Pseudallescheria boydii and Meyerozyma guilliermondii

Environmental Science and Pollution Research https://doi.org/10.1007/s11356-018-3015-x

RESEARCH ARTICLE

Pseudallescheria boydii and Meyerozyma guilliermondii: behavior of deteriogenic fungi during simulated storage of diesel, biodiesel, and B10 blend in Brazil Gabriela Boelter 1 & Juciana Clarice Cazarolli 1 & Sabrina Anderson Beker 1 & Patrícia Dörr de Quadros 1 & Camila Correa 2 & Marco Flôres Ferrão 2 & Carolina Faganello Galeazzi 2 & Tânia Mara Pizzolato 2 & Fátima Menezes Bento 1 Received: 4 February 2018 / Accepted: 20 August 2018 # Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Due to their renewable and sustainable nature, biodiesel blends boost studies predicting their stability during storage. Besides chemical degradation, biodiesel is more susceptible to biodegradation due to its raw composition. The aim of this work was to evaluate the deteriogenic potential (growth and degradation) of Pseudallescheria boydii and Meyerozyma guilliermondii in degrading pure diesel (B0), pure biodiesel (B100), and a B10 blend in mineral medium during storage. The biodeterioration susceptibility at different fuel ratios and in BH minimal mineral medium were evaluated. The biomass measurements of P. boydii during 45 days indicated higher biomass production in the B10 blend. The growth curve of M. guilliermondii showed similar growth in B10 and B100. Although there was no significant production of biosurfactant, lipase production was detected in the tributyrin agar medium of both microorganisms. The main compounds identified in the aqueous phase by GC-MS were alcohols, esters, acids, sulfur, ketones, and phenols. The results showed that P. boydii grew at the expense of fuels, degrading biodiesel esters, and diesel hydrocarbons. M. guilliermondii grew in B100 and B10; however, degradation was not detected. Keywords Biomass . B10 . Nuclear magnetic resonance . Gas chromatography mass spectrometry . Fungal degradation . Biodeterioration

Introduction Oil-derived fuels, such as diesel and gasoline, have been the world’s leading energy source. However, the search for more sustainable and environmentally friendly alternatives has

encouraged researches into the biodiesel use. Brazil has been able to use biofuels in times of economic crises and booms to overcome difficulties and improve its vehicular energy matrix (Novato and Lacerda 2017). On this regard, the RenovaBio policy will ensure predictability for the expansion of production

Responsible editor: Philippe Garrigues * Gabriela Boelter [email protected]

Carolina Faganello Galeazzi [email protected] Tânia Mara Pizzolato [email protected]

Juciana Clarice Cazarolli [email protected]

Fátima Menezes Bento [email protected]

Sabrina Anderson Beker [email protected] Patrícia Dörr de Quadros [email protected] Camila Correa [email protected] Marco Flôres Ferrão [email protected]

1

LABBIO Biodeterioration of Fuel and Biofuel Laboratory, Institute of Basic Health Sciences, Department of Microbiology, Immunology and Parasitology, Universidade Federal do Rio Grande do Sul, Rua Sarmento Leite, 500, Porto Alegre, RS 90050170, Brazil

2

Chemistry Institute, Department of Inorganic Chemistry, Universidade Federal do Rio Grande do Sul, Av. Bento Gonçalves, 9500, Porto Alegre, RS, Brazil

Environ Sci Pollut Res

and the use of biofuels. According to the ANP (the Brazilian National Agency of Petroleum), there are two types of diesel currently sold in Brazil, marine and road diesel. Diesel oil is classified as grade A (no addition of biodiesel) or grade B (addition of biodiesel). Currently, 10% of blend content is composed of biodiesel (B10) and the perspective is that this percentage will increase in the country (ANP 2016). The pure biodiesel used in Brazil is composed of long-chain fatty acid methyl esters derived from the transesterification of vegetable oils (mainly soya bean-84%) or tallow beef. According to Passman (2013), the microbial diversity of fuel systems includes bacteria, fungi, and archaea. De Azambuja et al. (2017) identified by high-throughput sequencing different phyla isolated from the interfacial biomass formed during simulated storage with different sulfur content diesel. The phyla detected were Firmicutes, Bacteroidetes, Actinobacteria, Ascomycota, Euryarchaeota, Thaumarchaeota, and Crenarchaeota. Some conditions are required to the fuel deterioration by microorganisms: water (accumulated in the tank bottom or dispersed in the oil phase); nutrients such as phosphorus, sulfur, potassium, and calcium; diesel or biodiesel as a carbon source; oxygen; and appropriate temperature and pH (Hill 1987; Chung et al. 2000; Passman 2003, 2013). The presence of a microbial community with the capacity of using fuel as a carbon source to produce enzymes is essential to biodegradation occurrence. Biosurfactants are usually a group of secondary metabolites that reduce the surface and interfacial tension properties which some microorganisms are capable to produce, contributing on the biodeterioration (Perfumo et al. 2009). Several groups of enzymes produced by deteriogenic microorganisms act in the degradation of both diesel and biodiesel compounds. In the stage of degradation of fossil fuels, such as diesel, in aerobic situations, we highlight the oxidation process. Oxigenases are involved in the process of activation and incorporation of oxygen into the substrate (Das and Chandran 2011; Pepper et al. 2011). The biodegradation of biodiesel can involve enzymes that act first in the catabolism of esters, conducting the hydrolysis of methyl or ethyl ester. This reaction is catalyzed by a lipase which results in a fatty acid and an alcohol. After this reaction, the fatty acids are incorporated into the microbial cells by processes of β-oxidation (Chapelle 2001; Pepper et al. 2011). The biodegradation of fuels by microorganisms during storage is associated with damage to the final product, equipment, and parts of the fuel system. This process may also cause clogging of filters (press and motor) mainly due to the biomass (biological sludge) accumulated in the oil-water interface (Bento et al. 2004; Cazarolli et al. 2016; Zimmer et al. 2017; De Azambuja et al. 2017). There may be water accumulation in the bottom of trunks during transportation and in the bottom of tanks during storage. Water infiltration can occur due to sealing problems or moisture condensation in the

