Elisa Korenblum1*, Rachel Chavão1, Lauro M. de Souza3, Guilherme Sassaki3, Érika Valoni 2, Gina Sebastián Vasquez 2, Thaïs Souto Padrón1, Eliana Barreto-Bergter 1& Lucy Seldin1

1

AMS T6-5 was obtained by ammonium sulfate precipitation and the crude protein extract applied on a DE-52 ion exchange chromatography to achieve a purified AMS. The other substance, AMS H2O-1, was extracted with chloroform–methanol (2:1, v/v) from the culture supernatant adjusted to pH 2.0 with concentrated HCl and partially purified on a silica-gel 60 column chromatography and eluted with chloroform–methanol 9:1 v/v. The structure of the AMS H2O-1 was determined

Instituto de Microbiologia Prof. Paulo de Góes, UFRJ, Rio de Janeiro, RJ 1CENPES-PETROBRAS 2

Departamento de Bioquimica, Universidade Federal do Paraná, PR (UFPR), Brasil 3

*elisa@micro.ufrj.br

INTRODUCTION METHODS

Biogenic hydrogen sulfide (H2S) produced by sulfate reducing bacteria (SRB) is often found associatedwith petroleum secondary recovery. When seawater is injected into the reservoir, souring is typicallyobserved. This metabolite of sulfate reduction also promotes corrosion of metal surface (Figure 1). A cost

ti ti f billi f d ll t i B il d t t i l l t d it i d

Isolation and purification of surfactin-like molecules from Bacillus strains• AMS T6-5 was obtained by ammonium sulfate precipitation and the crude protein extract applied on a DE-52ion exchange chromatography to achieve a purified AMS.• AMS H2O-1 was extracted with chloroform–methanol (2:1 v/v) from the culture supernatant adjusted to pH 2 0estimation of billions of dollars per year are spent in Brazil due to material lost and monitoring and

preventing sulfide production, including biocide treatment. For that reason, alternatives of preventing andcontrolling the biogenic H2S, for example the search for new antimicrobial substances (AMS), have beeninvestigated to reduce the cost and the environmental impact. Two AMS produced by Bacillus strains,which were previously demonstrated as capable of inhibition SRB growth (Figure 2) and removing SRBbiofilm, were partially characterized.

AMS H2O 1 was extracted with chloroform methanol (2:1, v/v) from the culture supernatant adjusted to pH 2.0with concentrated HCl and partially purified on a silica-gel 60 column chromatography and eluted withchloroform–methanol 9:1 v/v (Monteiro et al. 2007. Chemistry and Physics of Lipids 147: 1–13)• Mass spectrometry (MS) was acquired in an ESI-MS source, with a triple quadrupole, Quattro LC (Micromass-Waters), at atmospheric pressure ionization (API). For detecting the positive ions, the energy setup was: cone35V (low), 63V (medium), 85V (high) and the capillary at 2.6kV.Nitrogen was used as nebuliser and desolvationgas, the source block temperature was held at 80 °C and desolvation gas at 200°C. For tandem-MSexperiments, each precursor ion was selected by MS1 and fragmented by collision-induced dissociation(CID)with argon as collision gas (de Souza et al.,2008. J. Chromatography 1207:101-109).

Transmission Electron Microscopy• Transmission electron microscopic (TEM) examination was used to study the biocidal action of AMS Afterincubation (24h) of the SRB suspensions with SAM T6-5 at 30°C, 15% glutaraldehyde (pH 7.0) in 0.1 M sodiumcacodylate buffer (SCB) was added to each tube to minimize the impact of salts on bacterial ultrastructure duringcentrifugation, and the suspension was centrifuged (2,360 g for 5 min at 4°C) with a Biofuge 17R centrifuge

RESULTS RESULTS

Figure 2: Inhibition zone of SRB growth by AMS T6-5. Korenblum, et al. 2005. J. Appl. Microbiol.

