Biosurfactants are surface-active compounds that have sparked interest in recent years because of their environmental advantages over conventional surfactants. The aim of this study was to investigate the production of biosurfactants by soil fungi isolated from the Amazon forest. Fungi colonies were isolated from soil samples and screened for biosurfactant production in submerged fermentation. In addition, the influences of bioprocess factors (carbon source, nitrogen source, pH, and fermentation time) were investigated. Finally, the biosurfactant produced was semipurified and submitted to stability tests. One hundred fungal cultures were obtained from the soil samples, identified by micromorphology, and submitted to screening for biosurfactant production. Sixty-one strains produced biosurfactants. The strain
Surfactants are among the most versatile materials in chemical and process industry. Its amphiphilic nature, containing both hydrophilic and lipophilic functional groups in one molecule, plays an important role in numerous chemical applications (dispersion systems, as emulsions and colloids, personal hygiene, detergents, fabric softeners, emulsions, and paints over food texture) [
Biosurfactants are mainly produced by bacteria and yeast, but, in recent years, studies have highlighted their production by filamentous fungi as well [
Despite intensive efforts to study the Amazon, we lack knowledge of the microbial diversity and the production of substances of biotechnological interest by the filamentous fungi that make up this biome. Amazonian bioprospecting studies such as this one can result in the discovery of fungi with high productivity and new biosurfactants. In the current study, we investigated the production of biosurfactants by fungi isolated from soil samples of the Amazonian forest. Analyses were performed related to the following: (i) isolation and identification of biosurfactant-producing fungi, (ii) optimization and kinetic parameters related to the production of the biosurfactant selected, and (iii) comparison of the emulsion stability of the biosurfactant of fungal origin compared to that of a conventional surfactant.
The samples were collected at six locations in the woods of the National Institute of Amazonian Research (INPA) (3.10′39′′S; 59.96′′77′′W), Amazonas, Brazil. For isolation, 1 g of soil was transferred to a tube containing 9 mL of water, and a 1 : 100 dilution was prepared. Then, 100
The selection of biosurfactant producers was performed in Erlenmeyer flasks (125 mL) containing 25 mL of culture medium (with 40 g L−1 soybean and 20 g L−1 peptone) as previously described by Accorsini et al., 2012; however, the mineral solution was replaced with peptone [
In the drop collapse test, a polystyrene plate with 96 microwells (8.5 × 12.7 cm) was used. The plate was washed with hot water, ethanol, and distilled water. Then, 5
To test the emulsification index (E24), a 4 mL aliquot of the culture filtrate (cell-free) was mixed with 6 mL of toluene in a screw tube. The mixture was shaken vigorously for 2 min on a tube shaker-type vortex (Phoenix®). After 24 h, the ratio of emulsified toluene was compared with the total volume. The emulsification index was calculated using the following formula:
The influences of different carbon (20 g L−1) and nitrogen (10 g L−1) sources on biosurfactant production were evaluated. The experimental conditions were the same as those described in the Selection of Biosurfactant Producers, and the results for each substrate were analyzed using univariate analysis. The carbon sources investigated were soybean oil, starch, sucrose, cellulose, and xylose, and the nitrogen sources investigated were peptone, yeast extract, meat extract, sodium nitrate, and malt.
We evaluated the influence of soybean oil, yeast extract, pH, and bioprocess time through a
Semipurification of the biosurfactant was performed with 30 repetitions under optimized conditions. The resulting solutions were pooled, filtered, and precipitated with ethanol (1 : 4 v/v, 4°C, 48 h). The mixture was subjected to centrifugation (5000 rev/min for 20 min), and the precipitate obtained was used for stability testing.
The effect of the addition of NaCl (30% w/v) was evaluated. After the addition of salt to the 1% w/v solution of the precipitate containing the biosurfactant, the emulsifying activity was tested using the emulsification index.
The effect of temperature on the biosurfactant activity was investigated by keeping 1% w/v of the precipitate containing the biosurfactant at 100°C in a water bath for 60 min and verifying the emulsification index. The synthetic surfactant Tween 80 (1% w/v) was used as a control substance in these assays.
Statistical analyses were performed using STATISTICA versions 5.0 and 6.0 (STATGRAPHICS, Statpoint Technologies, Inc., Warrenton, VA, USA). All experiments were performed in triplicate for the calculation of mean and standard deviation.
One hundred fungal strains were screened for their biosurfactant production. We obtained colonies from the genera
To select the biosurfactant-producing fungi, we performed analyses of bioprocessing in submerged fermentation and evaluated the emulsification index and drop collapse test (Table
Fungal cultures isolated from soil samples and results for the best producers of biosurfactants, obtained using the drop collapse test and emulsification index (E24).