inner walls of trucks and tanks (Bento et al. 2010; Passman 2013; ASTM 2014). Studies about the microbial composition of biological sludges have demonstrated the importance of deteriogenic filamentous fungi (Ulfig et al. 2006; Bücker et al. 2011; Stanley et al. 2016; Leuchtle et al. 2017; De Azambuja et al. 2017; Bücker et al. 2018). Fungi and yeasts show better performance in degrading oils than bacteria. The advantage of filamentous fungi is related to the presence of hyphae and their large surface area, which allows the penetration of hydrocarbons and charged aggregates of anoxic hydrocarbons into the fungus’ cells (Passman 2003). The use of tools that evaluate the impact of microbial growth in fuel can help creating mitigation measures to avoid the problems derived from microbial contamination. The degradation of esters and hydrocarbons and the production of metabolites by microorganisms are a few of the possible evaluation tools. The aim of this study was to evaluate the deteriogenic potential (growth and degradation) of Pseudallescheria boydii and Meyerozyma guilliermondii in degrading the Brazilian fuels pure diesel (B0), pure biodiesel (B100), and B10 blend in mineral medium during storage.

Materials and methods Fuels Biodiesel (B100) and diesel (B0) S10 were provided by Ipiranga Products of Petroleum Inc. Biodiesel was produced from soybean oil. The B10 blend was prepared in the laboratory. The fuels were sterilized by vacuum filtration in a Kitassato flask with a 0.22-μm pore size membrane (Milipore). Afterwards, the fuels were stored in dark autoclaved bottles to prevent photo-oxidation.

Microorganisms Pseudallescheria boydii strain was isolated from farm soil impacted by oil industry residues (Schultz 2010) and belongs to the laboratory collection. Molecular identification was carried out by the amplification of the ITS-5.8S-ITS2 region using the primers ITS-1 and ITS-4. Afterwards, automatic sequencing was performed on an Amersham MegaBACE1000 system using the standard protocols of the Brazilian Genome Network. The sequences generated by the process were compared to the BLAST database. Inocula were grown in malt agar (g L−1: 30.0 malt extract; 5.0 peptone; 15.0 agar) for 7 days at 30 °C. Suspensions were prepared with distilled water and counted in Neubauer chamber to obtain a final concentration of 104 spores mL−1. The yeast Meyerozyma guilliermondii was isolated from diesel oil by Bento (Bento and Gaylarde 2001).


Identification was carried out by the amplification of the ITS5.8S-ITS2 region using the primers ITS-1 and ITS-4. Afterwards, the strain was submitted to automatic sequencing using an AmershamMegaBACE 1000 system. Inocula were grown in GYMP agar (g L−1: 20.0 glucose; 5.0 yeast extract; 20.0 malt extract; 2.0 monobasic sodium phosphate; 15.0 agar all from Himedia Brand) for 48 h at 28 °C. Suspensions were prepared with distilled water and counted in Neubauer chamber to obtain a final concentration of 102 cells mL−1.

Simulated storage For fuel storage simulations with P. boydii, the aqueous phase was prepared with Bushnell & Haas (BH) mineral medium (g L−1: 1.0 KH2PO4, 0.2MgSO4, 0.02 CaCl2, 1.0 K2HPO4, 0.05 FeCl3, 1.0 NH4NO3) (Bushnell and Haas 1941). B0, B10, and B100 fuels comprised the oily phase as the only source of energy and carbon. Three different ratios were used in the oily/aqueous phase (1:6, 2:3, and 2:1). Five replicates of the mold inoculum were added in 150-mL flasks containing 30 mL of BH mineral medium and 5, 20, or 60 mL of the oily phase respective to ratios 1:6, 2:3, and 2:1. Non-inoculated microcosms and the BH mineral medium with inoculum were used as control conditions. The flasks were stored at 30 °C for 45 days. For storage simulations with M. guilliermondii, the BH mineral medium was used as the aqueous phase and B10 and B100 were used as the oily phase. For the simulations, 20% of oily phase (6 mL fuel and 30 mL BH mineral medium) was used in triplicates. Non-inoculated microcosms were used as control conditions. The flasks were stored at 28 °C, 120 rpm for 240 h.

Fungal growth After 10, 20, 30, and 45 days, the biomass of P. boydii formed between the oily and the aqueous phase was retained on filter paper (total volume); 10 mL of hexane was added to eliminate all the excess of fuel accumulated during filtration. The biomass was dried to a constant weight and the final weight was taken. After 3, 6, 9, 12, 24, 48, 72, 96, 120, 144, 168, and 240 h, samples of 100 μL of M. guilliermondii were collected and serially diluted in distilled water and enumerated as colonyforming units (CFU) on GYMP agar.