98(3): 667-75.Figure 1: Schematic representation of the

sulfate-reducing reaction

g , p g ( , g ) g g(Heraeus Sepatech GmbH, Osterode, Germany).• The pellet was then fixed with a mixture of 3% glutaraldehyde, 0.2% ruthenium red, and 0.05 M CaCl2, also in0.1 M SCB (pH 7.3), for 2.5 h at room temperature. Gels of 1 to 2 mm3 were prepared by adding 10% bovineserum albumin and 5% glutaraldehyde in SCB to the pellet. After thorough rinsing with 0.1 M SCB, the gels werepostfixed with 2% osmium tetroxide in 0.1 M SCB for 2 h at room temperature, dehydrated by using a series ofincreasing ethanol concentrations, and embedded in PolyBed 812 resin. Ultrathin (90-nm) sections wereobserved with a transmission electron microscope (model FEI Morgagni 268), after contrasting with uranylacetate and lead citrate. For each treatment, the test was repeated twice, and at least two blocks by replicatewere examined.

The structure of the AMS H O 1 was determined by electrospray ionization mass P d t t f f ti lik l l id tifi d i B ill i t d

Figure 3: MS/MS spectra

The structure of the AMS H2O-1 was determined by electrospray ionization mass spectrometry (ESI-MS) and identified as a surfactin-like lipopeptide.

Proposed structure of a surfactin-like molecule identified in Bacillus using tandem mass spectrometry (MS/MS)

g pobtained from the

protonated surfactins (a) [M+H]+ at m/z 1037 and (b)

[M+H]+ at m/z 1023.

Peptide fragment ions * [M+H]+ m/z 1037 [M+H]+ m/z 1023[H-R2R7-OH+H]+ 685 685[H-R5R7-OH+H]+ 360 360[H-R6R7 -OH+H]+ 245 245b1 (- H2O) 370 356b2 (- H2O) 483 469b3 (- H2O; -CO) 596 582b4 695 681

Table 1: Main fragment ions observed for the

RESULTS

Bacillus species are known to produce efficiently AMS that show a wide range of antimicrobial activity. The AMS T6-5 and H2O-1 AMS T6-5 treated and untreated cells observed by TEM confirmed that

the treated D alaskensis cells were damaged showing low electron

CONCLUSION

4

Y6b3 (-CO) 227 227Y5b5 (-H2O) 328 328Y6b5 (-H2O;-CO) 441 441Y6b6 (-CO) 554 554

* The nomenclature of the peptide fragment íons correponds to that proposed by Biemann K. 1990. Methods in Enzymology, 193: 455

surfactin-like molecules, β-OH-C15 (m/z 1037) and

β-OH-C14 (m/z 1023) isolated from Bacillus

strains

have been studied as an alternative treatment to prevent and control the SRB growth and also H2S production. In this work, surfactin-like molecules were isolated and their structure partially elucidated.

The application of tandem mass spectrometry (MS/MS) to analyse separately the components of a complex mixture was of great help in this study as this type of natural products is usually difficult to obtain as a single homogeneous species from bacterial strains

the treated D. alaskensis cells were damaged, showing low electron dense regions and membrane projections in the cytoplasm.

Figure 3: Ultrastructural morphologyof D. alaskensis treated with differentconcentrations of AMS T6-5.(A) D. alaskensis cells treated with

AMS T6-5 (0.5 ml of the extract):10% of affected cells (showingmembrane alterations or lowelectron dense citosol.

(B) D alaskensis cells treated with AMS strains. The collision-induced dissociation of protonated molecules allows

the peptide sequence and the length of the fatty acyl part of the molecules to be established. In the future, GC/MS methods will be required for the identification of isomeric lipid side chains.

(B) D. alaskensis cells treated with AMST6-5 (1.0 ml of the extract): 40% ofaffected cells (showing membranealterations or low electron densecitosol.