production of biosurfactants by soil fungi isolated from...

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Research Article Production of Biosurfactants by Soil Fungi Isolated from the Amazon Forest Hellen Holanda Sena, 1 Michele Alves Sanches, 1 Diego Fernando Silva Rocha, 2 Walter Oliva Pinto Filho Segundo, 2 Érica Simplício de Souza, 3 and João Vicente Braga de Souza 2 1 Post-Graduate Program in Pharmaceutical Sciences, Federal University of Amazonas (UFAM), R. Alexandre Amorim 330, 69010-300 Manaus, AM, Brazil 2 Department of Medical Microbiology, National Institute of Amazonian Research (INPA), Av. Andr´ e Ara´ ujo 2936, 69080-971 Manaus, AM, Brazil 3 Superior School of Technology, Amazonas State University (UEA), Av. Darcy Vargas 1200, 69050-020 Manaus, AM, Brazil Correspondence should be addressed to Jo˜ ao Vicente Braga de Souza; [email protected] Received 11 September 2017; Revised 14 November 2017; Accepted 6 February 2018; Published 24 April 2018 Academic Editor: Giuseppe Comi Copyright © 2018 Hellen Holanda Sena et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Biosurfactants are surface-active compounds that have sparked interest in recent years because of their environmental advantages over conventional surfactants. e 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. e strain Penicillium 8CC2 showed the highest emulsification index (54.2%). e optimized bioprocess conditions for biosurfactant production by Penicillium 8CC2 were as follows: soybean oil, 20 g/L; yeast extract, 30 g/L; pH 9; duration of 9 days. e semipurified biosurfactant showed stability aſter heating at 100 C for 60 min and aſter the addition of 30% NaCl (w/v). Tween 80 (0.2% w/v), a conventional surfactant, was used as the control. 1. Introduction Surfactants are among the most versatile materials in chemi- cal 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 soſteners, emulsions, and paints over food texture) [1, 2]. Recently, literature has been demonstrating the advantages of biosurfactants (surfactant of biological origin) if compared with surfactants from chemical industry: lowest toxicity, higher biodegradability, and possi- ble biological activities [2, 3]. Biosurfactants are a structurally diverse group of compounds produced by organisms that have surface-active properties and emulsification action. Biosurfactant properties are similar to the surfactant; they have industrial applications in relation to detergency, emulsi- fication, lubrication, foaming capacity, ability, solubility, dis- persion phases [2–5], and another interesting application in catalysis, biosensing, and electronics using microstructures [6]. Biosurfactants are mainly produced by bacteria and yeast, but, in recent years, studies have highlighted their production by filamentous fungi as well [7, 8]. ese microorganisms are potential producers of various substances of biotechnological interest, such as pigment enzymes and antibiotics, and their main sources of isolation are plants and soil [9]. e pro- duction of biosurfactants by microorganisms is influenced by several factors, including the nature of the carbon source and the concentrations of nutrients such as nitrogen, phosphorus, Hindawi International Journal of Microbiology Volume 2018, Article ID 5684261, 8 pages https://doi.org/10.1155/2018/5684261

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Page 1: Production of Biosurfactants by Soil Fungi Isolated from ...downloads.hindawi.com/journals/ijmicro/2018/5684261.pdf · ResearchArticle Production of Biosurfactants by Soil Fungi Isolated

Research ArticleProduction of Biosurfactants by Soil Fungi Isolated fromthe Amazon Forest

Hellen Holanda Sena1 Michele Alves Sanches1

Diego Fernando Silva Rocha2 Walter Oliva Pinto Filho Segundo2

Eacuterica Simpliacutecio de Souza3 and Joatildeo Vicente Braga de Souza 2

1Post-Graduate Program in Pharmaceutical Sciences Federal University of Amazonas (UFAM)R Alexandre Amorim 330 69010-300 Manaus AM Brazil2Department of Medical Microbiology National Institute of Amazonian Research (INPA)Av Andre Araujo 2936 69080-971 Manaus AM Brazil3Superior School of Technology Amazonas State University (UEA) Av Darcy Vargas 1200 69050-020 Manaus AM Brazil

Correspondence should be addressed to Joao Vicente Braga de Souza joaovicentebragasouzayahoocombr

Received 11 September 2017 Revised 14 November 2017 Accepted 6 February 2018 Published 24 April 2018

Academic Editor Giuseppe Comi

Copyright copy 2018 Hellen Holanda Sena et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Biosurfactants are surface-active compounds that have sparked interest in recent years because of their environmental advantagesover conventional surfactants The aim of this study was to investigate the production of biosurfactants by soil fungi isolatedfrom the Amazon forest Fungi colonies were isolated from soil samples and screened for biosurfactant production in submergedfermentation In addition the influences of bioprocess factors (carbon source nitrogen source pH and fermentation time) wereinvestigated Finally the biosurfactant producedwas semipurified and submitted to stability tests One hundred fungal cultureswereobtained from the soil samples identified by micromorphology and submitted to screening for biosurfactant production Sixty-one strains produced biosurfactants The strain Penicillium 8CC2 showed the highest emulsification index (542) The optimizedbioprocess conditions for biosurfactant production by Penicillium 8CC2 were as follows soybean oil 20 gL yeast extract 30 gLpH 9 duration of 9 days The semipurified biosurfactant showed stability after heating at 100∘C for 60min and after the additionof 30 NaCl (wv) Tween 80 (02 wv) a conventional surfactant was used as the control

1 Introduction

Surfactants are among the most versatile materials in chemi-cal and process industry Its amphiphilic nature containingboth hydrophilic and lipophilic functional groups in onemolecule plays an important role in numerous chemicalapplications (dispersion systems as emulsions and colloidspersonal hygiene detergents fabric softeners emulsions andpaints over food texture) [1 2] Recently literature has beendemonstrating the advantages of biosurfactants (surfactant ofbiological origin) if comparedwith surfactants from chemicalindustry lowest toxicity higher biodegradability and possi-ble biological activities [2 3] Biosurfactants are a structurallydiverse group of compounds produced by organisms thathave surface-active properties and emulsification action

Biosurfactant properties are similar to the surfactant theyhave industrial applications in relation to detergency emulsi-fication lubrication foaming capacity ability solubility dis-persion phases [2ndash5] and another interesting application incatalysis biosensing and electronics using microstructures[6]

Biosurfactants aremainly produced by bacteria and yeastbut in recent years studies have highlighted their productionby filamentous fungi as well [7 8]These microorganisms arepotential producers of various substances of biotechnologicalinterest such as pigment enzymes and antibiotics and theirmain sources of isolation are plants and soil [9] The pro-duction of biosurfactants bymicroorganisms is influenced byseveral factors including the nature of the carbon source andthe concentrations of nutrients such as nitrogen phosphorus

HindawiInternational Journal of MicrobiologyVolume 2018 Article ID 5684261 8 pageshttpsdoiorg10115520185684261

2 International Journal of Microbiology

magnesium iron sulfur and manganese as well as the pHtemperature agitation and available oxygen [2 10 11] Thesefactors can make the production of biosurfactants moreexpensive than that of synthetic surfactants [12] Moreoverseveral studies have been performed to make the price of thebioprocess more competitive [13]

Despite intensive efforts to study the Amazon we lackknowledge of the microbial diversity and the production ofsubstances of biotechnological interest by the filamentousfungi that make up this biome Amazonian bioprospectingstudies such as this one can result in the discovery offungi with high productivity and new biosurfactants In thecurrent study we investigated the production of biosurfac-tants by fungi isolated from soil samples of the Amazonianforest Analyses were performed related to the following(i) isolation and identification of biosurfactant-producingfungi (ii) optimization and kinetic parameters related to theproduction of the biosurfactant selected and (iii) comparisonof the emulsion stability of the biosurfactant of fungal origincompared to that of a conventional surfactant

2 Materials and Methods

21 Isolation and Identification of Soil Fungi The sam-ples were collected at six locations in the woods of theNational Institute of AmazonianResearch (INPA) (31010158403910158401015840S5996101584010158407710158401015840W) Amazonas Brazil For isolation 1 g of soil wastransferred to a tube containing 9mL of water and a 1 100dilution was prepared Then 100 120583L of the diluted samplewas plated on a Petri dish containing Sabouraud agar withchloramphenicol (250mg Lminus1) After 72 h of incubation at25∘C the colonies that developed were transferred to tubeswith the same medium the test was performed in triplicateA streaking technique was performed to obtain monosporiccrops Fungal colonies were identified by observing macro-scopic aspects of the colonies and microscopic features asdescribed by Lacaz et al [14] and Barnett and Hunter [15]

22 Selection of Biosurfactant Producers The selection ofbiosurfactant producers was performed in Erlenmeyer flasks(125mL) containing 25mL of culture medium (with 40 g Lminus1soybean and 20 g Lminus1 peptone) as previously described byAccorsini et al 2012 however the mineral solution wasreplaced with peptone [16] The medium was inoculatedwith spores of the isolates at a final concentration of 1 times104 sporesmLminus1 Then the flasks were incubated withoutshaking at room temperature (25plusmn2∘C) for 7 days followed byfiltrationwith a quantitative filter paper (Oslash 125 cm pore size28120583m Quanty) for removal of fungal cells The filtrate wasseparated for use in detection of biosurfactants by the dropcollapse method described by Bodour and Miller-Maier [17]and the emulsification index described byCameron et al [18]

In the drop collapse test a polystyrene plate with 96microwells (85 times 127 cm) was used The plate was washedwith hot water ethanol and distilled water Then 5 120583L ofthe filtrate of each sample was inoculated separately intowells prefilled with 20 120583L of mineral oil previously left atroom temperature for 24 h After 1min of the reaction theresult was determined visually The result was considered

positive when the drop ofmineral oil collapsed A control wasprepared using SDS at a concentration of 25

To test the emulsification index (E24) a 4mL aliquot ofthe culture filtrate (cell-free) was mixed with 6mL of toluenein a screw tubeThemixture was shaken vigorously for 2minon a tube shaker-type vortex (Phoenix) After 24 h the ratioof emulsified toluene was compared with the total volumeThe emulsification index was calculated using the followingformula

E24 = (height of the emulsion layertotal height

) times 100 (1)

23 Influence of Carbon andNitrogen Sources on BiosurfactantProduction The influences of different carbon (20 g Lminus1) andnitrogen (10 g Lminus1) sources on biosurfactant production wereevaluated The experimental conditions were the same asthose described in the Selection of Biosurfactant Producersand the results for each substratewere analyzed using univari-ate analysis The carbon sources investigated were soybeanoil starch sucrose cellulose and xylose and the nitrogensources investigatedwere peptone yeast extractmeat extractsodium nitrate and malt

24 Effects of Concentration of Carbon and Nitrogen SourcespH and Treatment Time We evaluated the influence ofsoybean oil yeast extract pH and bioprocess time througha 2119896 factorial design (two levels) The chosen design was 24with four central points (evaluation of experimental error)as described by Neto et al [19] The other experimentalconditions were similar to those described in the Selectionof Biosurfactant Producers

25 Semipurification of Biosurfactant and Stability AnalysesSemipurification of the biosurfactant was performed with30 repetitions under optimized conditions The resultingsolutions were pooled filtered and precipitated with ethanol(1 4 vv 4∘C 48 h)The mixture was subjected to centrifuga-tion (5000 revmin for 20min) and the precipitate obtainedwas used for stability testing

The effect of the addition of NaCl (30wv) was eval-uated After the addition of salt to the 1wv solution ofthe precipitate containing the biosurfactant the emulsifyingactivity was tested using the emulsification index

The effect of temperature on the biosurfactant activitywasinvestigated by keeping 1wv of the precipitate containingthe biosurfactant at 100∘C in a water bath for 60min andverifying the emulsification index The synthetic surfactantTween 80 (1wv) was used as a control substance in theseassays

26 Statistical Analysis Statistical analyses were performedusing STATISTICA versions 50 and 60 (STATGRAPHICSStatpoint Technologies Inc Warrenton VA USA) Allexperiments were performed in triplicate for the calculationof mean and standard deviation

3 Results31 Isolation and Identification of Fungi One hundred fungalstrains were screened for their biosurfactant production We

International Journal of Microbiology 3

0

10

20

30

40

50

60

70

Soybean oil Starch Sucrose Cellobiose Xylose

Emul

sifica

tion

inde

x (

)

(a)

0

10

20

30

40

50

60

70

Peptone Yeastextract

Meatextract

Sodiumnitrate

Maltextract

Emul

sifica

tion

inde

x (

)

(b)

Figure 1 Univariate test measuring the influence of carbon and nitrogen sources in the production of biosurfactants by Penicillium 8CC2(a) Carbon source and (b) nitrogen source

Table 1 Fungal cultures isolated from soil samples and resultsfor the best producers of biosurfactants obtained using the dropcollapse test and emulsification index (E24)

Isolated organism Drop collapse test Emulsification indexFusarium sp 87LIV12 Neg 6428Fusarium sp 86LV272 Neg 5857Fusarium sp 85RV342 Neg 5714Penicillium sp 97VV74 Neg 5714Penicillium sp 8CC2 Neg 542Trichoderma sp 91BN312 Neg 5285Trichoderma sp 60AVR3 Pos 51Trichoderma sp 16AC2 Neg 4285Penicillium sp 62BLC2 Neg 403

obtained colonies from the genera Penicillium (46 isolates)Aspergillus (24 isolates) Fonsecaea Gliocladium (one isolate)Trichoderma (12 isolates) Fusarium (eight isolates) andAcremonium (two isolates) Additionally from the phylumZygomycota colonies from the genus Mucor (six isolates) ofthe class Zygomycetes were obtained

32 Selection of Fungal Producers of Biosurfactants To selectthe biosurfactant-producing fungi we performed analyses ofbioprocessing in submerged fermentation and evaluated theemulsification index and drop collapse test (Table 1) of theculture filtrate Nine isolates showed a high emulsificationindex (E24 gt 40) these belonged to the genera Penicillium(three isolates) Trichoderma (three isolates) and Fusarium(three isolates) The isolated Penicillium 8CC2 showed ahigh emulsification index (542) and was selected for theremaining stages of the study because its emulsion couldbe maintained for more than 15 days others have isolatedemulsification rates below 30

33 Effect of Carbon and Nitrogen Sources on BiosurfactantProduction To optimize the production of biosurfactants bythe isolated Penicillium 8CC2 we investigated the influenceof carbon and nitrogen sources by performing univariate

experiments (Figure 1)The carbon and nitrogen sources thatenabled the highest production rates were soybean oil (55)and yeast extract (647) respectively

34 Influence of Soybean Oil Yeast Extract pH and Time onBiosurfactant Production The influence of soybean oil andyeast extract concentrations pH and time on biosurfactantproduction by Penicillium 8CC2 was investigated using theexperimental design 24 as described with four replicatesadded at the central point (Table 2) All major factors andsome interactions (yeast extract times time and pH times time)showed statistical significance (Table 3) An analysis of vari-ance (ANOVA) was performed to validate the mathematicalmodel presented (see (2)) the related information is providedin Table 4 The mathematical model showed a significantregression (918) and its factors showed statistical signifi-cance The lack of fit was not significant (119901 gt 005)

E24 = 81375 minus 0128125 times Soybean Oil (gL)minus 0 534375 times Yeast Extract (gL) + 104063lowast pH + 45 lowast Time (h) + 0 184375times Yeast Extract (gL) times Time (h) minus 115625lowast pH lowast Time (h)

(2)

To represent the estimated response (E24) surfaces wereprepared (Figure 2) based on the data generated by themodelThe surfaces constructed on the basis of the model responsesshowed that the optimal conditions for maximal biosurfac-tant production were as follows soybean oil 20 g Lminus1 yeastextract 30 g Lminus1 pH 6 and a duration of 9 days These valueswere obtained with a theoretical E24 of 7982

35 Stability Studies The stability of the new biosurfactantwas evaluated under different physical conditions (Figure 3)Heating (100∘C for 60min) did not influence the biosurfac-tant activity A high NaCl concentration caused a reductionof 127 in the E24 value of the biosurfactant produced

4 International Journal of Microbiology

Table 2 Influence of soybean oil yeast extract pH and time onbiosurfactant production byPenicillium 8CC2 performed accordingto a factorial design

Soybean oil(g lminus1)