Aqueous phase analysis pH and surface tension The pH of the aqueous phase of both fungus species was determined after all sample times. The measurement of surface tension was determined by a Gibertini tensiometer after 10, 20, 30, and 45 days of incubation for P. boydii and after 72

and 240 h of incubation for M. guilliermondii. The aqueous phase was centrifuged at 6000 rpm for 10 min in quintuplicate (filamentous fungus) and triplicate (yeast) to evaluate conditions in medium without mycelium. GC-MS Sampling using headspace solid-phase microextraction (HSSPME) was performed in glass vials of 15-mL capacity with 2 mL of the aqueous phase. A PDMS fiber was heated to 60 °C for 5 min prior to fiber exposure during 15 min. The analyses were performed in triplicate using GC-MS, Shimadzu, model QP-2010 SE, equipped with a VA-1 column (60 m × 1 μm 0.5 × 0.5 mm). The materials found in the sample were identified based on the NIST library according to their mass models. Only samples with the lowest pH were analyzed. Lipase detection P. boydii: after 10, 20, 30, and 45 days,10 μL of the aqueous phase was inoculated in the center of an orifice made in a Petri dish containing tributyrin agar (g L−1: 2.5 meat peptone, 2.5 casein peptone, 3.0 yeast extract, 20.0 agar, and 10.0 mL L−1 tributyrin) (Carrasco et al. 2012). M. guilliermondii: after all incubation times, samples were stung with a platinum needle. In the tributyrin agar, the hydrolytic activity was identified as a clear zone around the fungus. Emulsification index (E24) For the determination of the emulsification index (E24), 2 mL of centrifuged and non-centrifuged aqueous phase was added to flasks containing 2 mL of oily phase and 200 μL of 0.05% rose bengal solution. Afterwards, the mixture was shaken with a vortex mixer. After 24 h, the measurement of the emulsified column divided by the total height (4 mL) and multiplied by 100 gave the index value (Cooper and Goldenberg 1987).

Fuel analysis FTIR/ATR Triplicates of the oily phase samples of treatments and controls after 0, 20, and 45 days (P. boydii) and 0, 48, 96, 168, and 240 h (M. guilliermondii) were analyzed by FTIR/ATR (Fourier transform infrared with attenuated total reflectance). This standard method is based on the use of a band related to the vibrational mode of the carbonyl group in esters. FTIR spectra were obtained using the Cary 630 spectrophotometer (Agilent Technologies) equipped with a horizontal attenuated total reflectance (HATR). Infrared data were analyzed using PLS-Toolbox (Zimmer et al. 2013).


NMR

Aqueous phase

Biodiesel triplicate samples, which had higher P. boydii biomass from day 20 to day 45, were selected. Spectra were determined on a Bruker device (400 MHz). Approximately 15 mg of each sample was solubilized in 550 μL of 99.8% chloroform-D (Cambridge Isotope Laboratories, Inc.). Spectra were determined at 90° and 3 s of pulse time relaxation (De Azambuja et al. 2017).

Biosurfactant production

Statistical analyses Experiments were carried out using five independent replicates. The results were analyzed by ANOVA with a confidence level of 95%. If ANOVA results were significant, the Duncan test was applied using the software XLSTAT 2015.1.

Results Growth curve Biomass formation was observed during the growth of P. boydii in B0, B10, and B100 during 45 days of evaluation. The biomass averages recovered of the three fuels were significantly different over the 45 days (p < 0.05) (B0 19.95 mg; B10 108.47 mg; and B100 66.13 mg). The results demonstrated the highest dry weight of the fungus when in contact with the B10 blend. When only the influence of the different oily/aqueous phase ratios was analyzed during the biomass formation, different results were observed. At the last day of incubation, there was a higher biomass production in the 1:6 ratio (166.61 mg), followed by ratios 2:3 (45.57 mg) and 2:1 (35.23 mg). Comparing the control condition with only BH mineral medium and inoculum, little biomass growth was observed, reaching the maximum value of 9.0 mg after 45 days. The B10 blend favored significantly (p < 0.05) the fungal growth, and it was independent of the oily/aqueous phase ratio. After 45 days, the fungal growth in B10 was 49% times higher than in B100 and 90% times higher than in B0 (Table 1). During the initial times of M. guilliermondii growth in B10 and in the BH mineral medium, an acclimation phase was observed in the first 9 h of incubation (Fig. 1). After this period, an exponential growth phase was observed up to 48 h, with a three-log increase. After 72 h, there was an increase up to 107 cells mL−1, maintaining the stationary phase until the end of the 240-h experiment. In B100, the acclimation phase was extended up to 12 h of incubation. Afterwards, the yeast population entered in the exponential phase, with the increase of three logs until reaching 106 cells mL−1. From 96 h, stationary phase was reached with 107 cells mL−1.

The surface tension of the aqueous phase in the presence of B0, B10, and B100 decreased over 45 days of experiment with P. boydii; it differed significantly (p < 0.05) between time zero and the final time. Over the 45-day experiment, the mean values reduced from 68.47 mN m−1 to 44.95 mN m−1 in B100, to 48.59 mN m−1 in B10, and to 54.91 mN m−1 in B0. There was no significant difference in the surface tension among treatments concerning the different ratios at the end of the experiment. The only situation in which the treatments differed significantly from the controls was when the aqueous phase was in contact with the B10 blend, indicating values of 48.59 mN m−1 for the treatment and 44.05 mN m−1 for the control. The surface tension measurements during the growth of M. guilliermondii in the aqueous phase of the B10 blend demonstrated a statistical difference (p < 0.05) between initial and final incubation times. The initial surface tension was 52.30 and 41.59 mN m−1 in the final time. However, comparing the surface tension between treatment and control after 240 h of incubation, no differences were seen (p > 0.05). The samples containing B100 as the oily phase differed significantly between the initial and final time, showing values between 50.56 and 40.82 mN m−1, respectively. The analysis between treatment and control at the final incubation time showed no significant difference; the same was observed between surface tension values of B10 and B100.

