Biodegradation of diesel and heating oil by Acinetobacter calcoaceticus MM5: its possible applications on bioremediation

International Biodeterioration & Biodegradation (1995) 269-285 Copyright 0 1995 Elsevier Science Limited Printed in Great Britain. All rights reserved

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0964-8305(95)00067-4

Biodegradation of Diesel and Heating Oil by Acinetobacter calcoaceticus MM5: its Possible

Applications on Bioremediation

Mercedes Marin, Ana Pedregosa, Santiago Rios, M. Luisa Ortiz & Fernando Laborda

Departamento de Microbiologia y Parasitologia, Universidad de Alcalh de Henares, Carretera Madrid-Barcelona, km 33.6, 28871 AlcalL de Henares, Madrid, Spain

ABSTRACT

Twenty aerobic bacterial strains were isolated from altered heating oil. Among them the strain catalogued as MM5 and identified as Acinetobacter calcoaceticus is able to grow on hydrocarbon substrates. When strain MM5 was grown on heating oil, crude oil and tetradecane, increases of protein concentration and of caprilate-lipase and acetate-esterase enzymatic activities were observed in the cultureJltrate, with a simultaneous pH drop. A strong emulstfication of petroleum by-products was also noticed. Degrada- tion of heating oil was followed by gas chromatography and infrared spectroscopy. Presence of available nitrogen and phosphorus sources were essentialfor hydrocarbon biodegradation. Intracellular electron transparent inclusions were observed by transmission electron microscopy when strain MM5 cells were grown on hydrocarbons. Light and scanning electron microscopy showed bacteria interconnected by an extracellular polymer and attached to hydrocarbon droplets and to sheets of polymeric material. A bioemulstfier was extractedfrom the cell-free culture supernatants of strain MM5 grown on tetradecane. The emulsifier is a high molecular weight product that comprises proteins, sugars andfatty acids and which is resistant to high temperature. Strain MM5 should be helpfulfor the design of strate- gies for the bioremediation of hydrocarbon contaminated sites.

INTRODUCTION

The environmental contamination by petroleum and its derivatives is a problem that appears to be increasing, with obvious ecological and

270 M. Marin et al.

economic implications. Considerable amounts of hydrocarbons are present in water and soil as a consequence of spills from oil tankers and activities carried out in oil-drilling sites, petroleum refineries and storage facilities, gas stations, factories and areas adjacent to leaking pipelines. Certain microorganisms are able to use petroleum derivatives as the sole source of carbon and energy (Bossert & Bartha, 1984; Rosenberg, 1992). Thus, biodegradation of hydrocarbons by microorganisms represents one of the primary mechanisms by which those pollutants could be eliminated from the environment (Leahy & Colwell, 1990; Atlas & Bartha, 1992).

The study of hydrocarbon-oxidizing microorganisms has also received considerable attention because of the negative effects caused by those organisms during their growth on petroleum derivatives. In the last few years there appears to have been an increase in the extent of microbial problems in fuel storage systems. Genner and Hill (1981) emphasized that spoilage only occurs when the petroleum products come in contact with water. Microbial growth in the water bottoms of storage tanks can cause a variety of deleter- ious effects, including pump and filters blockage, tank corrosion and changes in the characteristics of the fuel product (Shennan, 1988).

A serious problem in the biological oxidation of hydrocarbon compounds arises from their extremely low solubility in water. Many hydrocarbon-degrading microorganisms produce emulsifying agents that facilitate the utilization of hydrocarbons (Gutnick & Shabtai, 1987). In recent years, there has been a growing interest in bioemulsifiers and other microbial surface active agents. The potential applications of bioemulsitiers include their use in the field of bioremediation, industry, agriculture, medicine, cosmetics and for enhanced oil recovery from reservoir rocks, in substitution of synthetic surfactants. The advantages of those compounds produced by microorganisms include their biodegradability and controlled inactivation, the diversity of structure and function for different applications and the selectivity for specific hydrocarbon/water interfaces (Rosen- berg, 1993). In the present work, the isolation and identification of the bacteria isolated from altered fuel has been carried out. Furthermore, one of the isolates, which was able to utilize hydrocarbons and produce emulsifier polymers, was studied in more detail.