Yeast extract(g lminus1) pH Time

(d) E24

40 20 5 7 6160 30 4 9 7660 10 4 9 5340 20 5 7 6060 30 6 9 7620 30 6 9 8020 10 4 5 4740 20 5 7 5840 20 5 7 6260 30 4 5 5120 10 6 9 6220 10 6 5 5660 30 6 5 6020 30 6 5 6960 10 4 5 4820 10 4 9 5860 10 6 5 6020 30 4 5 6220 30 4 9 7860 10 6 9 47

Table 3 Effect of tested variables on biosurfactant production byPenicillium 8CC2

Factors EffectAverage 612 plusmn 0381881A soybean minus5125 plusmn 0853913lowastB yeast extract 15125 plusmn 0853913lowastC pH 4625 plusmn 0853913lowastD time 9625 plusmn 0853913lowastAB minus1375 plusmn 0853913AC minus0875 plusmn 0853913AD minus1375 plusmn 0853913BC minus0125 plusmn 0853913BD 7375 plusmn 0853913lowastCD minus4625 plusmn 0853913lowastThe effects that showed statistical significance (95 confidence level) areindicated by the symbol lowast

by Penicillium 8CC2 and completely inhibited the emulsionformation by the commercial surfactant Tween 80

4 Discussion

We found Penicillium 8CC2 (strain isolated fromAmazoniansoil) produced biosurfactant with high emulsification index(EI24gt 50) In addition we found that the biosurfactant from

Penicillium 8CC2 has similar thermal-stability and higherNaCl-stability if compared to the surfactant Tween 80 Thepresent work was the first to screen the potential of fungusobtained from soil samples of the Amazon forest to producebiosurfactant and the selected microorganisms and theirbiosurfactant have industrial potential

Among the fungi obtained from soil the genera thatshowed a strong ability to emulsify toluene were PenicilliumTrichoderma and Fusarium The potential of Penicillium spto produce biosurfactants has already been demonstrated[20 21] but few studies have described the biosurfactantpotentials of Trichoderma [22] and Fusarium

Carbon sources formicrobial production of biosurfactantcan be obtained from carbohydrates vegetable oils hydro-carbons and waste frying oils [23 24] In this study soybeanoil was the best carbon source for biosurfactant productionSoybean oil was also described as a suitable carbon sourcefor fungal production of biosurfactant by Accorsini et al[16] these last ones investigated more affordable culturemedium to produce biosurfactant by yeasts We believethat triglycerides composition is a natural inducer of thebiosurfactant production since the microorganisms producebiosurfactant to increase the access to lipid degradation and itis clear that the triglycerides can be directly used to anabolizethe production of the surfactant Starch despite being usedby other microorganisms as a carbon source [25] does notappear to be a suitable substrate for biosurfactant productionby Penicillium 8CC2

Biosurfactants are produced when there is nitrogen lim-itation during the stationary phase of growth of biomass[26ndash28] In the present work yeast extract was found to bethe most suitable nutrient source for the production of themetabolite of interest followed by peptone meat extractand malt The mineral nitrogen source sodium nitrate wasnot suitable for production of the biosurfactant Anotherstudy showed similar results it was observed that Torulopsisbombicola produced biosurfactants using yeast extract butwas not able to produce them using sodium nitrate

The results of this study showed that the factors studied(soybean oil peptone pH and time) and some of theirinteractions had a significant effect on the response vari-able (biosurfactant production) The validated mathematicalmodel demonstrated that an E24 of 7982 could be obtainedusing 20 g Lminus1 soybean oil 30 g Lminus1 yeast extract pH 6 anda 9-day duration of the bioprocess Maximum biosurfactantproduction occurred in the optimal pH range employedfor microorganism growth During the production of thisbiosurfactant as well as during any chemical reaction the pHdirectly affects the microbial activity because of the effects ofH+ ions on cell permeability and enzyme activity [29] Kiranet al [30] reported that an Aspergillus ustus isolate showedoptimal biosurfactant production at pH7Mucor circinelloideswas found to maximally produce biosurfactant at pH 8 usingapple peel vegetable oil and corn water as substrates [31]The time required for maximum biosurfactant productionby Penicillium sp 8CC2 in the current study was 9 daysAspergillus fumigatus was found to produce biosurfactantin 112 h in solid-state fermentation [32] Aspergillus ustus

International Journal of Microbiology 5

76

72

68

64

60

56

52

30

20

10 20

40

60

E24 E24

E24

E24

E24

E24

Yaest extract (gL)

60

58

56

54

52

50

6

55

5

45

4 20

40

60

pH

pH

Soybean oil (gL)

Soybean oil (gL)

Soybean oil (gL)

58

57

56

55

54

53

52

51

9

85

8

75

7 20

40

60

76

72

68

64

60

56

6

55

5

45

4 10

15

20

25

30

82

78

74

70

66

62

58

9

85

8

75

7 10

15

20

25

30

Time (d) pH

Time (d)

Time (d)

595

585

575

565

555

545

9

85

8

75

7 4

45

5

55

6

Yeast extract (gL)

Yeast extract (gL)

Figure 2 Response surface analysis for biosurfactant production by Penicillium 8CC2 with soybean oil yeast extract pH and productiontime

6 International Journal of Microbiology

Table 4 Analysis of variance for evaluating the statistical significance of the model for biosurfactant production by Penicillium 8CC2 (1198772 =918529)

Source Sum of squares Df Mean of Squares 119865 ratio 119875 valueA soybean oil 105063 1 105063 3602 00093B yeast extract 915063 1 915063 31374 00004C pH 855625 1 855625 2934 00123D time 370563 1 370563 12705 00015BD 217563 1 217563 7459 00033CD 855625 1 855625 2934 00123Lack of fit 149075 10 149075 511 01030Pure error 875 3 875Total 19372 19

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +heating

Tween 80 Tween 80 +heating

Emul

sifica

tion

inde

x (

)

(a)

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +NaCl (30 wv)

Tween 80 Tween 80 +NaCl (30 wv)

Emul

sifica

tion

inde

x (

)

(b)

Figure 3 Stability of biosurfactants from Penicillium 8CC2 under different physical conditions (a) after heating 100∘C (60min) and (b) withthe addition of NaCl (30wv) The synthetic surfactant Tween 80 (1wv) was used as a control substance in these assays

isolated from amarine sponge showed optimized productionat 120 h in Sabouraud dextrose broth [30]

The stability of the biosurfactant was evaluated at hightemperatures and high ionic strengths After subjecting thebiosurfactant to a temperature of 100∘C for 60min therewas no verified change in biosurfactant activity This resultdemonstrates the potential utility of the biosurfactant in thefood pharmaceutical and cosmetic industries where heatsterility is of great importance [32 33] In the case of ionicstrength there was a small reduction in the emulsificationability of Penicillium 8CC2 when NaCl (30) was added buta stable emulsion could still be maintained The commercialemulsifier Tween 80 used as a control did not remain stablewhen NaCl was added This result demonstrates that thebiosurfactant produced by Penicillium 8CC2 can be used inhigh salt-concentration conditions for example in tertiaryoil recovery activities

This study verified stability parameters for only onefungus from the 61 potential biosurfactant-producing fungithus ignoring possible potentiality in many others includingsome with high levels of emulsification In the stability testswe could have assessed the influence of pH The resultsdemonstrate that biotechnological approaches can be usedto improve biosurfactant production by Penicillium 8CC2The current results suggest the potential usefulness of thebiosurfactant produced by a filamentous fungus isolated fromsoil samples from the Amazon region

The data from the present work is important since asignificative number of isolates (100 cultures isolated fromsoil samples from the Amazon) were investigated for theproduction of biosurfactant The analytical methodologyused in the screening of biosurfactant producers did notquantify the biosurfactant directly in the culture filtratenowadays the screening methods have been developed thatrely on the interfacial activity of the biosurfactants but do notmeasure it directly We used methodology that quantifies theability of the biosurfactant present in the culture filtrate toproduce emulsificationwith toluene ldquoemulsification indexrdquo asclassically described by Mullingan et al [34] Unfortunatelyas a work limitation during the experimental conditions itwas not possible to identify the species of Penicillium 8CC2and the chemical composition of the biosurfactant Literaturedoes not have description of chemical composition of thebiosurfactants produced by Penicillium sp and further worksare necessary to elucidate it

The development for screening microbes from thousandsof potentially active organisms and the subsequent evaluationof surface activity holds the key to the discovery of newbiosurfactants or production strains

Conflicts of Interest

The authors declare that they have no conflicts of interest

International Journal of Microbiology 7

References

[1] Z A Raza A Rehman M T Hussain et al ldquoProduction ofrhamnolipid surfactant and its application in bioscouring ofcotton fabricrdquo Carbohydrate Research vol 391 no 1 pp 97ndash1052014

[2] I M Banat A Franzetti I Gandolfi et al ldquoMicrobial biosur-factants production applications and future potentialrdquo AppliedMicrobiology andBiotechnology vol 87 no 2 pp 427ndash444 2010

[3] M Pacwa-Płociniczak G A Płaza Z Piotrowska-Seget andS S Cameotra ldquoEnvironmental applications of biosurfactantsrecent advancesrdquo International Journal of Molecular Sciencesvol 12 no 1 pp 633ndash654 2011

[4] P Das S Mukherjee and R Sen ldquoImproved bioavailabilityand biodegradation of a model polyaromatic hydrocarbonby a biosurfactant producing bacterium of marine originrdquoChemosphere vol 72 no 9 pp 1229ndash1234 2008

[5] M Nitschke andGM Pastore ldquoBiossurfactantes propriedadese aplicacoesrdquo Quımica Nova vol 25 no 5 pp 772ndash776 2002

[6] A Rehman Z A Raza Saif-ur-Rehman et al ldquoSynthesis anduse of self-assembled rhamnolipid microtubules as templatesfor gold nanoparticles assembly to form gold microstructuresrdquoJournal of Colloid and Interface Science vol 347 no 2 pp 332ndash335 2010

[7] G L Castiglioni T E Bertolin and J A Costa ldquoProducao debiossurfactante por Aspergillus fumigatus utilizando resıduosagroindustriais como substratordquo Quımica Nova vol 32 no 2pp 292ndash295 2009

[8] Z Velioglu and R O Urek ldquoBiosurfactant production byPleurotus ostreatus in submerged and solid-state fermentationsystemsrdquo Turkish Journal of Biology vol 39 no 1 pp 160ndash1662015

[9] J L Adrio and A L Demain ldquoFungal biotechnologyrdquo Interna-tional Microbiology vol 6 no 3 pp 191ndash199 2003

[10] G C Fontes P F Amaral and M A Coelho ldquoProducao debiossurfactante por levedurardquo Quımica Nova vol 31 no 8 pp2091ndash2099 2008

[11] M Pirollo Estudo da Producao de Biossurfactantes UtilizandoHidrocarbonetos Universidade Estadual Paulista Sao PauloBrazil 2006

[12] RThavasi S Jayalakshmi T Balasubramanian and IM BanatldquoBiosurfactant production by Corynebacterium kutscheri fromwastemotor lubricant oil and peanut oil cakerdquo Letters in AppliedMicrobiology vol 45 no 6 pp 686ndash691 2007

[13] K Pattanathu M Rahman and M Gapke ldquoProductioncharacterisation and applications of biosurfactants - ReviewrdquoBiotechnology vol 2 pp 360ndash370 2008

[14] C Lacaz E Porto and J Martins Martins MicrobiologiaMedica Fungos Actinomicetos e Algas de Interesse Medico vol33 Sarvier Sao Paulo Brazil 8th edition 2001

[15] H L Barnett and B B Hunter IIlustrated Genera of ImperfectFungi vol 64 Burgess Publishing Co 4th edition 1998

[16] F R Accorsini M J R Mutton E G M Lemos and MBenincasa ldquoBiosurfactants production by yeasts using soybeanoil and glycerol as low cost substraterdquo Brazilian Journal ofMicrobiology vol 43 no 1 pp 116ndash125 2012

[17] A A Bodour and R M Miller-Maier ldquoApplication of a mod-ified drop-collapse technique for surfactant quantitation andscreening of biosurfactant-producingmicroorganismsrdquo Journalof Microbiological Methods vol 32 no 3 pp 273ndash280 1998

[18] D R Cameron D G Cooper and R J Neufeld ldquoThe manno-protein of Saccharomyces cerevisiae is an effective bioemulsi-fierrdquoApplied and Environmental Microbiology vol 54 no 6 pp1420ndash1425 1988

[19] B B Neto I S Scarminio and R E Bruns Planejamento eotimizacao de experimentos 1995

[20] M A Luna-Velasco F Esparza-Garcıa R O Canızares-Vil-lanueva and R Rodrıguez-Vazquez ldquoProduction and proper-ties of a bioemulsifier synthesized by phenanthrene-degradingPenicillium sprdquo Process Biochemistry vol 42 no 3 pp 310ndash3142007

[21] M M Camargo-de-Morais S A Ramos M C Pimentel andJ Morais ldquoProduction of an extracellular polysaccharide withemulsifier properties by Penicillium citrinumrdquoWorld Journal ofMicrobiology and Biotechnology vol 19 pp 191ndash194 2003

[22] S Askolin T Nakari-Setala and M Tenkanen ldquoOverproduc-tion purification and characterization of the Trichodermareesei hydrophobin HFBIrdquo Applied Microbiology and Biotech-nology vol 57 no 1-2 pp 124ndash130 2001

[23] Z A Raza M S Khan and ZM Khalid ldquoEvaluation of distantcarbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutantrdquo Process Biochemistryvol 42 no 4 pp 686ndash692 2007

[24] H-S Kim B-D Yoon C-H Lee et al ldquoProduction andproperties of a lipopeptide biosurfactant from Bacillus subtilisC9rdquo Journal of Fermentation and Bioengineering vol 84 no 1pp 41ndash46 1997

[25] R S Makkar and S S Cameotra ldquoProduction of biosurfac-tant at mesophilic and thermophilic conditions by a strainof Bacillus subtilisrdquo Journal of Industrial Microbiology andBiotechnology vol 20 no 1 pp 48ndash52 1998

[26] IM Banat andD J Desai ldquoMicrobial production of surfactantsand their commercial potentialrdquo Microbiology and MolecularBiology Reviews vol 61 no 1 pp 47ndash64 1997

[27] A Decesaro M R Rigon A Thome and L M CollaldquoProducao de biossurfactantes por microrganismos isolados desolo contaminado com oleo dieselrdquo Quımica Nova vol 36 no7 pp 947ndash954 2013

[28] H-S Kim J-W Jeon B-H Kim C-Y Ahn H-M Oh and B-D Yoon ldquoExtracellular production of a glycolipid biosurfactantmannosylerythritol lipid by Candida sp SY16 using fed-batchfermentationrdquo Applied Microbiology and Biotechnology vol 70no 4 pp 391ndash396 2006

[29] G L Castiglioni G Stanescu L A O Rocha and J A VCosta ldquoActa scientiarum analytical modeling and numericaloptimization of the biosurfactants production in solid-statefermentation by aspergillus fumigatusrdquo Acta Scientiarum vol36 no 1 pp 61ndash67 2014

[30] G S Kiran T A Hema R Gandhimathi et al ldquoOptimizationand production of a biosurfactant from the sponge-associatedmarine fungus Aspergillus ustus MSF3rdquo Colloids and SurfacesB Biointerfaces vol 73 no 2 pp 250ndash256 2009

[31] L M Acioly A Silveira M Anjos et al ldquoBiosurfactantproduction by Mucor circinelloides using apple peel vegetableoil and corn steep liquor as substraterdquo in Microbes in AppliedResearchndashCurrents Advances and Challenges A Mendez-VilasEd pp 344ndash347 World Science New York NY USA 2012

[32] A S Monteiro M R Q Bonfim V S Domingues et alldquoIdentification and characterization of bioemulsifier-producingyeasts isolated from effluents of a dairy industryrdquo BioresourceTechnology vol 101 no 14 pp 5186ndash5193 2010

8 International Journal of Microbiology

[33] A Khopade B Ren X-Y Liu K Mahadik L Zhang andC Kokare ldquoProduction and characterization of biosurfactantfrom marine Streptomyces species B3rdquo Journal of Colloid andInterface Science vol 367 no 1 pp 311ndash318 2012

[34] CMullinganDCooper andRNeufeld ldquoSelection ofmicrobesproducing biosurfactants in media without hydrocarbonsrdquoJournal of Fermentation Technology vol 4 pp 311ndash314 1984