Change of pH Measurements of pH indicated the production of acid or basic metabolites by the filamentous fungus and the yeast. Isolating the fuel variable in the experiment with P. boydii, the BH mineral medium had a pH reduction from 7.2 to 6.6 in the presence of B10 and from 7.2 to 6.6 in the presence of B100. However, these differences were not statistically significant. In the presence of B0, the pH reduced to 6.9, which was not significantly different from the results found for the B10 and B100 fuels. Comparing the oily and aqueous phase ratios at day 45, the ratio 2:1 had the lowest pH (6.5), followed by 1:6 (6.6) and 2:3 (6.6) (p > 0.05). The control treatments did not show a significant reduction of pH when compared with the initial pH (7.2). Both microcosms, containing only fuel and BH mineral medium and containing only BH mineral medium and inoculum, showed similar results. In the experiments with M. guilliermondii in the B10 blend, pH remained approximately constant, with an average of 6.7. The lowest pH was seen at 168 h (6.1) and the highest at 3 and 9 h (7.1). In the control condition, there was a decrease of initial pH from 7.2 to 6.6 in 96 h, remaining close to the initial value in all other times. The treatment and control conditions

Environ Sci Pollut Res Table 1 Pseudallescheria boydii dried biomass (mg) in different ratios (1:6, 2:3, and 2:1) of aqueous (BH medium) and oily phase (B0 diesel, B10 blend, and B100 biodiesel) during 45 days of experiment B0 2:3

B10 2:1

Time

1:6

B100

Time

1:6

2:3

2:1

Time

1:6

2:3

2:1

0

0.0 ± 0.0*

0.0 ± 0.0

0.0 ± 0.0

0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

0

0.0 ± 0.0

0.0 ± 0.0

0.0 ± 0.0

10

11.2 ± 1.6

24.4 ± 2.7

36.5 ± 2.6

10

43.5 ± 0.7

39.4 ± 2.3

35.7 ± 1.0

10

65.0 ± 2.6

55.2 ± 3.1

59.8 ± 2.3

20 30

15.6 ± 1.3 13.6 ± 2.2

21.5 ± 0.6 22.0 ± 4.0

32.5 ± 0.58 26.0 ± 4.3

20 30

138.3 ± 11.0 42.8 ± 3.6

48.5 ± 2.9 213.3 ± 17.9

92.0 ± 6.1 130.7 ± 6.4

20 30

55.6 ± 8.6 108.8 ± 23.1

50.4 ± 3.4 69.2 ± 11.0

49.4 ± 2.9 69.7 ± 20.6

45

9.0 ± 1.8

13.0 ± 1.0

20.2 ± 3.3

45

335.7 ± 16.9

38.2 ± 2.6

45.0 ± 16.2

45

87.0 ± 25.2

84.0 ± 22.2

44.2 ± 6.18

*Data are expressed as mean ± SD. p values were obtained by ANOVA and Duncan tests

had significant differences (p < 0.05) in the final time of incubation. In the B100, the pH remained constant up to 168 h, increasing only in the 24 h (7.3). At the final time (240 h), the pH decreased to 3.5, differing significantly from the control and the initial time; over time, the average pH was 6.6. In the control experiment, the average pH was 7.0. Comparing the pH of the initial and final times in the treatment and control conditions of the B10 and B100 fuels, only the sample incubated after 240 h in B100 with inoculum had a significant difference.

Lipase production Lipase production by yeast and filamentous fungus was detected by the presence of a translucent halo around the colony (Gopinath et al. 2005). In the control condition, the result was negative for all experimental times. P. boydii and M. guilliermondii showed the ability to produce extracellular lipase by the hydrolysis of tributyrin, a triacylglycerol that forms an opaque emulsion in agar. This surfactant is easily dispersed in the water by stirring and without addition of emulsifier (Gopinath et al. 2013).

Metabolites Using SPME and GC-MS, it was possible to detect which compounds were present in the aqueous phase. The results of the treatments with P. boydii in B10 and B100 from days 20 and 30 are shown since they had the lowest pH. At the final time, samples of M. guilliermondii in the aqueous phase were analyzed. The main compounds identified were alcohols, esters, acids, sulfur, ketones, and phenols (Tables 2, 3, 4, and 5). Fig. 1 Growth curve (CFU mL−1) of Meyerozyma guilliermondii in BH minimum mineral medium containing B10 and B100

Emulsification index (E24) Analyzing the emulsification index, emulsification was observed between the oily and aqueous phase only in M. guilliermondii treatments involving B10 without centrifugation (presence of cells) (59.09%) and B100 in the presence of cells (45.4%).

Environ Sci Pollut Res Table 2 Compounds in the aqueous phase in contact with B10 blend at days 20 and 30 in the ratios 1:6, 2:3, and 2:1 and their respective controls

Sample

Compounds

T20 1:6

Phenol, 2,6-bis(1,1-dimethylethyl)-4-(1-methylpropyl); geranyl formate; phenol, 2,4-bis(1,1-dimethylethyl); benzaldehyde, 2,4-dimethyl Benzaldehyde, 3,5-dimethyl; propanenitrile, 2,2′-azobis[2-methy]; geranyl formate; oxirane, 2-methyl-3-phenyl; nonanoic acid; dodecanoic acid; pentadecanoic acid; phenol, 2,4-bis(1,1-dimethylethyl)

T30 2:3

T30 2:1

1,2,3,7-Tetramethylindole; benzaldehyde, 3,4-dimethyl; acetic acid; propanoic acid; oxirane, 2-methyl-3-pheny; benzene, 1-methyl-2-(2-propenyl); tetralin; propanoic acid, phenol, 2,4-bis(1,1-dimethylethyl)

Control T20 1:6

Oxirane, 2-methyl-3-pheny; phenol, 2,4-bis(1,1-dimethylethyl); benzaldehyde, 2,4-dimethyl 2-Hydroxyethyl propyl sulfide; propanoic acid; dodecanoic acid; pentadecanoic acid; phenol, 2,4-bis(1,1-dimethylethyl)