MATERIALS AND METHODS

Isolation, identification and selection of hydrocarbon degrading bacteria

The strains of bacteria used in this study were isolated by the procedure indicated by Herbert et al. (1987) from samples of altered heating oil, collec-

Biodegradation of oil 271

ted in a storage tank. The bacterial isolates were named as MM. The isolates were identified at species level following the procedures recommended in the Bergey’s Manual of Systematic Bacteriology (Krieg & Holt, 1984; Sneath et al., 1986; Staley et al., 1989; Williams et al., 1989) and by Balows et al. (1992).

Isolates were tested for their ability to grow on gasoline, jet fuel, heating oil, crude oil, heating oil plus glucose, or glucose, using a liquid mineral medium (Bushnell & Haas, 1941) (BH medium), that contained (g/l): NH4N03, 1; K2HP04, 1; KH2P04, 1; MgS04a7H20, 0.2 and CaC12, 0.2. The medium also contained O-05 ml/l of a solution of trace elements (Bosch et al., 1988) and 1% of the carbon source. In this experiment, the BH medium was supplemented with 0.1% yeast extract. Growth was monitored by visual observation of turbidity in the medium after 20 days incubation at 28°C. A medium without any carbon source served as control. The emulsification of hydrocarbons was determined by examining the presence of droplets of emulsion in the cultures. The strain MM5 was selected for further studies.

Growth of the bacterial strain MM5 on diesel oil, crude oil and tetradecane

MM5 strain was grown in 500 ml flasks with 100 ml of liquid BH medium containing 1% diesel oil, crude oil or tetradecane as carbon and energy source. Incubations were carried out at 28°C in an orbital shaker (200 rpm) for 20 days. Samples were taken after 1, 2, 3, 5, 8, 10, 12 and 20 days incubation. Bacterial growth was estimated by determination of cell dry weight, optical density of the cultures at 620 nm (OD620) and by counting the colony forming units (CFU). Several fractions were obtained after centrifugation of the cultures at 10,000 g for 30 min: a pellet (formed by bacteria), a cell-free supernatant, an interface and an upper oily phase (which was discarded in this experiment). Emulsifying activity of those fractions was determined following the method of Bosch et al. (1988). The following determinations were carried out in the cell-free supernatants: pH was measured with a Crison pH-meter Model micropH 2000. Surface tension was measured with a Lauda Tensiometer Model TElC. Proteins were estimated by the commercial DC-Protein Assay (Bio-Rad Labs) and total sugars by the method described by Hanson and Phillips (1981). The enzymatic activities caprilate-lipase and acetate-esterase (Shabtai & Gutnick, 1985) were also measured.

Heating oil degradation by the bacterial strain MM5

To study the influence of the presence of a source of nitrogen and phosphorus on the degradation of heating oil, strain MM5 cells were incubated

212 IU. Marin et al.

on whole BH medium or BH medium in which nitrate, or phosphates or both kind of salts were omitted. Heating oil (0.5%) was used as carbon source. An uninoculated medium served as a control. Samples were taken after 20 days incubation. The residual diesel oil, after bacterial degradation was extracted with hexane (Kokub et al., 1990) and analyzed by gas chromatography and infrared spectrometry. The gas chromatography analyses were carried out using a Hewlett-Packard 5890 Series II gas chromatograph with a flame ionization detector. An Ultra 1 capillary column (25 m long x 0.2 mm diameter) was used. Operational temperature ranged from 80 to 280°C and helium was used as a carrier gas. The infrared spectra were carried out over a range of 4000 to 200/cm in a Perkin-Elmer 883 spectrometer.