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Page 2: Production of Biosurfactants by Soil Fungi Isolated from ...downloads.hindawi.com/journals/ijmicro/2018/5684261.pdf · ResearchArticle Production of Biosurfactants by Soil Fungi Isolated

2 International Journal of Microbiology

magnesium iron sulfur and manganese as well as the pHtemperature agitation and available oxygen [2 10 11] Thesefactors can make the production of biosurfactants moreexpensive than that of synthetic surfactants [12] Moreoverseveral studies have been performed to make the price of thebioprocess more competitive [13]

Despite intensive efforts to study the Amazon we lackknowledge of the microbial diversity and the production ofsubstances of biotechnological interest by the filamentousfungi that make up this biome Amazonian bioprospectingstudies such as this one can result in the discovery offungi with high productivity and new biosurfactants In thecurrent study we investigated the production of biosurfac-tants by fungi isolated from soil samples of the Amazonianforest Analyses were performed related to the following(i) isolation and identification of biosurfactant-producingfungi (ii) optimization and kinetic parameters related to theproduction of the biosurfactant selected and (iii) comparisonof the emulsion stability of the biosurfactant of fungal origincompared to that of a conventional surfactant

2 Materials and Methods

21 Isolation and Identification of Soil Fungi The sam-ples were collected at six locations in the woods of theNational Institute of AmazonianResearch (INPA) (31010158403910158401015840S5996101584010158407710158401015840W) Amazonas Brazil For isolation 1 g of soil wastransferred to a tube containing 9mL of water and a 1 100dilution was prepared Then 100 120583L of the diluted samplewas plated on a Petri dish containing Sabouraud agar withchloramphenicol (250mg Lminus1) After 72 h of incubation at25∘C the colonies that developed were transferred to tubeswith the same medium the test was performed in triplicateA streaking technique was performed to obtain monosporiccrops Fungal colonies were identified by observing macro-scopic aspects of the colonies and microscopic features asdescribed by Lacaz et al [14] and Barnett and Hunter [15]

22 Selection of Biosurfactant Producers The selection ofbiosurfactant producers was performed in Erlenmeyer flasks(125mL) containing 25mL of culture medium (with 40 g Lminus1soybean and 20 g Lminus1 peptone) as previously described byAccorsini et al 2012 however the mineral solution wasreplaced with peptone [16] The medium was inoculatedwith spores of the isolates at a final concentration of 1 times104 sporesmLminus1 Then the flasks were incubated withoutshaking at room temperature (25plusmn2∘C) for 7 days followed byfiltrationwith a quantitative filter paper (Oslash 125 cm pore size28120583m Quanty) for removal of fungal cells The filtrate wasseparated for use in detection of biosurfactants by the dropcollapse method described by Bodour and Miller-Maier [17]and the emulsification index described byCameron et al [18]

In the drop collapse test a polystyrene plate with 96microwells (85 times 127 cm) was used The plate was washedwith hot water ethanol and distilled water Then 5 120583L ofthe filtrate of each sample was inoculated separately intowells prefilled with 20 120583L of mineral oil previously left atroom temperature for 24 h After 1min of the reaction theresult was determined visually The result was considered

positive when the drop ofmineral oil collapsed A control wasprepared using SDS at a concentration of 25

To test the emulsification index (E24) a 4mL aliquot ofthe culture filtrate (cell-free) was mixed with 6mL of toluenein a screw tubeThemixture was shaken vigorously for 2minon a tube shaker-type vortex (Phoenix) After 24 h the ratioof emulsified toluene was compared with the total volumeThe emulsification index was calculated using the followingformula

E24 = (height of the emulsion layertotal height

) times 100 (1)

23 Influence of Carbon andNitrogen Sources on BiosurfactantProduction The influences of different carbon (20 g Lminus1) andnitrogen (10 g Lminus1) sources on biosurfactant production wereevaluated The experimental conditions were the same asthose described in the Selection of Biosurfactant Producersand the results for each substratewere analyzed using univari-ate analysis The carbon sources investigated were soybeanoil starch sucrose cellulose and xylose and the nitrogensources investigatedwere peptone yeast extractmeat extractsodium nitrate and malt

24 Effects of Concentration of Carbon and Nitrogen SourcespH and Treatment Time We evaluated the influence ofsoybean oil yeast extract pH and bioprocess time througha 2119896 factorial design (two levels) The chosen design was 24with four central points (evaluation of experimental error)as described by Neto et al [19] The other experimentalconditions were similar to those described in the Selectionof Biosurfactant Producers

25 Semipurification of Biosurfactant and Stability AnalysesSemipurification of the biosurfactant was performed with30 repetitions under optimized conditions The resultingsolutions were pooled filtered and precipitated with ethanol(1 4 vv 4∘C 48 h)The mixture was subjected to centrifuga-tion (5000 revmin for 20min) and the precipitate obtainedwas used for stability testing

The effect of the addition of NaCl (30wv) was eval-uated After the addition of salt to the 1wv solution ofthe precipitate containing the biosurfactant the emulsifyingactivity was tested using the emulsification index

The effect of temperature on the biosurfactant activitywasinvestigated by keeping 1wv of the precipitate containingthe biosurfactant at 100∘C in a water bath for 60min andverifying the emulsification index The synthetic surfactantTween 80 (1wv) was used as a control substance in theseassays

26 Statistical Analysis Statistical analyses were performedusing STATISTICA versions 50 and 60 (STATGRAPHICSStatpoint Technologies Inc Warrenton VA USA) Allexperiments were performed in triplicate for the calculationof mean and standard deviation

3 Results31 Isolation and Identification of Fungi One hundred fungalstrains were screened for their biosurfactant production We

International Journal of Microbiology 3

0

10

20

30

40

50

60

70

Soybean oil Starch Sucrose Cellobiose Xylose

Emul

sifica

tion

inde

x (

)

(a)

0

10

20

30

40

50

60

70

Peptone Yeastextract

Meatextract

Sodiumnitrate

Maltextract

Emul

sifica

tion

inde

x (

)

(b)

Figure 1 Univariate test measuring the influence of carbon and nitrogen sources in the production of biosurfactants by Penicillium 8CC2(a) Carbon source and (b) nitrogen source

Table 1 Fungal cultures isolated from soil samples and resultsfor the best producers of biosurfactants obtained using the dropcollapse test and emulsification index (E24)

Isolated organism Drop collapse test Emulsification indexFusarium sp 87LIV12 Neg 6428Fusarium sp 86LV272 Neg 5857Fusarium sp 85RV342 Neg 5714Penicillium sp 97VV74 Neg 5714Penicillium sp 8CC2 Neg 542Trichoderma sp 91BN312 Neg 5285Trichoderma sp 60AVR3 Pos 51Trichoderma sp 16AC2 Neg 4285Penicillium sp 62BLC2 Neg 403

obtained colonies from the genera Penicillium (46 isolates)Aspergillus (24 isolates) Fonsecaea Gliocladium (one isolate)Trichoderma (12 isolates) Fusarium (eight isolates) andAcremonium (two isolates) Additionally from the phylumZygomycota colonies from the genus Mucor (six isolates) ofthe class Zygomycetes were obtained

32 Selection of Fungal Producers of Biosurfactants To selectthe biosurfactant-producing fungi we performed analyses ofbioprocessing in submerged fermentation and evaluated theemulsification index and drop collapse test (Table 1) of theculture filtrate Nine isolates showed a high emulsificationindex (E24 gt 40) these belonged to the genera Penicillium(three isolates) Trichoderma (three isolates) and Fusarium(three isolates) The isolated Penicillium 8CC2 showed ahigh emulsification index (542) and was selected for theremaining stages of the study because its emulsion couldbe maintained for more than 15 days others have isolatedemulsification rates below 30

33 Effect of Carbon and Nitrogen Sources on BiosurfactantProduction To optimize the production of biosurfactants bythe isolated Penicillium 8CC2 we investigated the influenceof carbon and nitrogen sources by performing univariate

experiments (Figure 1)The carbon and nitrogen sources thatenabled the highest production rates were soybean oil (55)and yeast extract (647) respectively

34 Influence of Soybean Oil Yeast Extract pH and Time onBiosurfactant Production The influence of soybean oil andyeast extract concentrations pH and time on biosurfactantproduction by Penicillium 8CC2 was investigated using theexperimental design 24 as described with four replicatesadded at the central point (Table 2) All major factors andsome interactions (yeast extract times time and pH times time)showed statistical significance (Table 3) An analysis of vari-ance (ANOVA) was performed to validate the mathematicalmodel presented (see (2)) the related information is providedin Table 4 The mathematical model showed a significantregression (918) and its factors showed statistical signifi-cance The lack of fit was not significant (119901 gt 005)

E24 = 81375 minus 0128125 times Soybean Oil (gL)minus 0 534375 times Yeast Extract (gL) + 104063lowast pH + 45 lowast Time (h) + 0 184375times Yeast Extract (gL) times Time (h) minus 115625lowast pH lowast Time (h)

(2)

To represent the estimated response (E24) surfaces wereprepared (Figure 2) based on the data generated by themodelThe surfaces constructed on the basis of the model responsesshowed that the optimal conditions for maximal biosurfac-tant production were as follows soybean oil 20 g Lminus1 yeastextract 30 g Lminus1 pH 6 and a duration of 9 days These valueswere obtained with a theoretical E24 of 7982

35 Stability Studies The stability of the new biosurfactantwas evaluated under different physical conditions (Figure 3)Heating (100∘C for 60min) did not influence the biosurfac-tant activity A high NaCl concentration caused a reductionof 127 in the E24 value of the biosurfactant produced

4 International Journal of Microbiology

Table 2 Influence of soybean oil yeast extract pH and time onbiosurfactant production byPenicillium 8CC2 performed accordingto a factorial design

Soybean oil(g lminus1)

Yeast extract(g lminus1) pH Time

(d) E24

40 20 5 7 6160 30 4 9 7660 10 4 9 5340 20 5 7 6060 30 6 9 7620 30 6 9 8020 10 4 5 4740 20 5 7 5840 20 5 7 6260 30 4 5 5120 10 6 9 6220 10 6 5 5660 30 6 5 6020 30 6 5 6960 10 4 5 4820 10 4 9 5860 10 6 5 6020 30 4 5 6220 30 4 9 7860 10 6 9 47

Table 3 Effect of tested variables on biosurfactant production byPenicillium 8CC2

Factors EffectAverage 612 plusmn 0381881A soybean minus5125 plusmn 0853913lowastB yeast extract 15125 plusmn 0853913lowastC pH 4625 plusmn 0853913lowastD time 9625 plusmn 0853913lowastAB minus1375 plusmn 0853913AC minus0875 plusmn 0853913AD minus1375 plusmn 0853913BC minus0125 plusmn 0853913BD 7375 plusmn 0853913lowastCD minus4625 plusmn 0853913lowastThe effects that showed statistical significance (95 confidence level) areindicated by the symbol lowast

by Penicillium 8CC2 and completely inhibited the emulsionformation by the commercial surfactant Tween 80

4 Discussion

We found Penicillium 8CC2 (strain isolated fromAmazoniansoil) produced biosurfactant with high emulsification index(EI24gt 50) In addition we found that the biosurfactant from

Penicillium 8CC2 has similar thermal-stability and higherNaCl-stability if compared to the surfactant Tween 80 Thepresent work was the first to screen the potential of fungusobtained from soil samples of the Amazon forest to producebiosurfactant and the selected microorganisms and theirbiosurfactant have industrial potential

Among the fungi obtained from soil the genera thatshowed a strong ability to emulsify toluene were PenicilliumTrichoderma and Fusarium The potential of Penicillium spto produce biosurfactants has already been demonstrated[20 21] but few studies have described the biosurfactantpotentials of Trichoderma [22] and Fusarium

Carbon sources formicrobial production of biosurfactantcan be obtained from carbohydrates vegetable oils hydro-carbons and waste frying oils [23 24] In this study soybeanoil was the best carbon source for biosurfactant productionSoybean oil was also described as a suitable carbon sourcefor fungal production of biosurfactant by Accorsini et al[16] these last ones investigated more affordable culturemedium to produce biosurfactant by yeasts We believethat triglycerides composition is a natural inducer of thebiosurfactant production since the microorganisms producebiosurfactant to increase the access to lipid degradation and itis clear that the triglycerides can be directly used to anabolizethe production of the surfactant Starch despite being usedby other microorganisms as a carbon source [25] does notappear to be a suitable substrate for biosurfactant productionby Penicillium 8CC2

Biosurfactants are produced when there is nitrogen lim-itation during the stationary phase of growth of biomass[26ndash28] In the present work yeast extract was found to bethe most suitable nutrient source for the production of themetabolite of interest followed by peptone meat extractand malt The mineral nitrogen source sodium nitrate wasnot suitable for production of the biosurfactant Anotherstudy showed similar results it was observed that Torulopsisbombicola produced biosurfactants using yeast extract butwas not able to produce them using sodium nitrate

The results of this study showed that the factors studied(soybean oil peptone pH and time) and some of theirinteractions had a significant effect on the response vari-able (biosurfactant production) The validated mathematicalmodel demonstrated that an E24 of 7982 could be obtainedusing 20 g Lminus1 soybean oil 30 g Lminus1 yeast extract pH 6 anda 9-day duration of the bioprocess Maximum biosurfactantproduction occurred in the optimal pH range employedfor microorganism growth During the production of thisbiosurfactant as well as during any chemical reaction the pHdirectly affects the microbial activity because of the effects ofH+ ions on cell permeability and enzyme activity [29] Kiranet al [30] reported that an Aspergillus ustus isolate showedoptimal biosurfactant production at pH7Mucor circinelloideswas found to maximally produce biosurfactant at pH 8 usingapple peel vegetable oil and corn water as substrates [31]The time required for maximum biosurfactant productionby Penicillium sp 8CC2 in the current study was 9 daysAspergillus fumigatus was found to produce biosurfactantin 112 h in solid-state fermentation [32] Aspergillus ustus

International Journal of Microbiology 5

76

72

68

64

60

56

52

30

20

10 20

40

60

E24 E24

E24

E24

E24

E24

Yaest extract (gL)

60

58

56

54

52

50

6

55

5

45

4 20

40

60

pH

pH

Soybean oil (gL)

Soybean oil (gL)

Soybean oil (gL)

58

57

56

55

54

53

52

51

9

85

8

75

7 20

40

60

76

72

68

64

60

56

6

55

5

45

4 10

15

20

25

30

82

78

74

70

66

62

58

9

85

8

75

7 10

15

20

25

30

Time (d) pH

Time (d)

Time (d)

595

585

575

565

555

545

9

85

8

75

7 4

45

5

55

6

Yeast extract (gL)

Yeast extract (gL)

Figure 2 Response surface analysis for biosurfactant production by Penicillium 8CC2 with soybean oil yeast extract pH and productiontime

6 International Journal of Microbiology

Table 4 Analysis of variance for evaluating the statistical significance of the model for biosurfactant production by Penicillium 8CC2 (1198772 =918529)

Source Sum of squares Df Mean of Squares 119865 ratio 119875 valueA soybean oil 105063 1 105063 3602 00093B yeast extract 915063 1 915063 31374 00004C pH 855625 1 855625 2934 00123D time 370563 1 370563 12705 00015BD 217563 1 217563 7459 00033CD 855625 1 855625 2934 00123Lack of fit 149075 10 149075 511 01030Pure error 875 3 875Total 19372 19

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +heating

Tween 80 Tween 80 +heating

Emul

sifica

tion

inde

x (

)

(a)

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +NaCl (30 wv)

Tween 80 Tween 80 +NaCl (30 wv)

Emul

sifica

tion

inde

x (

)

(b)

Figure 3 Stability of biosurfactants from Penicillium 8CC2 under different physical conditions (a) after heating 100∘C (60min) and (b) withthe addition of NaCl (30wv) The synthetic surfactant Tween 80 (1wv) was used as a control substance in these assays

isolated from amarine sponge showed optimized productionat 120 h in Sabouraud dextrose broth [30]

The stability of the biosurfactant was evaluated at hightemperatures and high ionic strengths After subjecting thebiosurfactant to a temperature of 100∘C for 60min therewas no verified change in biosurfactant activity This resultdemonstrates the potential utility of the biosurfactant in thefood pharmaceutical and cosmetic industries where heatsterility is of great importance [32 33] In the case of ionicstrength there was a small reduction in the emulsificationability of Penicillium 8CC2 when NaCl (30) was added buta stable emulsion could still be maintained The commercialemulsifier Tween 80 used as a control did not remain stablewhen NaCl was added This result demonstrates that thebiosurfactant produced by Penicillium 8CC2 can be used inhigh salt-concentration conditions for example in tertiaryoil recovery activities