T30 2:3 T30 2:1

1,3,5-Trioxane; diethyl ether, 2,2′-bis(ethylthio); hydroquinone, 2,5-di-tert-butyl; 1,3,5-trimethylbenzen; 3-octanol, 3,7-dimethyl; caprylic ether; dinonylsebacate; benzil sulfide; 3-methyl-1-butanol; benzene, 1-methyl-2-(2-propenyl); tetralin; oxirane, 2-methyl-3-pheny; phenol, 2,4-bis(1,1-dimethylethyl); propanoic acid

Italics: different compounds in the treatment and control conditions. T20, 20 days; T30, 30 days

Oil phase Fuel degradation Ester degradation in pure biodiesel and in the portion of biodiesel present in the B10 blend were evaluated by infrared spectroscopy (Figs. 2 and 3). For P. boydii samples, we analyzed the band in the 1750 cm−1 region that corresponds to the axial deformation vibrations of the carbonyl group. Over time, Table 3 Compounds in the aqueous phase in contact with B100 biodiesel at day 30 in the ratios 1:6, 2:3, and 2:1 and their respective controls

there was a reduction of the carbonyl group. A new signal indicating the degradation product was also observed, mainly in the ratio 1:6, which appeared in the 1710 cm−1 region in B10 and B100. That observation was only possible in biodiesel and in the blend, not in B0. The spectra obtained from the control samples of P. boydii (B100 and B10) did not show secondary peaks or differences among sample times. During the experiment time, it was possible to monitor whether the biofuel and the B10 blend were degraded and how much it

Sample

Compounds

T30 1:6

Oxirane, 2-methyl-3-phenyl; benzaldehyde, 2,4-dimethyl; 3-methyl-1-butanol; Dinonylsebacate; phenol, 2,4-bis(1,1-dimethylethyl); propanoic acid; 1-decanol, 2-ethyl; caprylic ether

T30 2:3

1-Hexene, 3,5,5-trimethyl; cyclohexanol, 2-(1,1-dimethylethyl); (7a-isopropenyl-4,5-dimethyloctahydroinden-4-yl)methanol; 1-hexanotiol; phenol, 2,4-bis (1,1-dimethylethyl); acetic acid; 2-ethylhexyl salicylate; decanoic acid; propanoic acid; caprylicether; 3-methy-1-butanol; tiofen p-tert-Butyl-alpha-methylhydrocinnamaldehyde; ethylcyclohexylketone; 1-decanol, 2-ethyl; hexanotiol; tiofeno; 3-mehyl-1-butanol; nonanoicacid; tert-butyl-p-benzoquinone; phenol, 2,4-bis(1,1-dimethylethyl); propanoic acid; phenol, 2,6-bis(1,1-dimethylethyl)-4-(1-methylpropyl); decanoic acid

T30 2:1

Control T30 1:6

1,3,5-Trioxane; tiofen; 2-ethylhexyl salicylate; homomenthylsalicylate; 3-methyl-1-butanol; decanoic acid;1-decanol, 2-ethyl; phenol, 2,4-bis(1,1-dimethylethyl); propanoic acid; caprylic ether

T30 2:3

3-Octanol, 3,7-dimethyl; 4-tert-butylcyclohexyl acetate; isozonarol; 1-hexanol, 2-ethyl; 2-octanol, 8,8-dimethoxy; phenol, 2,6-bis(1,1-dimethylethyl)-4-(1-methylpropyl); hexanotiol; 3,7-dimethyl-3-octanol; tert-butyl-p-benzoquinone; homomenthylsalicylate; nonanoic acid; acetic acid; propanoic acid; phenol, 2,4-bis(1,1-dimethylethyl); caprylic ether; 2-ethylhexyl salicylate; 3-methyl-1-butanol; decanoic acid; tiofen

T30 2:1

1-Hexanol, 2-ethyl; 3-octanol, 3,7-dimethyl; 2-octanol, 8,8-dimethoxy; phenol, 2,6-bis(1,1-dimethylethyl)-4-(1-methylpropyl); tert-butyl-p-benzoquinone; phenol, 2,4-bis(1,1-dimethylethyl); propanoic acid; decanoic acid

Italics: different compounds in the treatment and control conditions. T30, 30 days

Environ Sci Pollut Res Table 4 Compounds in the aqueous phase in contact with B10 blend after 240 h and their respective controls

Sample

Compounds

T240

Methyl 11,14-eicosadienoate; 1-heptanotiol; 1-methyl-piperazine; 1-octanotiol; 2,5-dimethylthiophene; 3-mehyl-1-butanol; 4-hexeno-3-ol, 2-methyl; 4-methyl-cyclohexanol; acetic acid cyclohexanol; butanoic acid; phtalic acid; sulfurous acid; dodecane; eicosane; phenol, 2,5-bis(1,1-dimethylethyl); phenol, 2,6-bis(1,1-dimethylethyl)-4-(1-methylpropyl); heptadecane; hexadecane; methyl-cyclohexanol; naphthalene, 1,2,3,4-tetrahydro-5,6-dimethyl; naphthalene, 1,2,3,4-tetrahydro-6-methyl; naphthalene, decahydro-2,6-dimetil; pentadecane, 2,6,10,14-tetramethyl; benzil sulfide; tetradecane; tiofen; triacontane; tridecane; undecane, 4,7-dimethyl; 1-ethyl-4methyl-ciclohexane; decanoic acid; pentanoic acid; propanoic acid; cyclohexanethiol; hexanethiol; methyl elaidate; naphtalene, 1,2,3,4-tetrahydro-5-methyl; octanethiol

T 240 control

8,11-octadecadienoic acid; methyl-ciclohexanol; naphthalene, 1,2,3,4-tetrahydro; 1-hexanethiol; stearic acid; linoleic acid; palmitic acid; pentanedioic acid (2,4-di-t-butylphenyl); benzenethiol; phenol, 2,4-bis(1,1-dimethylethyl); 1-ethyl-4methyl-ciclohexane; decanoic acid; pentanoic acid; propanoic acid; cyclohexanethiol; hexanethiol; methyl elaidate; naphtalene, 1,2,3,4-tetrahydro-5-methyl; octanethiol