Isolated organism | Drop collapse test | Emulsification index |
---|---|---|
| Neg | 64.28 |
| Neg | 58.57 |
| Neg | 57.14 |
| Neg | 57.14 |
| Neg | 54.2 |
| Neg | 52.85 |
| Pos | 51 |
| Neg | 42.85 |
| Neg | 40.3 |
To optimize the production of biosurfactants by the isolated
Univariate test measuring the influence of carbon and nitrogen sources in the production of biosurfactants by
The influence of soybean oil and yeast extract concentrations, pH, and time on biosurfactant production by
Influence of soybean oil, yeast extract, pH, and time on biosurfactant production by
Soybean oil (g l−1) | Yeast extract (g l−1) | pH | Time | E24 |
---|---|---|---|---|
40 | 20 | 5 | 7 | 61 |
60 | 30 | 4 | 9 | 76 |
60 | 10 | 4 | 9 | 53 |
40 | 20 | 5 | 7 | 60 |
60 | 30 | 6 | 9 | 76 |
20 | 30 | 6 | 9 | 80 |
20 | 10 | 4 | 5 | 47 |
40 | 20 | 5 | 7 | 58 |
40 | 20 | 5 | 7 | 62 |
60 | 30 | 4 | 5 | 51 |
20 | 10 | 6 | 9 | 62 |
20 | 10 | 6 | 5 | 56 |
60 | 30 | 6 | 5 | 60 |
20 | 30 | 6 | 5 | 69 |
60 | 10 | 4 | 5 | 48 |
20 | 10 | 4 | 9 | 58 |
60 | 10 | 6 | 5 | 60 |
20 | 30 | 4 | 5 | 62 |
20 | 30 | 4 | 9 | 78 |
60 | 10 | 6 | 9 | 47 |
Effect of tested variables on biosurfactant production by
Factors | Effect |
---|---|
Average | 61,2 ± 0,381881 |
A: soybean | −5,125 ± 0,853913 |
B: yeast extract | 15,125 ± 0,853913 |
C: pH | 4,625 ± 0,853913 |
D: time | 9,625 ± 0,853913 |
AB | −1,375 ± 0,853913 |
AC | −0,875 ± 0,853913 |
AD | −1,375 ± 0,853913 |
BC | −0,125 ± 0,853913 |
BD | 7,375 ± 0,853913 |
CD | −4,625 ± 0,853913 |
The effects that showed statistical significance (95% confidence level) are indicated by the symbol
Analysis of variance for evaluating the statistical significance of the model for biosurfactant production by
Source | Sum of squares | Df | Mean of Squares | | |
---|---|---|---|---|---|
A: soybean oil | 105,063 | 1 | 105,063 | 36,02 | 0,0093 |
B: yeast extract | 915,063 | 1 | 915,063 | 313,74 | 0,0004 |
C: pH | 85,5625 | 1 | 85,5625 | 29,34 | 0,0123 |
D: time | 370,563 | 1 | 370,563 | 127,05 | 0,0015 |
BD | 217,563 | 1 | 217,563 | 74,59 | 0,0033 |
CD | 85,5625 | 1 | 85,5625 | 29,34 | 0,0123 |
Lack of fit | 149,075 | 10 | 149,075 | 5,11 | 0,1030 |
Pure error | 8,75 | 3 | 8,75 | ||
Total | 1937,2 | 19 |
To represent the estimated response (E24), surfaces were prepared (Figure
Response surface analysis for biosurfactant production by
The stability of the new biosurfactant was evaluated under different physical conditions (Figure
Stability of biosurfactants from
We found
Among the fungi obtained from soil, the genera that showed a strong ability to emulsify toluene were
Carbon sources for microbial production of biosurfactant can be obtained from carbohydrates, vegetable oils, hydrocarbons, and waste frying oils [
Biosurfactants are produced when there is nitrogen limitation during the stationary phase of growth of biomass [
The results of this study showed that the factors studied (soybean oil, peptone, pH, and time) and some of their interactions had a significant effect on the response variable (biosurfactant production). The validated mathematical model demonstrated that an E24 of 79.82 could be obtained using 20 g L−1 soybean oil, 30 g L−1 yeast extract, pH 6, and a 9-day duration of the bioprocess. Maximum biosurfactant production occurred in the optimal pH range employed for microorganism growth. During the production of this biosurfactant, as well as during any chemical reaction, the pH directly affects the microbial activity because of the effects of H+ ions on cell permeability and enzyme activity [
The stability of the biosurfactant was evaluated at high temperatures and high ionic strengths. After subjecting the biosurfactant to a temperature of 100°C for 60 min, there was no verified change in biosurfactant activity. This result demonstrates the potential utility of the biosurfactant in the food, pharmaceutical, and cosmetic industries where heat sterility is of great importance [
This study verified stability parameters for only one fungus from the 61 potential biosurfactant-producing fungi, thus ignoring possible potentiality in many others, including some with high levels of emulsification. In the stability tests, we could have assessed the influence of pH. The results demonstrate that biotechnological approaches can be used to improve biosurfactant production by
The data from the present work is important since a significative number of isolates (100 cultures isolated from soil samples from the Amazon) were investigated for the production of biosurfactant. The analytical methodology used in the screening of biosurfactant producers did not quantify the biosurfactant directly in the culture filtrate; nowadays, the screening methods have been developed that rely on the interfacial activity of the biosurfactants but do not measure it directly. We used methodology that quantifies the ability of the biosurfactant present in the culture filtrate to produce emulsification with toluene “emulsification index” as classically described by Mullingan et al. [
The development for screening microbes from thousands of potentially active organisms and the subsequent evaluation of surface activity holds the key to the discovery of new biosurfactants or production strains.
The authors declare that they have no conflicts of interest.