This study verified stability parameters for only onefungus from the 61 potential biosurfactant-producing fungithus ignoring possible potentiality in many others includingsome with high levels of emulsification In the stability testswe could have assessed the influence of pH The resultsdemonstrate that biotechnological approaches can be usedto improve biosurfactant production by Penicillium 8CC2The current results suggest the potential usefulness of thebiosurfactant produced by a filamentous fungus isolated fromsoil samples from the Amazon region

The data from the present work is important since asignificative number of isolates (100 cultures isolated fromsoil samples from the Amazon) were investigated for theproduction of biosurfactant The analytical methodologyused in the screening of biosurfactant producers did notquantify the biosurfactant directly in the culture filtratenowadays the screening methods have been developed thatrely on the interfacial activity of the biosurfactants but do notmeasure it directly We used methodology that quantifies theability of the biosurfactant present in the culture filtrate toproduce emulsificationwith toluene ldquoemulsification indexrdquo asclassically described by Mullingan et al [34] Unfortunatelyas a work limitation during the experimental conditions itwas not possible to identify the species of Penicillium 8CC2and the chemical composition of the biosurfactant Literaturedoes not have description of chemical composition of thebiosurfactants produced by Penicillium sp and further worksare necessary to elucidate it

The development for screening microbes from thousandsof potentially active organisms and the subsequent evaluationof surface activity holds the key to the discovery of newbiosurfactants or production strains

Conflicts of Interest

The authors declare that they have no conflicts of interest

International Journal of Microbiology 7

References

[1] Z A Raza A Rehman M T Hussain et al ldquoProduction ofrhamnolipid surfactant and its application in bioscouring ofcotton fabricrdquo Carbohydrate Research vol 391 no 1 pp 97ndash1052014

[2] I M Banat A Franzetti I Gandolfi et al ldquoMicrobial biosur-factants production applications and future potentialrdquo AppliedMicrobiology andBiotechnology vol 87 no 2 pp 427ndash444 2010

[3] M Pacwa-Płociniczak G A Płaza Z Piotrowska-Seget andS S Cameotra ldquoEnvironmental applications of biosurfactantsrecent advancesrdquo International Journal of Molecular Sciencesvol 12 no 1 pp 633ndash654 2011

[4] P Das S Mukherjee and R Sen ldquoImproved bioavailabilityand biodegradation of a model polyaromatic hydrocarbonby a biosurfactant producing bacterium of marine originrdquoChemosphere vol 72 no 9 pp 1229ndash1234 2008

[5] M Nitschke andGM Pastore ldquoBiossurfactantes propriedadese aplicacoesrdquo Quımica Nova vol 25 no 5 pp 772ndash776 2002

[6] A Rehman Z A Raza Saif-ur-Rehman et al ldquoSynthesis anduse of self-assembled rhamnolipid microtubules as templatesfor gold nanoparticles assembly to form gold microstructuresrdquoJournal of Colloid and Interface Science vol 347 no 2 pp 332ndash335 2010

[7] G L Castiglioni T E Bertolin and J A Costa ldquoProducao debiossurfactante por Aspergillus fumigatus utilizando resıduosagroindustriais como substratordquo Quımica Nova vol 32 no 2pp 292ndash295 2009

[8] Z Velioglu and R O Urek ldquoBiosurfactant production byPleurotus ostreatus in submerged and solid-state fermentationsystemsrdquo Turkish Journal of Biology vol 39 no 1 pp 160ndash1662015

[9] J L Adrio and A L Demain ldquoFungal biotechnologyrdquo Interna-tional Microbiology vol 6 no 3 pp 191ndash199 2003

[10] G C Fontes P F Amaral and M A Coelho ldquoProducao debiossurfactante por levedurardquo Quımica Nova vol 31 no 8 pp2091ndash2099 2008

[11] M Pirollo Estudo da Producao de Biossurfactantes UtilizandoHidrocarbonetos Universidade Estadual Paulista Sao PauloBrazil 2006

[12] RThavasi S Jayalakshmi T Balasubramanian and IM BanatldquoBiosurfactant production by Corynebacterium kutscheri fromwastemotor lubricant oil and peanut oil cakerdquo Letters in AppliedMicrobiology vol 45 no 6 pp 686ndash691 2007

[13] K Pattanathu M Rahman and M Gapke ldquoProductioncharacterisation and applications of biosurfactants - ReviewrdquoBiotechnology vol 2 pp 360ndash370 2008

[14] C Lacaz E Porto and J Martins Martins MicrobiologiaMedica Fungos Actinomicetos e Algas de Interesse Medico vol33 Sarvier Sao Paulo Brazil 8th edition 2001

[15] H L Barnett and B B Hunter IIlustrated Genera of ImperfectFungi vol 64 Burgess Publishing Co 4th edition 1998

[16] F R Accorsini M J R Mutton E G M Lemos and MBenincasa ldquoBiosurfactants production by yeasts using soybeanoil and glycerol as low cost substraterdquo Brazilian Journal ofMicrobiology vol 43 no 1 pp 116ndash125 2012

[17] A A Bodour and R M Miller-Maier ldquoApplication of a mod-ified drop-collapse technique for surfactant quantitation andscreening of biosurfactant-producingmicroorganismsrdquo Journalof Microbiological Methods vol 32 no 3 pp 273ndash280 1998

[18] D R Cameron D G Cooper and R J Neufeld ldquoThe manno-protein of Saccharomyces cerevisiae is an effective bioemulsi-fierrdquoApplied and Environmental Microbiology vol 54 no 6 pp1420ndash1425 1988

[19] B B Neto I S Scarminio and R E Bruns Planejamento eotimizacao de experimentos 1995

[20] M A Luna-Velasco F Esparza-Garcıa R O Canızares-Vil-lanueva and R Rodrıguez-Vazquez ldquoProduction and proper-ties of a bioemulsifier synthesized by phenanthrene-degradingPenicillium sprdquo Process Biochemistry vol 42 no 3 pp 310ndash3142007

[21] M M Camargo-de-Morais S A Ramos M C Pimentel andJ Morais ldquoProduction of an extracellular polysaccharide withemulsifier properties by Penicillium citrinumrdquoWorld Journal ofMicrobiology and Biotechnology vol 19 pp 191ndash194 2003

[22] S Askolin T Nakari-Setala and M Tenkanen ldquoOverproduc-tion purification and characterization of the Trichodermareesei hydrophobin HFBIrdquo Applied Microbiology and Biotech-nology vol 57 no 1-2 pp 124ndash130 2001

[23] Z A Raza M S Khan and ZM Khalid ldquoEvaluation of distantcarbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutantrdquo Process Biochemistryvol 42 no 4 pp 686ndash692 2007

[24] H-S Kim B-D Yoon C-H Lee et al ldquoProduction andproperties of a lipopeptide biosurfactant from Bacillus subtilisC9rdquo Journal of Fermentation and Bioengineering vol 84 no 1pp 41ndash46 1997

[25] R S Makkar and S S Cameotra ldquoProduction of biosurfac-tant at mesophilic and thermophilic conditions by a strainof Bacillus subtilisrdquo Journal of Industrial Microbiology andBiotechnology vol 20 no 1 pp 48ndash52 1998

[26] IM Banat andD J Desai ldquoMicrobial production of surfactantsand their commercial potentialrdquo Microbiology and MolecularBiology Reviews vol 61 no 1 pp 47ndash64 1997

[27] A Decesaro M R Rigon A Thome and L M CollaldquoProducao de biossurfactantes por microrganismos isolados desolo contaminado com oleo dieselrdquo Quımica Nova vol 36 no7 pp 947ndash954 2013

[28] H-S Kim J-W Jeon B-H Kim C-Y Ahn H-M Oh and B-D Yoon ldquoExtracellular production of a glycolipid biosurfactantmannosylerythritol lipid by Candida sp SY16 using fed-batchfermentationrdquo Applied Microbiology and Biotechnology vol 70no 4 pp 391ndash396 2006

[29] G L Castiglioni G Stanescu L A O Rocha and J A VCosta ldquoActa scientiarum analytical modeling and numericaloptimization of the biosurfactants production in solid-statefermentation by aspergillus fumigatusrdquo Acta Scientiarum vol36 no 1 pp 61ndash67 2014

[30] G S Kiran T A Hema R Gandhimathi et al ldquoOptimizationand production of a biosurfactant from the sponge-associatedmarine fungus Aspergillus ustus MSF3rdquo Colloids and SurfacesB Biointerfaces vol 73 no 2 pp 250ndash256 2009

[31] L M Acioly A Silveira M Anjos et al ldquoBiosurfactantproduction by Mucor circinelloides using apple peel vegetableoil and corn steep liquor as substraterdquo in Microbes in AppliedResearchndashCurrents Advances and Challenges A Mendez-VilasEd pp 344ndash347 World Science New York NY USA 2012

[32] A S Monteiro M R Q Bonfim V S Domingues et alldquoIdentification and characterization of bioemulsifier-producingyeasts isolated from effluents of a dairy industryrdquo BioresourceTechnology vol 101 no 14 pp 5186ndash5193 2010

8 International Journal of Microbiology

[33] A Khopade B Ren X-Y Liu K Mahadik L Zhang andC Kokare ldquoProduction and characterization of biosurfactantfrom marine Streptomyces species B3rdquo Journal of Colloid andInterface Science vol 367 no 1 pp 311ndash318 2012

[34] CMullinganDCooper andRNeufeld ldquoSelection ofmicrobesproducing biosurfactants in media without hydrocarbonsrdquoJournal of Fermentation Technology vol 4 pp 311ndash314 1984

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Page 3: Production of Biosurfactants by Soil Fungi Isolated from ...downloads.hindawi.com/journals/ijmicro/2018/5684261.pdf · ResearchArticle Production of Biosurfactants by Soil Fungi Isolated

International Journal of Microbiology 3

0

10

20

30

40

50

60

70

Soybean oil Starch Sucrose Cellobiose Xylose

Emul

sifica

tion

inde

x (

)

(a)

0

10

20

30

40

50

60

70

Peptone Yeastextract

Meatextract

Sodiumnitrate

Maltextract

Emul

sifica

tion

inde

x (

)

(b)

Figure 1 Univariate test measuring the influence of carbon and nitrogen sources in the production of biosurfactants by Penicillium 8CC2(a) Carbon source and (b) nitrogen source

Table 1 Fungal cultures isolated from soil samples and resultsfor the best producers of biosurfactants obtained using the dropcollapse test and emulsification index (E24)

Isolated organism Drop collapse test Emulsification indexFusarium sp 87LIV12 Neg 6428Fusarium sp 86LV272 Neg 5857Fusarium sp 85RV342 Neg 5714Penicillium sp 97VV74 Neg 5714Penicillium sp 8CC2 Neg 542Trichoderma sp 91BN312 Neg 5285Trichoderma sp 60AVR3 Pos 51Trichoderma sp 16AC2 Neg 4285Penicillium sp 62BLC2 Neg 403

obtained colonies from the genera Penicillium (46 isolates)Aspergillus (24 isolates) Fonsecaea Gliocladium (one isolate)Trichoderma (12 isolates) Fusarium (eight isolates) andAcremonium (two isolates) Additionally from the phylumZygomycota colonies from the genus Mucor (six isolates) ofthe class Zygomycetes were obtained

32 Selection of Fungal Producers of Biosurfactants To selectthe biosurfactant-producing fungi we performed analyses ofbioprocessing in submerged fermentation and evaluated theemulsification index and drop collapse test (Table 1) of theculture filtrate Nine isolates showed a high emulsificationindex (E24 gt 40) these belonged to the genera Penicillium(three isolates) Trichoderma (three isolates) and Fusarium(three isolates) The isolated Penicillium 8CC2 showed ahigh emulsification index (542) and was selected for theremaining stages of the study because its emulsion couldbe maintained for more than 15 days others have isolatedemulsification rates below 30

33 Effect of Carbon and Nitrogen Sources on BiosurfactantProduction To optimize the production of biosurfactants bythe isolated Penicillium 8CC2 we investigated the influenceof carbon and nitrogen sources by performing univariate

experiments (Figure 1)The carbon and nitrogen sources thatenabled the highest production rates were soybean oil (55)and yeast extract (647) respectively

34 Influence of Soybean Oil Yeast Extract pH and Time onBiosurfactant Production The influence of soybean oil andyeast extract concentrations pH and time on biosurfactantproduction by Penicillium 8CC2 was investigated using theexperimental design 24 as described with four replicatesadded at the central point (Table 2) All major factors andsome interactions (yeast extract times time and pH times time)showed statistical significance (Table 3) An analysis of vari-ance (ANOVA) was performed to validate the mathematicalmodel presented (see (2)) the related information is providedin Table 4 The mathematical model showed a significantregression (918) and its factors showed statistical signifi-cance The lack of fit was not significant (119901 gt 005)

E24 = 81375 minus 0128125 times Soybean Oil (gL)minus 0 534375 times Yeast Extract (gL) + 104063lowast pH + 45 lowast Time (h) + 0 184375times Yeast Extract (gL) times Time (h) minus 115625lowast pH lowast Time (h)

(2)

To represent the estimated response (E24) surfaces wereprepared (Figure 2) based on the data generated by themodelThe surfaces constructed on the basis of the model responsesshowed that the optimal conditions for maximal biosurfac-tant production were as follows soybean oil 20 g Lminus1 yeastextract 30 g Lminus1 pH 6 and a duration of 9 days These valueswere obtained with a theoretical E24 of 7982

35 Stability Studies The stability of the new biosurfactantwas evaluated under different physical conditions (Figure 3)Heating (100∘C for 60min) did not influence the biosurfac-tant activity A high NaCl concentration caused a reductionof 127 in the E24 value of the biosurfactant produced

4 International Journal of Microbiology

Table 2 Influence of soybean oil yeast extract pH and time onbiosurfactant production byPenicillium 8CC2 performed accordingto a factorial design

Soybean oil(g lminus1)

Yeast extract(g lminus1) pH Time

(d) E24

40 20 5 7 6160 30 4 9 7660 10 4 9 5340 20 5 7 6060 30 6 9 7620 30 6 9 8020 10 4 5 4740 20 5 7 5840 20 5 7 6260 30 4 5 5120 10 6 9 6220 10 6 5 5660 30 6 5 6020 30 6 5 6960 10 4 5 4820 10 4 9 5860 10 6 5 6020 30 4 5 6220 30 4 9 7860 10 6 9 47

Table 3 Effect of tested variables on biosurfactant production byPenicillium 8CC2

Factors EffectAverage 612 plusmn 0381881A soybean minus5125 plusmn 0853913lowastB yeast extract 15125 plusmn 0853913lowastC pH 4625 plusmn 0853913lowastD time 9625 plusmn 0853913lowastAB minus1375 plusmn 0853913AC minus0875 plusmn 0853913AD minus1375 plusmn 0853913BC minus0125 plusmn 0853913BD 7375 plusmn 0853913lowastCD minus4625 plusmn 0853913lowastThe effects that showed statistical significance (95 confidence level) areindicated by the symbol lowast

by Penicillium 8CC2 and completely inhibited the emulsionformation by the commercial surfactant Tween 80

4 Discussion

We found Penicillium 8CC2 (strain isolated fromAmazoniansoil) produced biosurfactant with high emulsification index(EI24gt 50) In addition we found that the biosurfactant from

Penicillium 8CC2 has similar thermal-stability and higherNaCl-stability if compared to the surfactant Tween 80 Thepresent work was the first to screen the potential of fungusobtained from soil samples of the Amazon forest to producebiosurfactant and the selected microorganisms and theirbiosurfactant have industrial potential

Among the fungi obtained from soil the genera thatshowed a strong ability to emulsify toluene were PenicilliumTrichoderma and Fusarium The potential of Penicillium spto produce biosurfactants has already been demonstrated[20 21] but few studies have described the biosurfactantpotentials of Trichoderma [22] and Fusarium