Microscopical study of the bacterial strain MM5

Bacteria were incubated on liquid BH medium containing 1% yeast extract or heating oil as carbon source. For light microscopy (LM) studies, samples of the bacterial cultures were mounted in methylene blue and observed with a Zeiss Universal microscope. For scanning electron microscopy (SEM), samples were fixed with 5% glutaraldehyde and dehydrated in a graded series of ethanol or acetone. Then samples were critical-point dried, mounted on aluminium stubs and sputter-coated with gold. Specimens were examined with an IS1 microscope model SX-25. For transmission electron microscopy (TEM), samples were fixed with 3% glutaraldehyde, post-fixed with 1% Os04, dehydrated in a graded acetone series and embedded in Spurr’s resin (1969). Sections were stained with uranyl acetate and lead citrate and examined in a Zeiss EM-1OC microscope.

Extraction and characterization of the emulsifier produced by the bacterial strain MM5

To produce the emulsifier of the MM5 strain, the microorganism was incubated on BH medium containing 1% tetradecane as carbon source. Culture supernatants obtained after 5 days incubation were concentrated by ultrafiltration (exclusion molecular size, 300 kDa). Extraction of the emulsifier from the concentrate was made as described by Bligh and Dyer (1959). Different determinations were carried out to characterize the MM5 emulsifier: proteins (DC-Protein Assay, Bio-Rad), sugars (Hanson & Phillips, 1981) reducing sugars (Moppar & Gindler, 1973) lipids (Merkotest, Merck) and o-acyl-ester linked fatty acids (Kates, 1972). The effect of several treatments on the emulsifier activity was studied: the


effect on the pH was investigated from the pH range 2-12. The temperature stability was analyzed over the range -2O-120°C. Cation requirements for emulsification were determined by adding 100 mM EDTA, MgS04 or NaCl in the emulsification assay. The effect of enzyme treatments (lipase, /?-glucosidase, esterase and protease) on emulsifying activity was analyzed by the method of Wasko and Bratt (1991).

RESULTS

Isolation, identification and selection of hydrocarbon-utilizing bacteria

Twenty bacterial isolates were obtained from contaminated heating oil and examined for biochemical cultural, morphological and physiologi- cal characteristics. The bacteria were included in the genera Pseudomo- nas, Acinetobacter, Staphylococcus, Micrococcus and Bacillus, and the majority of the microorganisms were identified at species level. One strain was not identified (Table 1). The isolates were screened for growth on hydrocarbons. All the strains isolated were able to tolerate the presence of hydrocarbons, as they were able to grow on heating oil plus glucose. One of the bacterial isolates (Acinetobacter calcoaceticus MM5) was selected for further studies. This strain failed to grow on glucose but strongly developed on diesel oil, heating oil and crude oil and produced the emulsification of those products in the culture medium (Table 1).

Growth of Acinetobacter ealcoaceticus MM5 on diesel oil, crude oil and tetradecane

The growth behaviour of A. calcoaceticus MM5 on diesel oil, crude oil and tetracecane was studied over a period of 20 days. The results obtained are reflected in Figs l-3, respectively. The maximum growth (expressed as CFU) was observed after 5 days incubation. However, the 0D6z0 and the dry weight of the cultures increased progressively up to 5 days, and remained constant until the end of the period studied [Figs l(A), 2(A) and 3(A)]. A significant acidification of the culture medium was recorded within five days after inoculation. In general, no marked changes in the pH were recorded over the remainder of the monitored period [Figs l(B), 2(B) and 3(B)].

Since A. calcoaceticus MM5 produces an emulsitication of hydrocarbons in the culture medium, the presence of bioemulsitiers was investigated in three fractions obtained after centrifugation of the

214 M. Marin et al.

TABLE 1 Growth on Petroleum Derivatives of the Bacterial Strains Isolated from an Altered

Heating Oil”

Strain Carbon source

Gas Jet Al Heat Crude oil Heat + glu Ghl

MM 1 Pseudomonas jluorescens MM2 Staphylococcus epiakrmidis MM3 Pseudomonas fluorescens MM4 Pseudomonas jluorescens MM5 Acinetobacter calcoaceticus MM6 Pseudomonasjluorescens MM7 Pseudomonas putida MM8 Pseudomonasjluorescens MM9 Pseudomonasjluorescens MM10 Micrococcus sp. MM1 1 Staphylococcus xylosus MM 12 Micrococcus luteus MM 13 Staphylococcus xylosus MM 14 Staphylococcus aureus MM 15 Staphyloccus epidermidis MM 16 Bacillus pumilus MM 17 Pseudomonas putida MM 18 Not identified MM19 Micrococcus luteus MM20 Staphylococcus xylosus