Italics: different compounds in the treatment and control conditions. T 240, 240 h

was degraded. This was possible due to the maximum absorbance values of the carbonyl peak and the percentage of degradation obtained. These analyses showed a higher degradation rate in B10 in the lowest oil column (1:6) at days 20 and 45 (34.90 and 48.58%, respectively). The highest degradation values for the B100 fuel were 3.36 and 4.41% at days 20 and 45, respectively, under the same conditions. For M. guilliermondii samples, the spectra were generated by infrared spectroscopy for the treatment and control samples at 0, 48, 96, 168, and 240 h in all fuels. The bands of regions 4000 to 800 cm−1 in B10 are shown in Fig. 4. The spectral regions from 1500 to 800 cm −1 were evaluated and correspond to the B100 fuel (Fig. 5). The PCA analysis of the B10 samples showed that two main components were capable to explain 92.53% of the data variance (Fig. 6). PC1 samples after 48 and 96 h had positive Table 5 Compounds in the aqueous phase in contact with B100 biodiesel after 240 h and their respective controls

scores; samples after 168 and 240 h had negative scores. Evaluating the PC1, the biotic degradation was predominant since loadings indicated a change in the unsaturation region of the methyl esters of the biodiesel; their corresponding peaks decreased according to the experimental time. On the other hand, PC2 separated the control samples with negative scores from the inoculated samples. Based on this result, we can assume that the degradation by M. guilliermondii was limited. The PC2 loadings showed that the control samples preserved the carbonyl group, which did not occur in the inoculated samples. The PCA analysis of the B100 samples showed that two main components were capable to explain 77.48% of the data variance (Fig. 7). In the PC1, the samples were separated according to the experimental time. Weight graphs indicated that there was a small change in the ester group region. The

Sample

Compounds

T240

E)-2-decenal; 2-undecenal; 3-methyl-1-butanol; adipic acid; butanoic acid; heptadecanoic acid; propanoic acid; salicylic acid, 2-ethylhexyl ester; hexanol; methyl elaidate; methyl palmitoleate; 9,15-octadecadienoic acid; 2-ethylhexanoic acid; dodecanoic acid; eicosadienoic acid; stearic acid; nonanoic acid; octadecanoic acid; palmitic acid; pentadecanoic acid; pentanoic acid; tetradecanoic acid; benzenethiol; caprylic ether; phenol, 2,4-bis(1,1-dimethylethyl); phenol, 2,5-bis(1,1-dimethylethyl); methyl 5,13-docosadienoate; methyl hexadecanoate; benzil sulfide Lauric aldehyde; cyclohexylmethanol; phenol, 2,6-bis(1,1-dimethylethyl)-4-(1-methylpropyl); hexanol; methyl octadecadienoate; benzoquinone, 2-tert-butyl; 9,15-octadecadienoic acid; 2-ethylhexanoic acid; dodecanoic acid; eicosadienoic acid; stearic acid; nonanoic acid; octadecanoic acid; palmitic acid; pentadecanoic acid; pentanoic acid; tetradecanoic acid; benzenethiol; caprylic ether; phenol, 2,4-bis(1,1-dimethylethyl); phenol, 2,5-bis (1,1-dimethylethyl); methyl 5,13-docosadienoate; methyl hexadecanoate; benzil sulfide

T 240 control

Italics: different compounds in the treatment and control conditions. T 240, 240 h


Fig. 2 Infrared spectra of the B10 blend at days 20 and 45. Time 0—blue; control—black; 1:6—red; 2:1—green; 2.3—cyano

PCA analysis grouped the samples from experimental time 0, 48, 96, 168, and 240 h. Therefore, there was a standardized change as the hours went by. The 1H NMR analyses of P. boydii indicated the degradation of ethyl, methyl, and aromatic groups. Using NMR, we observed that the regions of the OCH3 and CH2 = CH2 groups showed a higher degradation rate in the biodiesel esters present in B10 at day 20 in the ratio 1:6 than at day 45 in the ratio 2:1. Analyzing the B100 fuel, only the OCH3 and CH2-C-O regions showed some degree of degradation (7 and 14%, respectively) at the final time. In the B0 fuel, there was a slight degradation of the methyl and ethyl compounds; the aromatic compounds were not degraded. Comparing the 13C NMR spectrum of B100 from day 0 to day 45, a peak between 178 and 179 ppm was observed. This peak is characteristic of the carbon of the carboxylic acid functional group. Therefore, it is

the degradation product of the fatty acid esters present in biodiesel.

Discussion Fungal growth It is known that the oily phase height might influence the amount of oxygen available for microorganisms in the aqueous phase, considering a real condition in a storage tank or in microcosms as well. The biomass differed significantly along the growth curve among the proportion with the lowest oil column (1:6) and the other proportions. The biomass average of the three oily/aqueous phase ratios was analyzed and as we expected, the condition that the fungus had more availability of