Carbon sources formicrobial production of biosurfactantcan be obtained from carbohydrates vegetable oils hydro-carbons and waste frying oils [23 24] In this study soybeanoil was the best carbon source for biosurfactant productionSoybean oil was also described as a suitable carbon sourcefor fungal production of biosurfactant by Accorsini et al[16] these last ones investigated more affordable culturemedium to produce biosurfactant by yeasts We believethat triglycerides composition is a natural inducer of thebiosurfactant production since the microorganisms producebiosurfactant to increase the access to lipid degradation and itis clear that the triglycerides can be directly used to anabolizethe production of the surfactant Starch despite being usedby other microorganisms as a carbon source [25] does notappear to be a suitable substrate for biosurfactant productionby Penicillium 8CC2

Biosurfactants are produced when there is nitrogen lim-itation during the stationary phase of growth of biomass[26ndash28] In the present work yeast extract was found to bethe most suitable nutrient source for the production of themetabolite of interest followed by peptone meat extractand malt The mineral nitrogen source sodium nitrate wasnot suitable for production of the biosurfactant Anotherstudy showed similar results it was observed that Torulopsisbombicola produced biosurfactants using yeast extract butwas not able to produce them using sodium nitrate

The results of this study showed that the factors studied(soybean oil peptone pH and time) and some of theirinteractions had a significant effect on the response vari-able (biosurfactant production) The validated mathematicalmodel demonstrated that an E24 of 7982 could be obtainedusing 20 g Lminus1 soybean oil 30 g Lminus1 yeast extract pH 6 anda 9-day duration of the bioprocess Maximum biosurfactantproduction occurred in the optimal pH range employedfor microorganism growth During the production of thisbiosurfactant as well as during any chemical reaction the pHdirectly affects the microbial activity because of the effects ofH+ ions on cell permeability and enzyme activity [29] Kiranet al [30] reported that an Aspergillus ustus isolate showedoptimal biosurfactant production at pH7Mucor circinelloideswas found to maximally produce biosurfactant at pH 8 usingapple peel vegetable oil and corn water as substrates [31]The time required for maximum biosurfactant productionby Penicillium sp 8CC2 in the current study was 9 daysAspergillus fumigatus was found to produce biosurfactantin 112 h in solid-state fermentation [32] Aspergillus ustus

International Journal of Microbiology 5

76

72

68

64

60

56

52

30

20

10 20

40

60

E24 E24

E24

E24

E24

E24

Yaest extract (gL)

60

58

56

54

52

50

6

55

5

45

4 20

40

60

pH

pH

Soybean oil (gL)

Soybean oil (gL)

Soybean oil (gL)

58

57

56

55

54

53

52

51

9

85

8

75

7 20

40

60

76

72

68

64

60

56

6

55

5

45

4 10

15

20

25

30

82

78

74

70

66

62

58

9

85

8

75

7 10

15

20

25

30

Time (d) pH

Time (d)

Time (d)

595

585

575

565

555

545

9

85

8

75

7 4

45

5

55

6

Yeast extract (gL)

Yeast extract (gL)

Figure 2 Response surface analysis for biosurfactant production by Penicillium 8CC2 with soybean oil yeast extract pH and productiontime

6 International Journal of Microbiology

Table 4 Analysis of variance for evaluating the statistical significance of the model for biosurfactant production by Penicillium 8CC2 (1198772 =918529)

Source Sum of squares Df Mean of Squares 119865 ratio 119875 valueA soybean oil 105063 1 105063 3602 00093B yeast extract 915063 1 915063 31374 00004C pH 855625 1 855625 2934 00123D time 370563 1 370563 12705 00015BD 217563 1 217563 7459 00033CD 855625 1 855625 2934 00123Lack of fit 149075 10 149075 511 01030Pure error 875 3 875Total 19372 19

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +heating

Tween 80 Tween 80 +heating

Emul

sifica

tion

inde

x (

)

(a)

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +NaCl (30 wv)

Tween 80 Tween 80 +NaCl (30 wv)

Emul

sifica

tion

inde

x (

)

(b)

Figure 3 Stability of biosurfactants from Penicillium 8CC2 under different physical conditions (a) after heating 100∘C (60min) and (b) withthe addition of NaCl (30wv) The synthetic surfactant Tween 80 (1wv) was used as a control substance in these assays

isolated from amarine sponge showed optimized productionat 120 h in Sabouraud dextrose broth [30]

The stability of the biosurfactant was evaluated at hightemperatures and high ionic strengths After subjecting thebiosurfactant to a temperature of 100∘C for 60min therewas no verified change in biosurfactant activity This resultdemonstrates the potential utility of the biosurfactant in thefood pharmaceutical and cosmetic industries where heatsterility is of great importance [32 33] In the case of ionicstrength there was a small reduction in the emulsificationability of Penicillium 8CC2 when NaCl (30) was added buta stable emulsion could still be maintained The commercialemulsifier Tween 80 used as a control did not remain stablewhen NaCl was added This result demonstrates that thebiosurfactant produced by Penicillium 8CC2 can be used inhigh salt-concentration conditions for example in tertiaryoil recovery activities

This study verified stability parameters for only onefungus from the 61 potential biosurfactant-producing fungithus ignoring possible potentiality in many others includingsome with high levels of emulsification In the stability testswe could have assessed the influence of pH The resultsdemonstrate that biotechnological approaches can be usedto improve biosurfactant production by Penicillium 8CC2The current results suggest the potential usefulness of thebiosurfactant produced by a filamentous fungus isolated fromsoil samples from the Amazon region

The data from the present work is important since asignificative number of isolates (100 cultures isolated fromsoil samples from the Amazon) were investigated for theproduction of biosurfactant The analytical methodologyused in the screening of biosurfactant producers did notquantify the biosurfactant directly in the culture filtratenowadays the screening methods have been developed thatrely on the interfacial activity of the biosurfactants but do notmeasure it directly We used methodology that quantifies theability of the biosurfactant present in the culture filtrate toproduce emulsificationwith toluene ldquoemulsification indexrdquo asclassically described by Mullingan et al [34] Unfortunatelyas a work limitation during the experimental conditions itwas not possible to identify the species of Penicillium 8CC2and the chemical composition of the biosurfactant Literaturedoes not have description of chemical composition of thebiosurfactants produced by Penicillium sp and further worksare necessary to elucidate it

The development for screening microbes from thousandsof potentially active organisms and the subsequent evaluationof surface activity holds the key to the discovery of newbiosurfactants or production strains

Conflicts of Interest

The authors declare that they have no conflicts of interest

International Journal of Microbiology 7

References

[1] Z A Raza A Rehman M T Hussain et al ldquoProduction ofrhamnolipid surfactant and its application in bioscouring ofcotton fabricrdquo Carbohydrate Research vol 391 no 1 pp 97ndash1052014

[2] I M Banat A Franzetti I Gandolfi et al ldquoMicrobial biosur-factants production applications and future potentialrdquo AppliedMicrobiology andBiotechnology vol 87 no 2 pp 427ndash444 2010

[3] M Pacwa-Płociniczak G A Płaza Z Piotrowska-Seget andS S Cameotra ldquoEnvironmental applications of biosurfactantsrecent advancesrdquo International Journal of Molecular Sciencesvol 12 no 1 pp 633ndash654 2011

[4] P Das S Mukherjee and R Sen ldquoImproved bioavailabilityand biodegradation of a model polyaromatic hydrocarbonby a biosurfactant producing bacterium of marine originrdquoChemosphere vol 72 no 9 pp 1229ndash1234 2008

[5] M Nitschke andGM Pastore ldquoBiossurfactantes propriedadese aplicacoesrdquo Quımica Nova vol 25 no 5 pp 772ndash776 2002

[6] A Rehman Z A Raza Saif-ur-Rehman et al ldquoSynthesis anduse of self-assembled rhamnolipid microtubules as templatesfor gold nanoparticles assembly to form gold microstructuresrdquoJournal of Colloid and Interface Science vol 347 no 2 pp 332ndash335 2010

[7] G L Castiglioni T E Bertolin and J A Costa ldquoProducao debiossurfactante por Aspergillus fumigatus utilizando resıduosagroindustriais como substratordquo Quımica Nova vol 32 no 2pp 292ndash295 2009

[8] Z Velioglu and R O Urek ldquoBiosurfactant production byPleurotus ostreatus in submerged and solid-state fermentationsystemsrdquo Turkish Journal of Biology vol 39 no 1 pp 160ndash1662015

[9] J L Adrio and A L Demain ldquoFungal biotechnologyrdquo Interna-tional Microbiology vol 6 no 3 pp 191ndash199 2003

[10] G C Fontes P F Amaral and M A Coelho ldquoProducao debiossurfactante por levedurardquo Quımica Nova vol 31 no 8 pp2091ndash2099 2008

[11] M Pirollo Estudo da Producao de Biossurfactantes UtilizandoHidrocarbonetos Universidade Estadual Paulista Sao PauloBrazil 2006

[12] RThavasi S Jayalakshmi T Balasubramanian and IM BanatldquoBiosurfactant production by Corynebacterium kutscheri fromwastemotor lubricant oil and peanut oil cakerdquo Letters in AppliedMicrobiology vol 45 no 6 pp 686ndash691 2007

[13] K Pattanathu M Rahman and M Gapke ldquoProductioncharacterisation and applications of biosurfactants - ReviewrdquoBiotechnology vol 2 pp 360ndash370 2008

[14] C Lacaz E Porto and J Martins Martins MicrobiologiaMedica Fungos Actinomicetos e Algas de Interesse Medico vol33 Sarvier Sao Paulo Brazil 8th edition 2001

[15] H L Barnett and B B Hunter IIlustrated Genera of ImperfectFungi vol 64 Burgess Publishing Co 4th edition 1998

[16] F R Accorsini M J R Mutton E G M Lemos and MBenincasa ldquoBiosurfactants production by yeasts using soybeanoil and glycerol as low cost substraterdquo Brazilian Journal ofMicrobiology vol 43 no 1 pp 116ndash125 2012

[17] A A Bodour and R M Miller-Maier ldquoApplication of a mod-ified drop-collapse technique for surfactant quantitation andscreening of biosurfactant-producingmicroorganismsrdquo Journalof Microbiological Methods vol 32 no 3 pp 273ndash280 1998

[18] D R Cameron D G Cooper and R J Neufeld ldquoThe manno-protein of Saccharomyces cerevisiae is an effective bioemulsi-fierrdquoApplied and Environmental Microbiology vol 54 no 6 pp1420ndash1425 1988

[19] B B Neto I S Scarminio and R E Bruns Planejamento eotimizacao de experimentos 1995

[20] M A Luna-Velasco F Esparza-Garcıa R O Canızares-Vil-lanueva and R Rodrıguez-Vazquez ldquoProduction and proper-ties of a bioemulsifier synthesized by phenanthrene-degradingPenicillium sprdquo Process Biochemistry vol 42 no 3 pp 310ndash3142007

[21] M M Camargo-de-Morais S A Ramos M C Pimentel andJ Morais ldquoProduction of an extracellular polysaccharide withemulsifier properties by Penicillium citrinumrdquoWorld Journal ofMicrobiology and Biotechnology vol 19 pp 191ndash194 2003

[22] S Askolin T Nakari-Setala and M Tenkanen ldquoOverproduc-tion purification and characterization of the Trichodermareesei hydrophobin HFBIrdquo Applied Microbiology and Biotech-nology vol 57 no 1-2 pp 124ndash130 2001

[23] Z A Raza M S Khan and ZM Khalid ldquoEvaluation of distantcarbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutantrdquo Process Biochemistryvol 42 no 4 pp 686ndash692 2007

[24] H-S Kim B-D Yoon C-H Lee et al ldquoProduction andproperties of a lipopeptide biosurfactant from Bacillus subtilisC9rdquo Journal of Fermentation and Bioengineering vol 84 no 1pp 41ndash46 1997

[25] R S Makkar and S S Cameotra ldquoProduction of biosurfac-tant at mesophilic and thermophilic conditions by a strainof Bacillus subtilisrdquo Journal of Industrial Microbiology andBiotechnology vol 20 no 1 pp 48ndash52 1998

[26] IM Banat andD J Desai ldquoMicrobial production of surfactantsand their commercial potentialrdquo Microbiology and MolecularBiology Reviews vol 61 no 1 pp 47ndash64 1997

[27] A Decesaro M R Rigon A Thome and L M CollaldquoProducao de biossurfactantes por microrganismos isolados desolo contaminado com oleo dieselrdquo Quımica Nova vol 36 no7 pp 947ndash954 2013

[28] H-S Kim J-W Jeon B-H Kim C-Y Ahn H-M Oh and B-D Yoon ldquoExtracellular production of a glycolipid biosurfactantmannosylerythritol lipid by Candida sp SY16 using fed-batchfermentationrdquo Applied Microbiology and Biotechnology vol 70no 4 pp 391ndash396 2006

[29] G L Castiglioni G Stanescu L A O Rocha and J A VCosta ldquoActa scientiarum analytical modeling and numericaloptimization of the biosurfactants production in solid-statefermentation by aspergillus fumigatusrdquo Acta Scientiarum vol36 no 1 pp 61ndash67 2014

[30] G S Kiran T A Hema R Gandhimathi et al ldquoOptimizationand production of a biosurfactant from the sponge-associatedmarine fungus Aspergillus ustus MSF3rdquo Colloids and SurfacesB Biointerfaces vol 73 no 2 pp 250ndash256 2009

[31] L M Acioly A Silveira M Anjos et al ldquoBiosurfactantproduction by Mucor circinelloides using apple peel vegetableoil and corn steep liquor as substraterdquo in Microbes in AppliedResearchndashCurrents Advances and Challenges A Mendez-VilasEd pp 344ndash347 World Science New York NY USA 2012

[32] A S Monteiro M R Q Bonfim V S Domingues et alldquoIdentification and characterization of bioemulsifier-producingyeasts isolated from effluents of a dairy industryrdquo BioresourceTechnology vol 101 no 14 pp 5186ndash5193 2010

8 International Journal of Microbiology

[33] A Khopade B Ren X-Y Liu K Mahadik L Zhang andC Kokare ldquoProduction and characterization of biosurfactantfrom marine Streptomyces species B3rdquo Journal of Colloid andInterface Science vol 367 no 1 pp 311ndash318 2012

[34] CMullinganDCooper andRNeufeld ldquoSelection ofmicrobesproducing biosurfactants in media without hydrocarbonsrdquoJournal of Fermentation Technology vol 4 pp 311ndash314 1984

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Page 4: Production of Biosurfactants by Soil Fungi Isolated from ...downloads.hindawi.com/journals/ijmicro/2018/5684261.pdf · ResearchArticle Production of Biosurfactants by Soil Fungi Isolated

4 International Journal of Microbiology

Table 2 Influence of soybean oil yeast extract pH and time onbiosurfactant production byPenicillium 8CC2 performed accordingto a factorial design

Soybean oil(g lminus1)

Yeast extract(g lminus1) pH Time

(d) E24

40 20 5 7 6160 30 4 9 7660 10 4 9 5340 20 5 7 6060 30 6 9 7620 30 6 9 8020 10 4 5 4740 20 5 7 5840 20 5 7 6260 30 4 5 5120 10 6 9 6220 10 6 5 5660 30 6 5 6020 30 6 5 6960 10 4 5 4820 10 4 9 5860 10 6 5 6020 30 4 5 6220 30 4 9 7860 10 6 9 47

Table 3 Effect of tested variables on biosurfactant production byPenicillium 8CC2

Factors EffectAverage 612 plusmn 0381881A soybean minus5125 plusmn 0853913lowastB yeast extract 15125 plusmn 0853913lowastC pH 4625 plusmn 0853913lowastD time 9625 plusmn 0853913lowastAB minus1375 plusmn 0853913AC minus0875 plusmn 0853913AD minus1375 plusmn 0853913BC minus0125 plusmn 0853913BD 7375 plusmn 0853913lowastCD minus4625 plusmn 0853913lowastThe effects that showed statistical significance (95 confidence level) areindicated by the symbol lowast

by Penicillium 8CC2 and completely inhibited the emulsionformation by the commercial surfactant Tween 80

4 Discussion

We found Penicillium 8CC2 (strain isolated fromAmazoniansoil) produced biosurfactant with high emulsification index(EI24gt 50) In addition we found that the biosurfactant from

Penicillium 8CC2 has similar thermal-stability and higherNaCl-stability if compared to the surfactant Tween 80 Thepresent work was the first to screen the potential of fungusobtained from soil samples of the Amazon forest to producebiosurfactant and the selected microorganisms and theirbiosurfactant have industrial potential