kb _ _ _ _ - f f f - - It -

-

_

_

It _

f It

+EC _

+E -

zt -

+ +E + _

f - - - _

+ + - _

f

It -

f -

++ _

-

zt * _ _ - - - _ _ _ - _ f

+ ++E - ++ f ++E * ++E

++E ++E + ++E + ++ + ++E + ++ - +

++ - ++ - ++ - ++

++ - ++ l ++

f ++

zt ++

++ ++ ++ ++ _

++ ++ ++ ++ +

++ ++ ++ f-t ++ ++ ++ +

++ ++

OBacteria were incubated for 20 days at 28°C with occasional agitation in tubes with 10 ml of BH medium supplemented with 0.01% yeast extract and containing different carbon sources (each 1 X). bGrowth was determined by visual observation of turbidity in the medium. Symbols:

negative growth; Z!Z, weak growth; +, positive growth; + +, strongly positive growth. ‘B indicates emulsification of the hydrocarbons in the culture medium. Abbreviations: gas, gasoline; jet-Al, jet fuel; heat, heating oil; heat + glu, heating oil plus glucose; glu, glucose.

bacterial cultures (pellet, cell-free supernatant and interface). When the MM5 strain was incubated on diesel oil as the sole carbon source, maximal levels of emulsifier were determined in 5 days culture pellets; from then, the emulsifying activity in this fraction remained constant until the end of the period studied. A strong activity was also detected in the interfaces, from 3 to 9 days of incubation. However, lower levels of emulsifying activity were measured in the cell-free supernatants from 5 to 10 days of growth [Fig. l(B)]. Similar results were obtained when A. calcoaceticus MM5 was grown on crude oil [Fig. Z(B)]. However, in this case, the interfaces formed between the oily and aqueous phases of

Fig. 1. Growth of Acinetobacter calcoaceticus MM5 on heating oil as the only carbon source. (A) Growth; (B) bioemulsifier production; (C) protein and enzyme production.

Biodegradation of oil

Incubation time (days)



275

the cultures were not recovered because of the high viscosity of the petroleum. During the incubation of strain MM5 on tetradecane, the behavior of the emulsifying activity in the pellet and interface was in general similar to that reported for diesel oil. In this medium, high values of emulsifying activity were measured in the cell-free supernatants [Fig. 3(B)]. On the other hand, no appreciable reduction of the

276 M. Marin et al.

Fig.


B -5

u 0 2 4 5 5 10 12 14 15 15 20

incubation time (days)

10 i2 14 15 15 S


2. Growth of Acinetobacter calcoaceticus MM5 on crude oil as the only carbon source. (A) Growth; (B) bioemulsifier production; (C) protein and enzyme production.

surface tension of the culture medium was observed during the bacterial growth on heating oil, crude oil and tetradecane.

The concentration of proteins in the culture medium increased during incubation of A. calcoaceticus MM5 on heating oil, crude oil and tetradecane. Extracellular caprilate-esterase and acetate-lipase activities were recorded after one day incubation on the above carbon sources. The higher levels of those enzymes were detected in a period that was coin- cident with the late-exponential and early-stationary growth [Figs l(C), 2(C) and 3(C)].





Fig. 3. Growth of Acinetobacter calcoaceticus MM5 on tetradecane as the only carbon source. (A) Growth; (B) bioemulsifier production; (C) protein and enzyme production.

Heating oil degradation by Acinetobacter cukoaceticus MM5

Gas chromatography studies showed that, after 20 days incubation in the presence of a source of nitrogen and phosphorus, A. calcoaceticus MM5 was able to degrade hydrocarbons (mainly n-alkanes) of heating oil. However, without addition of nitrate or phosphate salts to the culture medium, no changes in the chromatographic profiles of the heating oil, compared with those of sterile controls, were observed (Fig. 4).