Fig. 3 Infrared spectra of B100 at days 20 and 45. Time 0—blue; control—black; 1:6—red; 2:1—green; 2.3—cyano

oxygen corresponds to the lowest proportion (1:6) and more biomass was observed. Our results are in concordance with Cai et al. (2015) that investigated the aerobic and anaerobic conditions of oil storage tanks. The authors concluded that the microbiota was able to degrade the most labile oil fractions in 7 days. However, in the deepest part of the reservoir, there was no aerobic biodegradation due to the low oxygen rate, stimulating the growth of anaerobic microorganisms. Considering the growth curve of P. boydii in B0, until day 10, the log phase was observed in ratios 2:3 and 2 1 and up to day 20 in 1:6, presenting decline/cell death from day 30 in 1:6 and 2:3 and in the first 10 days in 2:1. In B10, in the ratio 1:6, the log phase was presented until day 20 and growth was shown until day 45. In 2:3 and 2:1 the same growth behavior was observed until day 30, followed by decline phase. In B100, in all proportions, we obtained log phase until day 10, followed by decline, returning the growth to 30 days. In this

fuel, the phase of decline after time 30 in 1:6 and 2:1 and log phase at 45 days in 2:3 was different. The results show distinct characteristics among the conditions under which the same microorganism was evaluated. P. boydii (Division Ascomycota) is a deteriogenic fungus commonly used in studies concerning biodiesel deterioration (Janda-Ulfig et al. 2008; Schultz 2010; Cazarolli et al. 2014). The majority of the studies shows microorganisms growing better in contact with pure biodiesel (Pasqualino et al. 2006; Mariano et al. 2008; Bücker et al. 2011). Schleicher et al. (2009) studying aerated systems demonstrated higher microbial growth, from a humus soil sample of an obviously uncontaminated grass landsite, in B20 and B5 blends and lower growth in B100. As soon as we know, our study is the first to describe a major growth of filamentous fungi in a blend, in this case, B10, then in pure biodiesel, simulating storage fuel. This result is associated with the capability of P. boydii to


Fig. 4 Infrared spectra of the B10 blend of treatment, control, and initial time samples

Fig. 5 Infrared spectra of the B100 of treatment, control, and initial time samples


Fig. 6 PCA scores of B10 of treatment and control samples

metabolize the amount of biodiesel in the blend because it consists of fatty acids, the most labile portion. It shows that 10% of biodiesel interacted with 90% of diesel was sufficient to mycelia growth. Some studies have already detected M. guilliermondii (Division Ascomycota) degradation capability (Gaylarde et al. 1999; Liu et al. 2014; Ma et al. 2015). Using M. guilliermondii, 48 h were sufficient to obtain an increase of three logs in the growth curve in both B100 and B10. No differences were seen between the growth rate of M. guilliermondii in the two fuels. Therefore, the yeast was efficient in metabolizing fatty acids and hydrocarbons. The microorganisms evaluated in this study were able to grow using diesel and biodiesel blends as the only source of carbon and energy. In simulated storage conditions, the stipulated proportions of the BH mineral medium contributed to evaluate the aeration of a storage tank for 45 days and 240 h, depending on the microorganism.

Aqueous phase The point of measuring the surface tension was to detect surfactant compounds during the cell growth. These compounds facilitate microorganisms’ growth because the reduction of the surface tension provides available substrate to hydrocarbon solubilization (Singh et al. 2007) and increase cellular hydrophobicity (Paria 2008). In our study, there was no production

of biosurfactants by P. boydii and M. guilliermondii in the experimental conditions. The similar results concerning the reduction of surface tension in the treatments and in the controls with biodiesel are associated to the esters of fatty acids, resembling the structure of some surfactants (Bücker et al. 2011, 2014). Therefore, fatty acids can simulate the action of biosurfactants produced by microorganisms (Bognolo 1999). The pH of the aqueous phase did not significantly reduce between the treatments. Depending on the sample time (20, 30, and 45 days), a reduction in pH was detected, indicating the possible production of acid metabolites by P. boydii. The pH reduction may also be associated with cell lysis, polymer products, and organic acids (Parbey 1970). The fungus showed ability to produce biomass, but no significant reductions in pH of the aqueous phase were observed. This result can be explained by the concentrations of the phosphates KH2PO2 and K2HPO4 that comprise the BH mineral medium. The phosphates could cause a buffering effect. To supply a nutrient balance (source of phosphorus) to the microbial cell, the phosphate should always comprise the culture and minimal media. The constancy of the pH of M. guilliermondii can be explained by the concentration of phosphate in the BH mineral medium; or because the yeast was not able to produce such metabolite. However, the pH in the final time of the experiment with B100 (decreased from 7.2 to 3.48) can be interpreted as a high production of acid metabolites.


Fig. 7 PCA scores of B100 of treatment and initial time samples

The compounds found in the aqueous phase by GC-MS could have migrated from the fuels during their degradation, been originated from the biodiesel raw material or the refining petroleum in diesel. During the degradation of the fuels and biofuels, some carboxylic acids were detected, probably making the pH of the BH mineral medium more acidic. Therefore, the hydrocarbons or fatty acids of the fuels became available in the aqueous phase, and were probably easily metabolized by the fungus. The compounds identified by GC-MS found exclusively in the control samples may have been consumed by the fungus. In contrast, other compounds were present in the control and in the treatments, indicating that there was abiotic degradation and the compounds were probably not consumed. Propanoic acid and phenol, 2,4-bis(1,1-dimethylethyl) were present in both conditions (control and treatments). However, the lipid oxidation must be considered. There were compounds, such as hydroperoxides, formed from secondary oxidation products including aldehydes and short-chain fatty acids (Schneider et al. 2008). GC-MS has been used for several purposes during the composition, evaluation, or degradation related to oils (Gieg et al.