Among the fungi obtained from soil the genera thatshowed a strong ability to emulsify toluene were PenicilliumTrichoderma and Fusarium The potential of Penicillium spto produce biosurfactants has already been demonstrated[20 21] but few studies have described the biosurfactantpotentials of Trichoderma [22] and Fusarium

Carbon sources formicrobial production of biosurfactantcan be obtained from carbohydrates vegetable oils hydro-carbons and waste frying oils [23 24] In this study soybeanoil was the best carbon source for biosurfactant productionSoybean oil was also described as a suitable carbon sourcefor fungal production of biosurfactant by Accorsini et al[16] these last ones investigated more affordable culturemedium to produce biosurfactant by yeasts We believethat triglycerides composition is a natural inducer of thebiosurfactant production since the microorganisms producebiosurfactant to increase the access to lipid degradation and itis clear that the triglycerides can be directly used to anabolizethe production of the surfactant Starch despite being usedby other microorganisms as a carbon source [25] does notappear to be a suitable substrate for biosurfactant productionby Penicillium 8CC2

Biosurfactants are produced when there is nitrogen lim-itation during the stationary phase of growth of biomass[26ndash28] In the present work yeast extract was found to bethe most suitable nutrient source for the production of themetabolite of interest followed by peptone meat extractand malt The mineral nitrogen source sodium nitrate wasnot suitable for production of the biosurfactant Anotherstudy showed similar results it was observed that Torulopsisbombicola produced biosurfactants using yeast extract butwas not able to produce them using sodium nitrate

The results of this study showed that the factors studied(soybean oil peptone pH and time) and some of theirinteractions had a significant effect on the response vari-able (biosurfactant production) The validated mathematicalmodel demonstrated that an E24 of 7982 could be obtainedusing 20 g Lminus1 soybean oil 30 g Lminus1 yeast extract pH 6 anda 9-day duration of the bioprocess Maximum biosurfactantproduction occurred in the optimal pH range employedfor microorganism growth During the production of thisbiosurfactant as well as during any chemical reaction the pHdirectly affects the microbial activity because of the effects ofH+ ions on cell permeability and enzyme activity [29] Kiranet al [30] reported that an Aspergillus ustus isolate showedoptimal biosurfactant production at pH7Mucor circinelloideswas found to maximally produce biosurfactant at pH 8 usingapple peel vegetable oil and corn water as substrates [31]The time required for maximum biosurfactant productionby Penicillium sp 8CC2 in the current study was 9 daysAspergillus fumigatus was found to produce biosurfactantin 112 h in solid-state fermentation [32] Aspergillus ustus

International Journal of Microbiology 5

76

72

68

64

60

56

52

30

20

10 20

40

60

E24 E24

E24

E24

E24

E24

Yaest extract (gL)

60

58

56

54

52

50

6

55

5

45

4 20

40

60

pH

pH

Soybean oil (gL)

Soybean oil (gL)

Soybean oil (gL)

58

57

56

55

54

53

52

51

9

85

8

75

7 20

40

60

76

72

68

64

60

56

6

55

5

45

4 10

15

20

25

30

82

78

74

70

66

62

58

9

85

8

75

7 10

15

20

25

30

Time (d) pH

Time (d)

Time (d)

595

585

575

565

555

545

9

85

8

75

7 4

45

5

55

6

Yeast extract (gL)

Yeast extract (gL)

Figure 2 Response surface analysis for biosurfactant production by Penicillium 8CC2 with soybean oil yeast extract pH and productiontime

6 International Journal of Microbiology

Table 4 Analysis of variance for evaluating the statistical significance of the model for biosurfactant production by Penicillium 8CC2 (1198772 =918529)

Source Sum of squares Df Mean of Squares 119865 ratio 119875 valueA soybean oil 105063 1 105063 3602 00093B yeast extract 915063 1 915063 31374 00004C pH 855625 1 855625 2934 00123D time 370563 1 370563 12705 00015BD 217563 1 217563 7459 00033CD 855625 1 855625 2934 00123Lack of fit 149075 10 149075 511 01030Pure error 875 3 875Total 19372 19

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +heating

Tween 80 Tween 80 +heating

Emul

sifica

tion

inde

x (

)

(a)

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +NaCl (30 wv)

Tween 80 Tween 80 +NaCl (30 wv)

Emul

sifica

tion

inde

x (

)

(b)

Figure 3 Stability of biosurfactants from Penicillium 8CC2 under different physical conditions (a) after heating 100∘C (60min) and (b) withthe addition of NaCl (30wv) The synthetic surfactant Tween 80 (1wv) was used as a control substance in these assays

isolated from amarine sponge showed optimized productionat 120 h in Sabouraud dextrose broth [30]

The stability of the biosurfactant was evaluated at hightemperatures and high ionic strengths After subjecting thebiosurfactant to a temperature of 100∘C for 60min therewas no verified change in biosurfactant activity This resultdemonstrates the potential utility of the biosurfactant in thefood pharmaceutical and cosmetic industries where heatsterility is of great importance [32 33] In the case of ionicstrength there was a small reduction in the emulsificationability of Penicillium 8CC2 when NaCl (30) was added buta stable emulsion could still be maintained The commercialemulsifier Tween 80 used as a control did not remain stablewhen NaCl was added This result demonstrates that thebiosurfactant produced by Penicillium 8CC2 can be used inhigh salt-concentration conditions for example in tertiaryoil recovery activities

This study verified stability parameters for only onefungus from the 61 potential biosurfactant-producing fungithus ignoring possible potentiality in many others includingsome with high levels of emulsification In the stability testswe could have assessed the influence of pH The resultsdemonstrate that biotechnological approaches can be usedto improve biosurfactant production by Penicillium 8CC2The current results suggest the potential usefulness of thebiosurfactant produced by a filamentous fungus isolated fromsoil samples from the Amazon region

The data from the present work is important since asignificative number of isolates (100 cultures isolated fromsoil samples from the Amazon) were investigated for theproduction of biosurfactant The analytical methodologyused in the screening of biosurfactant producers did notquantify the biosurfactant directly in the culture filtratenowadays the screening methods have been developed thatrely on the interfacial activity of the biosurfactants but do notmeasure it directly We used methodology that quantifies theability of the biosurfactant present in the culture filtrate toproduce emulsificationwith toluene ldquoemulsification indexrdquo asclassically described by Mullingan et al [34] Unfortunatelyas a work limitation during the experimental conditions itwas not possible to identify the species of Penicillium 8CC2and the chemical composition of the biosurfactant Literaturedoes not have description of chemical composition of thebiosurfactants produced by Penicillium sp and further worksare necessary to elucidate it

The development for screening microbes from thousandsof potentially active organisms and the subsequent evaluationof surface activity holds the key to the discovery of newbiosurfactants or production strains

Conflicts of Interest

The authors declare that they have no conflicts of interest

International Journal of Microbiology 7

References

[1] Z A Raza A Rehman M T Hussain et al ldquoProduction ofrhamnolipid surfactant and its application in bioscouring ofcotton fabricrdquo Carbohydrate Research vol 391 no 1 pp 97ndash1052014

[2] I M Banat A Franzetti I Gandolfi et al ldquoMicrobial biosur-factants production applications and future potentialrdquo AppliedMicrobiology andBiotechnology vol 87 no 2 pp 427ndash444 2010

[3] M Pacwa-Płociniczak G A Płaza Z Piotrowska-Seget andS S Cameotra ldquoEnvironmental applications of biosurfactantsrecent advancesrdquo International Journal of Molecular Sciencesvol 12 no 1 pp 633ndash654 2011

[4] P Das S Mukherjee and R Sen ldquoImproved bioavailabilityand biodegradation of a model polyaromatic hydrocarbonby a biosurfactant producing bacterium of marine originrdquoChemosphere vol 72 no 9 pp 1229ndash1234 2008

[5] M Nitschke andGM Pastore ldquoBiossurfactantes propriedadese aplicacoesrdquo Quımica Nova vol 25 no 5 pp 772ndash776 2002

[6] A Rehman Z A Raza Saif-ur-Rehman et al ldquoSynthesis anduse of self-assembled rhamnolipid microtubules as templatesfor gold nanoparticles assembly to form gold microstructuresrdquoJournal of Colloid and Interface Science vol 347 no 2 pp 332ndash335 2010

[7] G L Castiglioni T E Bertolin and J A Costa ldquoProducao debiossurfactante por Aspergillus fumigatus utilizando resıduosagroindustriais como substratordquo Quımica Nova vol 32 no 2pp 292ndash295 2009

[8] Z Velioglu and R O Urek ldquoBiosurfactant production byPleurotus ostreatus in submerged and solid-state fermentationsystemsrdquo Turkish Journal of Biology vol 39 no 1 pp 160ndash1662015

[9] J L Adrio and A L Demain ldquoFungal biotechnologyrdquo Interna-tional Microbiology vol 6 no 3 pp 191ndash199 2003

[10] G C Fontes P F Amaral and M A Coelho ldquoProducao debiossurfactante por levedurardquo Quımica Nova vol 31 no 8 pp2091ndash2099 2008

[11] M Pirollo Estudo da Producao de Biossurfactantes UtilizandoHidrocarbonetos Universidade Estadual Paulista Sao PauloBrazil 2006

[12] RThavasi S Jayalakshmi T Balasubramanian and IM BanatldquoBiosurfactant production by Corynebacterium kutscheri fromwastemotor lubricant oil and peanut oil cakerdquo Letters in AppliedMicrobiology vol 45 no 6 pp 686ndash691 2007

[13] K Pattanathu M Rahman and M Gapke ldquoProductioncharacterisation and applications of biosurfactants - ReviewrdquoBiotechnology vol 2 pp 360ndash370 2008

[14] C Lacaz E Porto and J Martins Martins MicrobiologiaMedica Fungos Actinomicetos e Algas de Interesse Medico vol33 Sarvier Sao Paulo Brazil 8th edition 2001

[15] H L Barnett and B B Hunter IIlustrated Genera of ImperfectFungi vol 64 Burgess Publishing Co 4th edition 1998

[16] F R Accorsini M J R Mutton E G M Lemos and MBenincasa ldquoBiosurfactants production by yeasts using soybeanoil and glycerol as low cost substraterdquo Brazilian Journal ofMicrobiology vol 43 no 1 pp 116ndash125 2012

[17] A A Bodour and R M Miller-Maier ldquoApplication of a mod-ified drop-collapse technique for surfactant quantitation andscreening of biosurfactant-producingmicroorganismsrdquo Journalof Microbiological Methods vol 32 no 3 pp 273ndash280 1998

[18] D R Cameron D G Cooper and R J Neufeld ldquoThe manno-protein of Saccharomyces cerevisiae is an effective bioemulsi-fierrdquoApplied and Environmental Microbiology vol 54 no 6 pp1420ndash1425 1988

[19] B B Neto I S Scarminio and R E Bruns Planejamento eotimizacao de experimentos 1995

[20] M A Luna-Velasco F Esparza-Garcıa R O Canızares-Vil-lanueva and R Rodrıguez-Vazquez ldquoProduction and proper-ties of a bioemulsifier synthesized by phenanthrene-degradingPenicillium sprdquo Process Biochemistry vol 42 no 3 pp 310ndash3142007

[21] M M Camargo-de-Morais S A Ramos M C Pimentel andJ Morais ldquoProduction of an extracellular polysaccharide withemulsifier properties by Penicillium citrinumrdquoWorld Journal ofMicrobiology and Biotechnology vol 19 pp 191ndash194 2003

[22] S Askolin T Nakari-Setala and M Tenkanen ldquoOverproduc-tion purification and characterization of the Trichodermareesei hydrophobin HFBIrdquo Applied Microbiology and Biotech-nology vol 57 no 1-2 pp 124ndash130 2001

[23] Z A Raza M S Khan and ZM Khalid ldquoEvaluation of distantcarbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutantrdquo Process Biochemistryvol 42 no 4 pp 686ndash692 2007

[24] H-S Kim B-D Yoon C-H Lee et al ldquoProduction andproperties of a lipopeptide biosurfactant from Bacillus subtilisC9rdquo Journal of Fermentation and Bioengineering vol 84 no 1pp 41ndash46 1997

[25] R S Makkar and S S Cameotra ldquoProduction of biosurfac-tant at mesophilic and thermophilic conditions by a strainof Bacillus subtilisrdquo Journal of Industrial Microbiology andBiotechnology vol 20 no 1 pp 48ndash52 1998

[26] IM Banat andD J Desai ldquoMicrobial production of surfactantsand their commercial potentialrdquo Microbiology and MolecularBiology Reviews vol 61 no 1 pp 47ndash64 1997

[27] A Decesaro M R Rigon A Thome and L M CollaldquoProducao de biossurfactantes por microrganismos isolados desolo contaminado com oleo dieselrdquo Quımica Nova vol 36 no7 pp 947ndash954 2013

[28] H-S Kim J-W Jeon B-H Kim C-Y Ahn H-M Oh and B-D Yoon ldquoExtracellular production of a glycolipid biosurfactantmannosylerythritol lipid by Candida sp SY16 using fed-batchfermentationrdquo Applied Microbiology and Biotechnology vol 70no 4 pp 391ndash396 2006

[29] G L Castiglioni G Stanescu L A O Rocha and J A VCosta ldquoActa scientiarum analytical modeling and numericaloptimization of the biosurfactants production in solid-statefermentation by aspergillus fumigatusrdquo Acta Scientiarum vol36 no 1 pp 61ndash67 2014

[30] G S Kiran T A Hema R Gandhimathi et al ldquoOptimizationand production of a biosurfactant from the sponge-associatedmarine fungus Aspergillus ustus MSF3rdquo Colloids and SurfacesB Biointerfaces vol 73 no 2 pp 250ndash256 2009

[31] L M Acioly A Silveira M Anjos et al ldquoBiosurfactantproduction by Mucor circinelloides using apple peel vegetableoil and corn steep liquor as substraterdquo in Microbes in AppliedResearchndashCurrents Advances and Challenges A Mendez-VilasEd pp 344ndash347 World Science New York NY USA 2012

[32] A S Monteiro M R Q Bonfim V S Domingues et alldquoIdentification and characterization of bioemulsifier-producingyeasts isolated from effluents of a dairy industryrdquo BioresourceTechnology vol 101 no 14 pp 5186ndash5193 2010

8 International Journal of Microbiology

[33] A Khopade B Ren X-Y Liu K Mahadik L Zhang andC Kokare ldquoProduction and characterization of biosurfactantfrom marine Streptomyces species B3rdquo Journal of Colloid andInterface Science vol 367 no 1 pp 311ndash318 2012

[34] CMullinganDCooper andRNeufeld ldquoSelection ofmicrobesproducing biosurfactants in media without hydrocarbonsrdquoJournal of Fermentation Technology vol 4 pp 311ndash314 1984

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Page 5: Production of Biosurfactants by Soil Fungi Isolated from ...downloads.hindawi.com/journals/ijmicro/2018/5684261.pdf · ResearchArticle Production of Biosurfactants by Soil Fungi Isolated

International Journal of Microbiology 5

76

72

68

64

60

56

52

30

20

10 20

40

60

E24 E24

E24

E24

E24

E24

Yaest extract (gL)

60

58

56

54

52

50

6

55

5

45

4 20

40

60

pH

pH

Soybean oil (gL)

Soybean oil (gL)

Soybean oil (gL)

58

57

56

55

54

53

52

51

9

85

8

75

7 20

40

60

76

72

68

64

60

56

6

55

5

45

4 10

15

20

25

30

82

78

74

70

66

62

58

9

85

8

75

7 10

15

20

25

30

Time (d) pH

Time (d)

Time (d)

595

585

575

565

555

545

9

85

8

75

7 4

45

5

55

6

Yeast extract (gL)

Yeast extract (gL)

Figure 2 Response surface analysis for biosurfactant production by Penicillium 8CC2 with soybean oil yeast extract pH and productiontime

6 International Journal of Microbiology

Table 4 Analysis of variance for evaluating the statistical significance of the model for biosurfactant production by Penicillium 8CC2 (1198772 =918529)

Source Sum of squares Df Mean of Squares 119865 ratio 119875 valueA soybean oil 105063 1 105063 3602 00093B yeast extract 915063 1 915063 31374 00004C pH 855625 1 855625 2934 00123D time 370563 1 370563 12705 00015BD 217563 1 217563 7459 00033CD 855625 1 855625 2934 00123Lack of fit 149075 10 149075 511 01030Pure error 875 3 875Total 19372 19