278 M. Marin et al.

- Nitrate - Nitrate

+ Nitrate + Phosphate

Fig. 4. Influence of nitrogen and phosphorus source on heating oil degradation by A. calcoaceticus MM5.

No disappearance of any peak in the i.r. spectra was observed when the MM5 strain was incubated in the conditions described above. However, when A. calcoaceticus MM5 was incubated in the presence of nitrogen and phosphorus salts, the i.r. spectrum revealed the appearance of an intense band at 1710/cm and a minor band at 174O/cm (spectra not presented).

Microscopical study of Acinetobucter calcoaceticus MM5 cultures

TEM studies showed that the growth of the MM5 strain on hydrocarbons results in the presence of intracellular electron transparent inclusions [Fig. 5(A)]. These inclusions were not observed when the MM5 strain was grown on water soluble substrates such as yeast extract [Fig. 5(B)].

Fig. 5.

Biodegradation of oil

Fig. 6.

280 M. Marin et al.

TABLE 2 Main Characteristics of MM5 Bioemulsilier

Characteristics

Molecular weight >300,000 D

Partial chemical composition Total sugars 15.5% Reducing sugars 3% Proteins 20% Fatty acids 1% (o-acil-ester)

Insoluble in Chloroform, methanol, ethanol, ethyl acetate, benzene and hydrocarbons C& 16

pH influence Maximum activity at pH 7-10

Temperature stability It is still active after several times freezing and thawing. It is active after autoclaving at 120°C for 30 min

Influence of cations Still active after treated with 100 mM EDTA and in presence of 100 mM MgClz or NaCl

Enzymatic treatments Still active after treatments with lipases, esterases or /?- glucosidase. It loses the activity after treatment with proteases

LM and SEM of A. calcoaceticus MM5 cultures showed the bacteria attached to themselves and to hydrocarbon droplets by an extracellular polymer [Fig. 6(A) and (B)]. Biofilms formed by the microorganisms adhering to sheets of polymeric material and connections through inter- cellular contacts were also observed [Fig. 6(C)].

Characterization of the emulsifier produced by Acinetobacfe~ calcuaceficus

MM5

An emulsifier was extracted from the cell-free culture supernatants of the MM5 strain incubated on tetradecane. Table 2 summarizes some characteristics of this bioemulsifier and the effect emulsifying activity.

DISCUSSION

of several treatments on its

Twenty bacterial isolates were obtained from altered heating oil. The predominant flora was composed of Pseudomonas spp. and Staphylo- coccus spp. Acinetobacter, Micrococcus and Bacillus were also isolated. Bacteria belonging to those genera have been described as contaminants in


petroleum derivatives or even as hydrocarbon-degraders (Rosenberg, 1992). All the bacteria isolated from heating oil tolerated the presence of hydrocarbons, as they were able to grow in a medium with glucose and heating oil. However, some of them were not able to use petroleum derivatives as the only carbon and energy source. Growth of those strains on the altered fuel could be supported by the organic matter produced by the hydrocarbon degraders or by the grit that become entrapped when biomass accumulates (Allsopp & Seal, 1986). Among the bacterial isolates, Acinetobacter cakoaceticus MM5 was selected for further studies on the basis of the growth and emulsification observed during incubation on hydrocarbons. Acinetobacter is among the bacterial genera most often found in petroleum-contaminated habitats and has been extensively used in studies of n-alkane oxidation (Asperger & Kleber, 1991). Furthermore, several Acinetobacter strains have important industrial applications (Gutnick et al., 1991).

The growth of MM5 on heating oil, crude oil and tetradecane was accompanied by a significant acidification of the culture medium. This fact has been described for other hydrocarbon-utilizing microorganisms and could be due to the release of organic acids as consequence of the degradation of hydrocarbons or to the production of extracellular acidic polymers (Walker et al., 1975; Kokub et al., 1990). However, the determination of the growth of MM5 on the petroleum by-products tested showed difficulties. The differences observed in the values of bacterial growth with the three methods of determination used could be due to the adherence of the microorganisms to polymers. Thus, the bacteria are not dispersed prior to enumeration, and a minor number of cells can be obtained (Rosenberg, 1992).

