obtained, it can be concluded that when Staphylococcus sp. was grown in the presence of visible light, no significant changes in the parameters studied were detected. Moreover, although a previous paper  confirmed that ion concentrations in mineral water (Na+, Ca2+, Mg2+ and HCO3-) comprise the ions which are, most probably, responsible for stimulating the lipolytic activity, the use of mineral concentrated water did not improve either cell growth or lipolytic enzyme secretion. On the other hand, some factors such as culture pH, culture temperature and the addition of lipid compounds and surfactants seemed to have great influence on the behaviour of the microorganism. Hence, these variables are studied in more detail in this work. An operational classification of enzyme activities as intracellular, extracellular or membrane lipolytic activity has been utilised. Thus, activity detected in the culture medium after biomass separation by centrifugation was considered as extracellular, while that recovered in solution after sonication of the buffer-resuspended cells and elimination of cell debris was considered as intracellular and membrane lipolytic activities, respectively [16, 17].
Effect of pH
Effect of temperature on growth and lipolytic activity
The studied strain was grown in shake flasks, within a wide range of temperature (from 30 to 55°C). The increase in temperature seemed to have a negative effect on biomass production and also lipolytic activity. Biomass production and total lipolytic activity reached their maximum, in flask culture incubated at 30°C after 24 h of incubation. For flask cultures incubated at temperature higher than 30°C, biomass production and total lipolytic activity were negligible after the same incubation time. The maximum biomass production and the total lipolytic activity levels were measured for 40 and 55°C at 48 h and 72 h of incubation, respectively (data not shown). One can say that in all cases, the highest final values were obtained when operating at a wide temperature range from 30 to 55°C. Cultures showed a significant decrease in cell growth and lipolytic activity at temperatures above 55°C. The cultures were stopped after 24 h of incubation at 30°C, since previous experiments indicated that, no significant increases in lipolytic enzyme activity were attained later in flask cultures .
Influence of lipid compounds and surfactants
Influence of the time of inducer addition
Using surfactants to favour the release of membrane bound Enzyme
Effect on biomass of Staphylococcus sp. by the addition of several surfactants after 24 h of growth.
Biomass-before addition (g L-1)
Biomass-after addition (g L-1)
Biomass-30 h (g L-1)
1.01 ± 0.04
1.00 ± 0.02
1.06 ± 0.01
1.01 ± 0.03
0.65 ± 0.03
0.35 ± 0.03
1.00 ± 0.04
1.00 ± 0.04
0.80 ± 0.04
1.00 ± 0.02
0.95 ± 0.06
0.70 ± 0.02
1.01 ± 0.01
0.98 ± 0.03
1.06 ± 0.02
1.00 ± 0.05
0.80 ± 0.04
0.25 ± 0.01
These results demonstrate that the compounds leading to a higher increase of extracellular lipolytic activity were Tween 80 and Triton X-100, each one by a different mechanism: the first by allowing a release of the membrane bound enzyme without causing too much cell damage, and the second by favouring lysis, which triggers the release of both membrane and intracellular protein. As a consequence, the extracellular lipolytic activity is considerably increased and, thus, it is not necessary to use another technique to achieve cell lysis (such as ultrasounds). Therefore, either one or the other surfactant could be selected depending on the operational system used as well as on the economic factors involved.
Simultaneous production of lipolytic activity and biosurfactant when using soapstocks as the sole carbon source
The results obtained in this study permit to conclude that pH is a highly significant factor in growth of a newly isolated Staphylococcus sp. strain, with an optimal growth and lipolytic enzyme production at pH 8.0 and a wide temperature range (from 30 to 55°C). Moreover, the effect of the addition of several inducers on enzyme production shows a different behaviour. This novel bacterium Staphylococcus sp., isolated from soil, was found to produce biosurfactants when grown on soapstocks, a co-product of the vegetable oil refining industry, as the sole carbon source.
This work is part of a post-doctoral thesis by Slim CHERIF. This work received financial support from "Ministère de l'enseignement supérieur et de la recherche scientifique" granted to the « Laboratoire des Bioprocédés, pôle d'excellence régional (PER, AUF), Centre de Biotechnologie de Sfax, Tunisie. Authors will to thank Pr. Abdelkarim Abousalham for his help.
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