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +heating

Tween 80 Tween 80 +heating

Emul

sifica

tion

inde

x (

)

(a)

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +NaCl (30 wv)

Tween 80 Tween 80 +NaCl (30 wv)

Emul

sifica

tion

inde

x (

)

(b)

Figure 3 Stability of biosurfactants from Penicillium 8CC2 under different physical conditions (a) after heating 100∘C (60min) and (b) withthe addition of NaCl (30wv) The synthetic surfactant Tween 80 (1wv) was used as a control substance in these assays

isolated from amarine sponge showed optimized productionat 120 h in Sabouraud dextrose broth [30]

The stability of the biosurfactant was evaluated at hightemperatures and high ionic strengths After subjecting thebiosurfactant to a temperature of 100∘C for 60min therewas no verified change in biosurfactant activity This resultdemonstrates the potential utility of the biosurfactant in thefood pharmaceutical and cosmetic industries where heatsterility is of great importance [32 33] In the case of ionicstrength there was a small reduction in the emulsificationability of Penicillium 8CC2 when NaCl (30) was added buta stable emulsion could still be maintained The commercialemulsifier Tween 80 used as a control did not remain stablewhen NaCl was added This result demonstrates that thebiosurfactant produced by Penicillium 8CC2 can be used inhigh salt-concentration conditions for example in tertiaryoil recovery activities

This study verified stability parameters for only onefungus from the 61 potential biosurfactant-producing fungithus ignoring possible potentiality in many others includingsome with high levels of emulsification In the stability testswe could have assessed the influence of pH The resultsdemonstrate that biotechnological approaches can be usedto improve biosurfactant production by Penicillium 8CC2The current results suggest the potential usefulness of thebiosurfactant produced by a filamentous fungus isolated fromsoil samples from the Amazon region

The data from the present work is important since asignificative number of isolates (100 cultures isolated fromsoil samples from the Amazon) were investigated for theproduction of biosurfactant The analytical methodologyused in the screening of biosurfactant producers did notquantify the biosurfactant directly in the culture filtratenowadays the screening methods have been developed thatrely on the interfacial activity of the biosurfactants but do notmeasure it directly We used methodology that quantifies theability of the biosurfactant present in the culture filtrate toproduce emulsificationwith toluene ldquoemulsification indexrdquo asclassically described by Mullingan et al [34] Unfortunatelyas a work limitation during the experimental conditions itwas not possible to identify the species of Penicillium 8CC2and the chemical composition of the biosurfactant Literaturedoes not have description of chemical composition of thebiosurfactants produced by Penicillium sp and further worksare necessary to elucidate it

The development for screening microbes from thousandsof potentially active organisms and the subsequent evaluationof surface activity holds the key to the discovery of newbiosurfactants or production strains

Conflicts of Interest

The authors declare that they have no conflicts of interest

International Journal of Microbiology 7

References

[1] Z A Raza A Rehman M T Hussain et al ldquoProduction ofrhamnolipid surfactant and its application in bioscouring ofcotton fabricrdquo Carbohydrate Research vol 391 no 1 pp 97ndash1052014

[2] I M Banat A Franzetti I Gandolfi et al ldquoMicrobial biosur-factants production applications and future potentialrdquo AppliedMicrobiology andBiotechnology vol 87 no 2 pp 427ndash444 2010

[3] M Pacwa-Płociniczak G A Płaza Z Piotrowska-Seget andS S Cameotra ldquoEnvironmental applications of biosurfactantsrecent advancesrdquo International Journal of Molecular Sciencesvol 12 no 1 pp 633ndash654 2011

[4] P Das S Mukherjee and R Sen ldquoImproved bioavailabilityand biodegradation of a model polyaromatic hydrocarbonby a biosurfactant producing bacterium of marine originrdquoChemosphere vol 72 no 9 pp 1229ndash1234 2008

[5] M Nitschke andGM Pastore ldquoBiossurfactantes propriedadese aplicacoesrdquo Quımica Nova vol 25 no 5 pp 772ndash776 2002

[6] A Rehman Z A Raza Saif-ur-Rehman et al ldquoSynthesis anduse of self-assembled rhamnolipid microtubules as templatesfor gold nanoparticles assembly to form gold microstructuresrdquoJournal of Colloid and Interface Science vol 347 no 2 pp 332ndash335 2010

[7] G L Castiglioni T E Bertolin and J A Costa ldquoProducao debiossurfactante por Aspergillus fumigatus utilizando resıduosagroindustriais como substratordquo Quımica Nova vol 32 no 2pp 292ndash295 2009

[8] Z Velioglu and R O Urek ldquoBiosurfactant production byPleurotus ostreatus in submerged and solid-state fermentationsystemsrdquo Turkish Journal of Biology vol 39 no 1 pp 160ndash1662015

[9] J L Adrio and A L Demain ldquoFungal biotechnologyrdquo Interna-tional Microbiology vol 6 no 3 pp 191ndash199 2003

[10] G C Fontes P F Amaral and M A Coelho ldquoProducao debiossurfactante por levedurardquo Quımica Nova vol 31 no 8 pp2091ndash2099 2008

[11] M Pirollo Estudo da Producao de Biossurfactantes UtilizandoHidrocarbonetos Universidade Estadual Paulista Sao PauloBrazil 2006

[12] RThavasi S Jayalakshmi T Balasubramanian and IM BanatldquoBiosurfactant production by Corynebacterium kutscheri fromwastemotor lubricant oil and peanut oil cakerdquo Letters in AppliedMicrobiology vol 45 no 6 pp 686ndash691 2007

[13] K Pattanathu M Rahman and M Gapke ldquoProductioncharacterisation and applications of biosurfactants - ReviewrdquoBiotechnology vol 2 pp 360ndash370 2008

[14] C Lacaz E Porto and J Martins Martins MicrobiologiaMedica Fungos Actinomicetos e Algas de Interesse Medico vol33 Sarvier Sao Paulo Brazil 8th edition 2001

[15] H L Barnett and B B Hunter IIlustrated Genera of ImperfectFungi vol 64 Burgess Publishing Co 4th edition 1998

[16] F R Accorsini M J R Mutton E G M Lemos and MBenincasa ldquoBiosurfactants production by yeasts using soybeanoil and glycerol as low cost substraterdquo Brazilian Journal ofMicrobiology vol 43 no 1 pp 116ndash125 2012

[17] A A Bodour and R M Miller-Maier ldquoApplication of a mod-ified drop-collapse technique for surfactant quantitation andscreening of biosurfactant-producingmicroorganismsrdquo Journalof Microbiological Methods vol 32 no 3 pp 273ndash280 1998

[18] D R Cameron D G Cooper and R J Neufeld ldquoThe manno-protein of Saccharomyces cerevisiae is an effective bioemulsi-fierrdquoApplied and Environmental Microbiology vol 54 no 6 pp1420ndash1425 1988

[19] B B Neto I S Scarminio and R E Bruns Planejamento eotimizacao de experimentos 1995

[20] M A Luna-Velasco F Esparza-Garcıa R O Canızares-Vil-lanueva and R Rodrıguez-Vazquez ldquoProduction and proper-ties of a bioemulsifier synthesized by phenanthrene-degradingPenicillium sprdquo Process Biochemistry vol 42 no 3 pp 310ndash3142007

[21] M M Camargo-de-Morais S A Ramos M C Pimentel andJ Morais ldquoProduction of an extracellular polysaccharide withemulsifier properties by Penicillium citrinumrdquoWorld Journal ofMicrobiology and Biotechnology vol 19 pp 191ndash194 2003

[22] S Askolin T Nakari-Setala and M Tenkanen ldquoOverproduc-tion purification and characterization of the Trichodermareesei hydrophobin HFBIrdquo Applied Microbiology and Biotech-nology vol 57 no 1-2 pp 124ndash130 2001

[23] Z A Raza M S Khan and ZM Khalid ldquoEvaluation of distantcarbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutantrdquo Process Biochemistryvol 42 no 4 pp 686ndash692 2007

[24] H-S Kim B-D Yoon C-H Lee et al ldquoProduction andproperties of a lipopeptide biosurfactant from Bacillus subtilisC9rdquo Journal of Fermentation and Bioengineering vol 84 no 1pp 41ndash46 1997

[25] R S Makkar and S S Cameotra ldquoProduction of biosurfac-tant at mesophilic and thermophilic conditions by a strainof Bacillus subtilisrdquo Journal of Industrial Microbiology andBiotechnology vol 20 no 1 pp 48ndash52 1998

[26] IM Banat andD J Desai ldquoMicrobial production of surfactantsand their commercial potentialrdquo Microbiology and MolecularBiology Reviews vol 61 no 1 pp 47ndash64 1997

[27] A Decesaro M R Rigon A Thome and L M CollaldquoProducao de biossurfactantes por microrganismos isolados desolo contaminado com oleo dieselrdquo Quımica Nova vol 36 no7 pp 947ndash954 2013

[28] H-S Kim J-W Jeon B-H Kim C-Y Ahn H-M Oh and B-D Yoon ldquoExtracellular production of a glycolipid biosurfactantmannosylerythritol lipid by Candida sp SY16 using fed-batchfermentationrdquo Applied Microbiology and Biotechnology vol 70no 4 pp 391ndash396 2006

[29] G L Castiglioni G Stanescu L A O Rocha and J A VCosta ldquoActa scientiarum analytical modeling and numericaloptimization of the biosurfactants production in solid-statefermentation by aspergillus fumigatusrdquo Acta Scientiarum vol36 no 1 pp 61ndash67 2014

[30] G S Kiran T A Hema R Gandhimathi et al ldquoOptimizationand production of a biosurfactant from the sponge-associatedmarine fungus Aspergillus ustus MSF3rdquo Colloids and SurfacesB Biointerfaces vol 73 no 2 pp 250ndash256 2009

[31] L M Acioly A Silveira M Anjos et al ldquoBiosurfactantproduction by Mucor circinelloides using apple peel vegetableoil and corn steep liquor as substraterdquo in Microbes in AppliedResearchndashCurrents Advances and Challenges A Mendez-VilasEd pp 344ndash347 World Science New York NY USA 2012

[32] A S Monteiro M R Q Bonfim V S Domingues et alldquoIdentification and characterization of bioemulsifier-producingyeasts isolated from effluents of a dairy industryrdquo BioresourceTechnology vol 101 no 14 pp 5186ndash5193 2010

8 International Journal of Microbiology

[33] A Khopade B Ren X-Y Liu K Mahadik L Zhang andC Kokare ldquoProduction and characterization of biosurfactantfrom marine Streptomyces species B3rdquo Journal of Colloid andInterface Science vol 367 no 1 pp 311ndash318 2012

[34] CMullinganDCooper andRNeufeld ldquoSelection ofmicrobesproducing biosurfactants in media without hydrocarbonsrdquoJournal of Fermentation Technology vol 4 pp 311ndash314 1984

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Page 6: Production of Biosurfactants by Soil Fungi Isolated from ...downloads.hindawi.com/journals/ijmicro/2018/5684261.pdf · ResearchArticle Production of Biosurfactants by Soil Fungi Isolated

6 International Journal of Microbiology

Table 4 Analysis of variance for evaluating the statistical significance of the model for biosurfactant production by Penicillium 8CC2 (1198772 =918529)

Source Sum of squares Df Mean of Squares 119865 ratio 119875 valueA soybean oil 105063 1 105063 3602 00093B yeast extract 915063 1 915063 31374 00004C pH 855625 1 855625 2934 00123D time 370563 1 370563 12705 00015BD 217563 1 217563 7459 00033CD 855625 1 855625 2934 00123Lack of fit 149075 10 149075 511 01030Pure error 875 3 875Total 19372 19

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +heating

Tween 80 Tween 80 +heating

Emul

sifica

tion

inde

x (

)

(a)

010203040506070

Biosurfactant(Penicillium

8CC2)

Biosurfactant +NaCl (30 wv)

Tween 80 Tween 80 +NaCl (30 wv)

Emul

sifica

tion

inde

x (

)

(b)

Figure 3 Stability of biosurfactants from Penicillium 8CC2 under different physical conditions (a) after heating 100∘C (60min) and (b) withthe addition of NaCl (30wv) The synthetic surfactant Tween 80 (1wv) was used as a control substance in these assays

isolated from amarine sponge showed optimized productionat 120 h in Sabouraud dextrose broth [30]

The stability of the biosurfactant was evaluated at hightemperatures and high ionic strengths After subjecting thebiosurfactant to a temperature of 100∘C for 60min therewas no verified change in biosurfactant activity This resultdemonstrates the potential utility of the biosurfactant in thefood pharmaceutical and cosmetic industries where heatsterility is of great importance [32 33] In the case of ionicstrength there was a small reduction in the emulsificationability of Penicillium 8CC2 when NaCl (30) was added buta stable emulsion could still be maintained The commercialemulsifier Tween 80 used as a control did not remain stablewhen NaCl was added This result demonstrates that thebiosurfactant produced by Penicillium 8CC2 can be used inhigh salt-concentration conditions for example in tertiaryoil recovery activities

This study verified stability parameters for only onefungus from the 61 potential biosurfactant-producing fungithus ignoring possible potentiality in many others includingsome with high levels of emulsification In the stability testswe could have assessed the influence of pH The resultsdemonstrate that biotechnological approaches can be usedto improve biosurfactant production by Penicillium 8CC2The current results suggest the potential usefulness of thebiosurfactant produced by a filamentous fungus isolated fromsoil samples from the Amazon region

The data from the present work is important since asignificative number of isolates (100 cultures isolated fromsoil samples from the Amazon) were investigated for theproduction of biosurfactant The analytical methodologyused in the screening of biosurfactant producers did notquantify the biosurfactant directly in the culture filtratenowadays the screening methods have been developed thatrely on the interfacial activity of the biosurfactants but do notmeasure it directly We used methodology that quantifies theability of the biosurfactant present in the culture filtrate toproduce emulsificationwith toluene ldquoemulsification indexrdquo asclassically described by Mullingan et al [34] Unfortunatelyas a work limitation during the experimental conditions itwas not possible to identify the species of Penicillium 8CC2and the chemical composition of the biosurfactant Literaturedoes not have description of chemical composition of thebiosurfactants produced by Penicillium sp and further worksare necessary to elucidate it

The development for screening microbes from thousandsof potentially active organisms and the subsequent evaluationof surface activity holds the key to the discovery of newbiosurfactants or production strains

Conflicts of Interest

The authors declare that they have no conflicts of interest

International Journal of Microbiology 7

References

[1] Z A Raza A Rehman M T Hussain et al ldquoProduction ofrhamnolipid surfactant and its application in bioscouring ofcotton fabricrdquo Carbohydrate Research vol 391 no 1 pp 97ndash1052014

[2] I M Banat A Franzetti I Gandolfi et al ldquoMicrobial biosur-factants production applications and future potentialrdquo AppliedMicrobiology andBiotechnology vol 87 no 2 pp 427ndash444 2010

[3] M Pacwa-Płociniczak G A Płaza Z Piotrowska-Seget andS S Cameotra ldquoEnvironmental applications of biosurfactantsrecent advancesrdquo International Journal of Molecular Sciencesvol 12 no 1 pp 633ndash654 2011

[4] P Das S Mukherjee and R Sen ldquoImproved bioavailabilityand biodegradation of a model polyaromatic hydrocarbonby a biosurfactant producing bacterium of marine originrdquoChemosphere vol 72 no 9 pp 1229ndash1234 2008

[5] M Nitschke andGM Pastore ldquoBiossurfactantes propriedadese aplicacoesrdquo Quımica Nova vol 25 no 5 pp 772ndash776 2002

[6] A Rehman Z A Raza Saif-ur-Rehman et al ldquoSynthesis anduse of self-assembled rhamnolipid microtubules as templatesfor gold nanoparticles assembly to form gold microstructuresrdquoJournal of Colloid and Interface Science vol 347 no 2 pp 332ndash335 2010

[7] G L Castiglioni T E Bertolin and J A Costa ldquoProducao debiossurfactante por Aspergillus fumigatus utilizando resıduosagroindustriais como substratordquo Quımica Nova vol 32 no 2pp 292ndash295 2009