The production of esterases and lipases seems to be frequent in the genus Acinetobacter (Breuil et al., 1978). Both activities have been detected in cell- free supernatants of MM5 incubated on petroleum by-products. However, although the involvement of those enzymes in the metabolism of hydrocarbons has been suggested, their exact role remains as an open question. Alkane metabolism leads to significant changes in cellular lipid composition and to the formation of several lipid products (Asperger dz Kleber, 1991); it is not then surprising that the production of those enzymes related to the metabolism of lipids. In addition, it has been proposed that the e&erase produced by A. calcoaceticus RAG-l has a role in the release of the bioemulsifier emulsan from the cell surface (Shabtai & Gutnick, 1985).

When the bacterial isolate MM5 was incubated on crude oil, diesel oil or tetradecane, a strong emulsification of those substrates in the aqueous medium was observed. This phenomenon has been reported for other Acinetobucter strains growing on hydrocarbons (Reisfeld et al., 1972;

282 M. Ma& et al.

Rosenberg & Rosenberg, 1981). Emulsification of hydrocarbons enhances the contact between bacteria and those water-insoluble compounds, and can provide a considerable ecological advantage for hydrocarbon-utilizing microorganisms (Rosenberg, 1992). The emulsifying activity has been detected in the three fractions obtained after centrifugation of the A. calcoaceticus MM5 cultures (pellet, cell-free supematant and interface). This fact indicates that MM5 produces emulsifiers that are extracellular products or integral compounds of the microbial surfaces, as has been described for other microorganisms that degrade hydrocarbons (Goldman et al., 1982).

During the growth on heating oil, A. calcoaceticus MM5 was able to degrade hydrocarbons (mainly n-alkanes) of this fuel, as was assessed by gas chromatography. Aliphatic hydrocarbon assimilation seems to be a common property of most Acinetobacter strains (Asperger & Kleber, 1991). Infrared spectroscopy, used to determine the chemical changes that occur during the diesel oil degradation by MM5, revealed the appearance of an intense band at 1710/cm and a minor band at 1740/cm. Those bands could correspond to carbonyl groups derived from the metabolism of hydrocarbons (Singer & Finnerty, 1984). The availability of a source of nitrogen and phosphorus in the incubation medium seems to be necessary for A. calcoaceticus MM5 to grow and degrade hydrocarbons. This fact has been recorded for mixtures of microorganisms growing on crude oil (Fedorak & Westlake, 1981), and for A. calcoaceticus RAG-l, also growing on this substrate (Reisfeld et al., 1972).

The growth of the MM5 strain on hydrocarbons results in the presence of intracellular electron transparent inclusions, as observed by TEM. Similar structures have been described in several hydrocarbon oxidizing microorganisms and could be related to the metabolism of those compounds (Kennedy et al., 1975; Scott & Finnerty, 1976). Microscopical studies also show that MM5 bacteria growing on hydrocarbons were interconnected by an extracellular polymer and attached to hydrocarbon droplets and to sheets of polymeric material. Similar sphere formation has been recorded in a strain of Acinetobacter (Kennedy et al., 1975), and may represent the oil-water emulsion of the liquid culture. A detailed study of the emulsions and biolilms produced by A. calcoaceticus MM5 is reported elsewhere (Marin et al., 1995).

A bioemulsifier was extracted from the cell-free culture supernatants of the MM5 strain incubated on tetradecane. This is a high molecular weight product, as the polymeric compounds produced by A. calcoaceticus RAG-l and BD4 (Gutnick & Shabtai, 1987). The stability of the MM5 bioemulsifier at high temperature is a characteristic that - to our knowledge - has not been described previously for the emulsifiers produced by other Acineto- batter strains, and could be an interesting property in order to consider its possible applications. On the basis of the ability of A. calcoaceticus MM5 to


grow on petroleum by-products, to degrade them and to produce an emulsifying agent on saline minimal media (without complex nutritional requirements), we consider that this strain could have a potential interest for the bioremediation of hydrocarbon contaminated sites.

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Biodegradation of diesel and heating oil by Acinetobacter calcoaceticus MM5: its possible applications on bioremediation