2010; Aktas et al. 2010, 2013; De Azambuja et al. 2017). Bento et al. (2005) detected metabolites with SPME after 60 days of incubation of Aspergillus fumigatus in contact with BH mineral medium and diesel oil. The metabolic products identified in the aqueous phase (pH 4.8) were propionic acid, series of alcohols, and ketones such as 2-butanol and 2pentanone. Pinho et al. (2014) used GC-MS to identify the oxidation products from soybean oil and castor oil biodiesel. The authors found several components, such as methyl 10oxo-8-decenoate and methyl 9-oxo-nonanoate, as products of the double bond of the methyl linoleate oxidation. A strong indication of the abiotic degradation in fuels is the increase of carbonyl bonds in the fuel and biofuel blends. Bian et al. (2015) analyzed the metabolic profile of samples collected from three different oil fields using GC-MS. Metabolites such as 2-benzylsuccinate and 2-naphthoatewere found in some of the samples and are indicators of anaerobic degradation of mono- or poly-aromatic hydrocarbons. Based on the tributyrin evaluation, we could detect that the biodegradation of biodiesel may involve enzymes that act first on the catabolism of the esters by hydrolyzing the methyl or ethyl ester. Hydrolytic enzymes, such as lipase and esterase,


are involved in this reaction, which action results in a fatty acid and an alcohol. After the reaction, the fatty acids are incorporated into the microbial cells by β-oxidation processes. Lipases catalyze the hydrolysis of triacylglyceride ester bonds of water-insoluble substrates, while the esterases act on water-soluble substrates, such as short-chain fatty acid esters and triglycerides (Pepper et al. 2011). The fungi were able to produce lipase, contributing to the degradation of the biodiesel portion and cell growth. Janda-Ulfig (2008) has already tested this feature of P. boydii isolated from sewage sludge and from clinical cases and detected the productions of this kind of enzymes during its growth in all substrates tested (agar containing tributyrin, rapeseed oil, biodiesel, and diesel). The emulsification index (EI24) indicated the ability of the yeast to produce bioemulsifiers. After 72 h of experiment, the cells in the aqueous phase were in contact with B10 and kept the emulsification after 24 h of rest. In the aqueous phase with B100, there was emulsion only in the presence of cells at the final experimental time. Thus, we assume that the emulsifier might have been attached to the yeast cell wall (Desai and Banat 1997; Banat et al. 2004; Mulligan 2005; Singh et al. 2007).

Fuel phase Infrared spectroscopy analysis of the P. boydii samples showed a decrease in the peak of the carbonyl function and the appearance of a secondary peak in the samples of B10 and B100. Observing the control samples, without addition of inoculum, the absence of secondary peaks and the similarity of the spectra at all sample times suggests that there was no evident abiotic degradation. In relation to the 1H NMR analysis of P. boydii, the B10 fuel presented a greater percentage of degradation of the aromatic compounds compared to pure diesel, and of OCH3 compared to pure biodiesel. As previously proposed, filamentous fungus developed better in B10. Their enzymes, such as monoxygenase and carboxylesterase, were probably more successful in degrading diesel and biodiesel together in the blend. In this way, the microorganism had the ability to metabolize the compounds of both fuels. In 13C NMR, the apparent peak indicates the occurrence of carboxylic acid as a degradation product corresponding to the peak in the infrared spectrum in the region of 1710 cm−1, not being consumed by the fungus in its metabolic pathway. These data suggest that the interaction between diesel and 10% biodiesel in the mixture provided adequate energy supply for P. boydii. In the samples of M. guilliermondii, abiotic degradation was verified in B10 and small degradation of the carbonyl function in treatment and control. Because of the decrease in the peak of unsaturation over time, probably, in the first hours, the biodiesel fraction of the mixture was degraded by factors such as oxidation or hydrolysis and not by

microbial action (Strömberg et al. 2013; Yaakob 2014). The microorganisms preferentially attack the portions in which single bonds are present in the biodiesel or metabolize the carbons that are already available in the aqueous phase (Khoury et al. 2011). Comparing the controls with the treatments, it was inferred that there was a small degradation of the carbonyl group in the yeast samples, although the growth curve showed a large increase in the CFU count. With the PCA results of B100, it was possible to visualize that the ester group region showed little change. However, a trend was observed by separating into groups for each incubation time of the treatment samples. The results obtained by infrared spectroscopy did not show fuel degradation as evident as in previous studies in the area (Zimmer et al. 2013; Bücker et al. 2014; De Azambuja et al. 2017). The results obtained in the determination of the degradation of the methyl esters of biodiesel over the experimental time were corroborated by other authors. De Azambuja et al. (2017) found similar results in treatments and control samples of several sulfur content diesel oils. The dendrograms of infrared spectra showed that all samples had modifications in their carbon chains, suggesting that the diesel hydrocarbon chains were degraded after 40 days by abiotic factors. Pinho et al. (2014) using the infrared spectra compared diesel and biodiesel B5 blends before and after aging by accelerated oxidation. The authors identified a wide absorbance in the region of C=O stretching after aged B5. The authors attributed these bands to the degradation of oxygenated products from the oxidation of C=C bonds in unsaturated compounds of biodiesel and diesel. Several studies have used infrared spectroscopy and NMR to analyze the oxidation, quality, or alteration of fuels and biofuels. Chuck et al. (2012) compared several techniques, such as Fourier transform infrared (FTIR) and 1H and 13C NMR, to evaluate the oxidation of biodiesel at 90 and 150 °C.

Conclusion The filamen to us fung us P. boyd ii a nd the y east M. guilliermondii used biodiesel and the B10 blend of diesel and biodiesel as a source of carbon for their growth. However, higher biomass production was observed in P. boydii simulation with biodiesel. Both fungal species were not efficient in producing biosurfactants. However, yeast was efficient in producing bioemulsifiers. The gas chromatographic analysis was able to detect the metabolites produced by fungi and compounds from the degradation of fuels. During simulations with P. boydii, there was degradation of pure biodiesel and B10 blend analyzed by the infrared technique and degradation of hydrocarbons analyzed by NMR. However, no significant degradation of biodiesel was observed when M. guilliermondii was used.

Environ Sci Pollut Res Acknowledgements The authors wish to thank LAB-BIO/UFRGS, Chemistry Institute/UFRGS, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the funds provided during the course of this study. Funding This study was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest.

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