[8] Z Velioglu and R O Urek ldquoBiosurfactant production byPleurotus ostreatus in submerged and solid-state fermentationsystemsrdquo Turkish Journal of Biology vol 39 no 1 pp 160ndash1662015

[9] J L Adrio and A L Demain ldquoFungal biotechnologyrdquo Interna-tional Microbiology vol 6 no 3 pp 191ndash199 2003

[10] G C Fontes P F Amaral and M A Coelho ldquoProducao debiossurfactante por levedurardquo Quımica Nova vol 31 no 8 pp2091ndash2099 2008

[11] M Pirollo Estudo da Producao de Biossurfactantes UtilizandoHidrocarbonetos Universidade Estadual Paulista Sao PauloBrazil 2006

[12] RThavasi S Jayalakshmi T Balasubramanian and IM BanatldquoBiosurfactant production by Corynebacterium kutscheri fromwastemotor lubricant oil and peanut oil cakerdquo Letters in AppliedMicrobiology vol 45 no 6 pp 686ndash691 2007

[13] K Pattanathu M Rahman and M Gapke ldquoProductioncharacterisation and applications of biosurfactants - ReviewrdquoBiotechnology vol 2 pp 360ndash370 2008

[14] C Lacaz E Porto and J Martins Martins MicrobiologiaMedica Fungos Actinomicetos e Algas de Interesse Medico vol33 Sarvier Sao Paulo Brazil 8th edition 2001

[15] H L Barnett and B B Hunter IIlustrated Genera of ImperfectFungi vol 64 Burgess Publishing Co 4th edition 1998

[16] F R Accorsini M J R Mutton E G M Lemos and MBenincasa ldquoBiosurfactants production by yeasts using soybeanoil and glycerol as low cost substraterdquo Brazilian Journal ofMicrobiology vol 43 no 1 pp 116ndash125 2012

[17] A A Bodour and R M Miller-Maier ldquoApplication of a mod-ified drop-collapse technique for surfactant quantitation andscreening of biosurfactant-producingmicroorganismsrdquo Journalof Microbiological Methods vol 32 no 3 pp 273ndash280 1998

[18] D R Cameron D G Cooper and R J Neufeld ldquoThe manno-protein of Saccharomyces cerevisiae is an effective bioemulsi-fierrdquoApplied and Environmental Microbiology vol 54 no 6 pp1420ndash1425 1988

[19] B B Neto I S Scarminio and R E Bruns Planejamento eotimizacao de experimentos 1995

[20] M A Luna-Velasco F Esparza-Garcıa R O Canızares-Vil-lanueva and R Rodrıguez-Vazquez ldquoProduction and proper-ties of a bioemulsifier synthesized by phenanthrene-degradingPenicillium sprdquo Process Biochemistry vol 42 no 3 pp 310ndash3142007

[21] M M Camargo-de-Morais S A Ramos M C Pimentel andJ Morais ldquoProduction of an extracellular polysaccharide withemulsifier properties by Penicillium citrinumrdquoWorld Journal ofMicrobiology and Biotechnology vol 19 pp 191ndash194 2003

[22] S Askolin T Nakari-Setala and M Tenkanen ldquoOverproduc-tion purification and characterization of the Trichodermareesei hydrophobin HFBIrdquo Applied Microbiology and Biotech-nology vol 57 no 1-2 pp 124ndash130 2001

[23] Z A Raza M S Khan and ZM Khalid ldquoEvaluation of distantcarbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutantrdquo Process Biochemistryvol 42 no 4 pp 686ndash692 2007

[24] H-S Kim B-D Yoon C-H Lee et al ldquoProduction andproperties of a lipopeptide biosurfactant from Bacillus subtilisC9rdquo Journal of Fermentation and Bioengineering vol 84 no 1pp 41ndash46 1997

[25] R S Makkar and S S Cameotra ldquoProduction of biosurfac-tant at mesophilic and thermophilic conditions by a strainof Bacillus subtilisrdquo Journal of Industrial Microbiology andBiotechnology vol 20 no 1 pp 48ndash52 1998

[26] IM Banat andD J Desai ldquoMicrobial production of surfactantsand their commercial potentialrdquo Microbiology and MolecularBiology Reviews vol 61 no 1 pp 47ndash64 1997

[27] A Decesaro M R Rigon A Thome and L M CollaldquoProducao de biossurfactantes por microrganismos isolados desolo contaminado com oleo dieselrdquo Quımica Nova vol 36 no7 pp 947ndash954 2013

[28] H-S Kim J-W Jeon B-H Kim C-Y Ahn H-M Oh and B-D Yoon ldquoExtracellular production of a glycolipid biosurfactantmannosylerythritol lipid by Candida sp SY16 using fed-batchfermentationrdquo Applied Microbiology and Biotechnology vol 70no 4 pp 391ndash396 2006

[29] G L Castiglioni G Stanescu L A O Rocha and J A VCosta ldquoActa scientiarum analytical modeling and numericaloptimization of the biosurfactants production in solid-statefermentation by aspergillus fumigatusrdquo Acta Scientiarum vol36 no 1 pp 61ndash67 2014

[30] G S Kiran T A Hema R Gandhimathi et al ldquoOptimizationand production of a biosurfactant from the sponge-associatedmarine fungus Aspergillus ustus MSF3rdquo Colloids and SurfacesB Biointerfaces vol 73 no 2 pp 250ndash256 2009

[31] L M Acioly A Silveira M Anjos et al ldquoBiosurfactantproduction by Mucor circinelloides using apple peel vegetableoil and corn steep liquor as substraterdquo in Microbes in AppliedResearchndashCurrents Advances and Challenges A Mendez-VilasEd pp 344ndash347 World Science New York NY USA 2012

[32] A S Monteiro M R Q Bonfim V S Domingues et alldquoIdentification and characterization of bioemulsifier-producingyeasts isolated from effluents of a dairy industryrdquo BioresourceTechnology vol 101 no 14 pp 5186ndash5193 2010

8 International Journal of Microbiology

[33] A Khopade B Ren X-Y Liu K Mahadik L Zhang andC Kokare ldquoProduction and characterization of biosurfactantfrom marine Streptomyces species B3rdquo Journal of Colloid andInterface Science vol 367 no 1 pp 311ndash318 2012

[34] CMullinganDCooper andRNeufeld ldquoSelection ofmicrobesproducing biosurfactants in media without hydrocarbonsrdquoJournal of Fermentation Technology vol 4 pp 311ndash314 1984

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Page 7: Production of Biosurfactants by Soil Fungi Isolated from ...downloads.hindawi.com/journals/ijmicro/2018/5684261.pdf · ResearchArticle Production of Biosurfactants by Soil Fungi Isolated

International Journal of Microbiology 7

References

[1] Z A Raza A Rehman M T Hussain et al ldquoProduction ofrhamnolipid surfactant and its application in bioscouring ofcotton fabricrdquo Carbohydrate Research vol 391 no 1 pp 97ndash1052014

[2] I M Banat A Franzetti I Gandolfi et al ldquoMicrobial biosur-factants production applications and future potentialrdquo AppliedMicrobiology andBiotechnology vol 87 no 2 pp 427ndash444 2010

[3] M Pacwa-Płociniczak G A Płaza Z Piotrowska-Seget andS S Cameotra ldquoEnvironmental applications of biosurfactantsrecent advancesrdquo International Journal of Molecular Sciencesvol 12 no 1 pp 633ndash654 2011

[4] P Das S Mukherjee and R Sen ldquoImproved bioavailabilityand biodegradation of a model polyaromatic hydrocarbonby a biosurfactant producing bacterium of marine originrdquoChemosphere vol 72 no 9 pp 1229ndash1234 2008

[5] M Nitschke andGM Pastore ldquoBiossurfactantes propriedadese aplicacoesrdquo Quımica Nova vol 25 no 5 pp 772ndash776 2002

[6] A Rehman Z A Raza Saif-ur-Rehman et al ldquoSynthesis anduse of self-assembled rhamnolipid microtubules as templatesfor gold nanoparticles assembly to form gold microstructuresrdquoJournal of Colloid and Interface Science vol 347 no 2 pp 332ndash335 2010

[7] G L Castiglioni T E Bertolin and J A Costa ldquoProducao debiossurfactante por Aspergillus fumigatus utilizando resıduosagroindustriais como substratordquo Quımica Nova vol 32 no 2pp 292ndash295 2009

[8] Z Velioglu and R O Urek ldquoBiosurfactant production byPleurotus ostreatus in submerged and solid-state fermentationsystemsrdquo Turkish Journal of Biology vol 39 no 1 pp 160ndash1662015

[9] J L Adrio and A L Demain ldquoFungal biotechnologyrdquo Interna-tional Microbiology vol 6 no 3 pp 191ndash199 2003

[10] G C Fontes P F Amaral and M A Coelho ldquoProducao debiossurfactante por levedurardquo Quımica Nova vol 31 no 8 pp2091ndash2099 2008

[11] M Pirollo Estudo da Producao de Biossurfactantes UtilizandoHidrocarbonetos Universidade Estadual Paulista Sao PauloBrazil 2006

[12] RThavasi S Jayalakshmi T Balasubramanian and IM BanatldquoBiosurfactant production by Corynebacterium kutscheri fromwastemotor lubricant oil and peanut oil cakerdquo Letters in AppliedMicrobiology vol 45 no 6 pp 686ndash691 2007

[13] K Pattanathu M Rahman and M Gapke ldquoProductioncharacterisation and applications of biosurfactants - ReviewrdquoBiotechnology vol 2 pp 360ndash370 2008

[14] C Lacaz E Porto and J Martins Martins MicrobiologiaMedica Fungos Actinomicetos e Algas de Interesse Medico vol33 Sarvier Sao Paulo Brazil 8th edition 2001

[15] H L Barnett and B B Hunter IIlustrated Genera of ImperfectFungi vol 64 Burgess Publishing Co 4th edition 1998

[16] F R Accorsini M J R Mutton E G M Lemos and MBenincasa ldquoBiosurfactants production by yeasts using soybeanoil and glycerol as low cost substraterdquo Brazilian Journal ofMicrobiology vol 43 no 1 pp 116ndash125 2012

[17] A A Bodour and R M Miller-Maier ldquoApplication of a mod-ified drop-collapse technique for surfactant quantitation andscreening of biosurfactant-producingmicroorganismsrdquo Journalof Microbiological Methods vol 32 no 3 pp 273ndash280 1998

[18] D R Cameron D G Cooper and R J Neufeld ldquoThe manno-protein of Saccharomyces cerevisiae is an effective bioemulsi-fierrdquoApplied and Environmental Microbiology vol 54 no 6 pp1420ndash1425 1988

[19] B B Neto I S Scarminio and R E Bruns Planejamento eotimizacao de experimentos 1995

[20] M A Luna-Velasco F Esparza-Garcıa R O Canızares-Vil-lanueva and R Rodrıguez-Vazquez ldquoProduction and proper-ties of a bioemulsifier synthesized by phenanthrene-degradingPenicillium sprdquo Process Biochemistry vol 42 no 3 pp 310ndash3142007

[21] M M Camargo-de-Morais S A Ramos M C Pimentel andJ Morais ldquoProduction of an extracellular polysaccharide withemulsifier properties by Penicillium citrinumrdquoWorld Journal ofMicrobiology and Biotechnology vol 19 pp 191ndash194 2003

[22] S Askolin T Nakari-Setala and M Tenkanen ldquoOverproduc-tion purification and characterization of the Trichodermareesei hydrophobin HFBIrdquo Applied Microbiology and Biotech-nology vol 57 no 1-2 pp 124ndash130 2001

[23] Z A Raza M S Khan and ZM Khalid ldquoEvaluation of distantcarbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutantrdquo Process Biochemistryvol 42 no 4 pp 686ndash692 2007

[24] H-S Kim B-D Yoon C-H Lee et al ldquoProduction andproperties of a lipopeptide biosurfactant from Bacillus subtilisC9rdquo Journal of Fermentation and Bioengineering vol 84 no 1pp 41ndash46 1997

[25] R S Makkar and S S Cameotra ldquoProduction of biosurfac-tant at mesophilic and thermophilic conditions by a strainof Bacillus subtilisrdquo Journal of Industrial Microbiology andBiotechnology vol 20 no 1 pp 48ndash52 1998

[26] IM Banat andD J Desai ldquoMicrobial production of surfactantsand their commercial potentialrdquo Microbiology and MolecularBiology Reviews vol 61 no 1 pp 47ndash64 1997

[27] A Decesaro M R Rigon A Thome and L M CollaldquoProducao de biossurfactantes por microrganismos isolados desolo contaminado com oleo dieselrdquo Quımica Nova vol 36 no7 pp 947ndash954 2013

[28] H-S Kim J-W Jeon B-H Kim C-Y Ahn H-M Oh and B-D Yoon ldquoExtracellular production of a glycolipid biosurfactantmannosylerythritol lipid by Candida sp SY16 using fed-batchfermentationrdquo Applied Microbiology and Biotechnology vol 70no 4 pp 391ndash396 2006

[29] G L Castiglioni G Stanescu L A O Rocha and J A VCosta ldquoActa scientiarum analytical modeling and numericaloptimization of the biosurfactants production in solid-statefermentation by aspergillus fumigatusrdquo Acta Scientiarum vol36 no 1 pp 61ndash67 2014

[30] G S Kiran T A Hema R Gandhimathi et al ldquoOptimizationand production of a biosurfactant from the sponge-associatedmarine fungus Aspergillus ustus MSF3rdquo Colloids and SurfacesB Biointerfaces vol 73 no 2 pp 250ndash256 2009

[31] L M Acioly A Silveira M Anjos et al ldquoBiosurfactantproduction by Mucor circinelloides using apple peel vegetableoil and corn steep liquor as substraterdquo in Microbes in AppliedResearchndashCurrents Advances and Challenges A Mendez-VilasEd pp 344ndash347 World Science New York NY USA 2012

[32] A S Monteiro M R Q Bonfim V S Domingues et alldquoIdentification and characterization of bioemulsifier-producingyeasts isolated from effluents of a dairy industryrdquo BioresourceTechnology vol 101 no 14 pp 5186ndash5193 2010

8 International Journal of Microbiology

[33] A Khopade B Ren X-Y Liu K Mahadik L Zhang andC Kokare ldquoProduction and characterization of biosurfactantfrom marine Streptomyces species B3rdquo Journal of Colloid andInterface Science vol 367 no 1 pp 311ndash318 2012

[34] CMullinganDCooper andRNeufeld ldquoSelection ofmicrobesproducing biosurfactants in media without hydrocarbonsrdquoJournal of Fermentation Technology vol 4 pp 311ndash314 1984

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Page 8: Production of Biosurfactants by Soil Fungi Isolated from ...downloads.hindawi.com/journals/ijmicro/2018/5684261.pdf · ResearchArticle Production of Biosurfactants by Soil Fungi Isolated

8 International Journal of Microbiology

[33] A Khopade B Ren X-Y Liu K Mahadik L Zhang andC Kokare ldquoProduction and characterization of biosurfactantfrom marine Streptomyces species B3rdquo Journal of Colloid andInterface Science vol 367 no 1 pp 311ndash318 2012

[34] CMullinganDCooper andRNeufeld ldquoSelection ofmicrobesproducing biosurfactants in media without hydrocarbonsrdquoJournal of Fermentation Technology vol 4 pp 311ndash314 1984

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom

Page 9: Production of Biosurfactants by Soil Fungi Isolated from ...downloads.hindawi.com/journals/ijmicro/2018/5684261.pdf · ResearchArticle Production of Biosurfactants by Soil Fungi Isolated

Hindawiwwwhindawicom

International Journal of

Volume 2018

Zoology

Hindawiwwwhindawicom Volume 2018

Anatomy Research International

PeptidesInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Journal of Parasitology Research

GenomicsInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom

The Scientific World Journal

Volume 2018

Hindawiwwwhindawicom Volume 2018

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Neuroscience Journal

Hindawiwwwhindawicom Volume 2018

BioMed Research International

Cell BiologyInternational Journal of

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Biochemistry Research International

ArchaeaHindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Genetics Research International

Hindawiwwwhindawicom Volume 2018

Advances in

Virolog y Stem Cells International

Hindawiwwwhindawicom Volume 2018

Hindawiwwwhindawicom Volume 2018

Enzyme Research

Hindawiwwwhindawicom Volume 2018

International Journal of

MicrobiologyHindawiwwwhindawicom

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwwwhindawicom