DEGRADATION OF PETROLEUM-DERIVED HYDROCARBONS BY ALGINATE-IMMOBILIZED CELLS OF CANDIDA LIPOLYTICA

Elżbieta Bogusławska-Wąs¹, Katarzyna Czeszejko¹, Artur Bartkowiak², Waldemar Dąbrowski¹, Agnieszka Michniewicz¹, Katarzyna Szameto^1
1 Department of Food Microbiology, Agricultural University of Szczecin, Poland
² Department of Packaging and Polymers, Agricultural University of Szczecin, Poland

ABSTRACT

The objective of the studies was to evaluate possibilities of petroleum oil biodegradation by free and immobilized cells of C. lipolytica. Immobilization was conducted in 2% sodium alginate coated/non-coated with 1.0% chitosane. The elimination process involved 94.1% of the total amount of introduced hydrocarbons in cultures containing free cells of C. lipolytica and 96.5% and 96.1% of hydrocarbons in cultures containing immobilized cells. Statistically significant differences were observed for the rate of hydrocarbon degradation. The remains of traced substratum were at the level of 5.1% and 5.4% in cultures with encapsulated yeasts whereas comparable amounts of petroleum hydrocarbons (4.9%) were detected on 21^st day of the experiment.

Key words: cell immobilization, C. lipolytica, biodegradation of hydrocarbons.

INTRODUCTION

The entire contemporary industrial civilization runs on petroleum and petroleum-based products. The alarming amounts of hydrocarbons delivered into water, air, and soil as well as hindrances encountered during their elimination have become more serious ecological problems for petrochemical industry. Anthropogenic petroleum-derived substances occurring in the natural environment exert a negative effect on all living organisms. Their mutagenic and carcinogenic influence is a result of accumulation in organisms at different trophic levels of food chains.

The most rational way of decontamination of the environment loaded with petroleum derivatives is an application of methods based mainly on metabolic activity of microorganisms [9,14,28,29]. Decontamination by means of biodegradation is of crucial ecological significance as its basic mechanisms are based on bioremediation processes. Two biodegradation mechanisms leading to the elimination of complex hydrocarbon substances are referred to. Either there are hydrophobic interactions developing from the structure of cell wall or secretion of intracellular emulgators [3]. The ability to actively decompose specified fractions of petroleum oil is expressed by many microorganisms. Studies were conducted both on microorganisms isolated from contaminated areas – Pseudomonas sp. [15], Bacillus sp. [18], Rhodococcus sp. [21], Mycobacterium sp. [16], Trichosporon sp. [22] – as well as on genetically modified strains [13].

Facing the grim future of the natural environment, there is an urgent need for methods accelerating decontamination processes. Most frequently the biodegradation rate is shaped by addition of a particular biosurfactant [19], application of capsule-immobilized cells [21,22] or cells immobilized inside ceramic carriers [27]. Yuan et al. [29] suggested introduction of mixed cultures of bacteria and fungi, especially that not all components of petroleum-derived hydrocarbon mixture are decomposed simultaneously. It could enable to employ microorganisms at individual stages of biodegradation and to avoid stage inhibition. Yet, introduction of such cultures may influence kinetics of biodegradation processes [23].

The aim of our studies was to isolate a microorganism whose enzymatic activity and rate of petroleum oil decomposition could facilitate its practical application in biodegradation processes. Particular cell properties, i.e. hydrophobicity of a cell wall and biosurfactant production were preliminary criterions of elimination of analyzed yeasts.

MATERIALS AND METHODS

Strain origin
Samples were collected from the superficial water layer within the port area intended for a tourist fleet. Qualitative and quantitative analyses of yeasts and yeast-like organisms was conducted according to Bogusławska-Wšs and Dšbrowski [5]. Tests ID32C and API 20C AUX (bioMérieux) were used to confirm species designation of isolates. Following strains were used for further studies: Candida lipolytica (P-115/HB), Rhodotorula mucilaginosa (P-153/BF), Geotrichum sp. (P-1003/HB) and petroleum-isolated Trichosporon mucoides (R-104/HF).

Microbial adhesion to hydrocarbons (MATH)
Yeasts strains used in tests for cell surface hydrophobicity (CSH) were cultured according to Bogusławska-Wšs et al. [4]. MATH test [25] was applied to evaluate CSH level with hexadecane and petroleum oil as non-polar solvents against which changes of absorbance in suspensions of analyzed strains were evaluated spectrophotometrically at 600 nm. Results served to prepare a graphical representation of a function (A_t/A_o100; A_o – initial extinction, A_t – extinction of suspension after vortexing a sample and measured against a blank – a 0.1M phosphoric buffer) which revealed changes in the affinity of cells to applied solvents.

Cell immobilization of C. lipolytica P-115/HB
Inoculum (10⁵cells/ml) was derived from a 48-h culture of C. lipolytica. Cells were immobilized by application of a 2% sodium alginate entrapped in a 0.9% CaCl₂ in a 0.95% of NaCl solution. Beside, the encapsulation of C. lipolytica cells in sodium alginate coated with a 1% chitosane solution was performed.

Decomposition of mixture of petroleum hydrocarbons
To evaluate the rate of decomposition of mixture of hydrocarbons by C. lipolytica (P-115/HB), R. mucilaginosa (P-153/BF), Geotrichum sp. (P-1003/HB) and T. mucoides (R-104/HF), a synthetic medium (SM) (KH₂PO₄ – 2.0 g dm^-3, Mg₂SO₄ – 0.17 g dm^-3, (NH₄)₂SO₄ – 2.0 g dm^-3, CaCl₂ – 0.3 g dm^-3, vitamins – 1 mL dm^-3, and microelements 0.25 mL dm^-3 [12] was prepared. The experiment was conducted according to the scheme presented in Table 1. Cultures (250 mL) were incubated constantly agitated for 35 days at 20-22°C. The rate of petroleum decomposition was evaluated at weekly intervals for 5 weeks by means of a gravimetric separation described by Hermanowicz [11] and in Standard Methods [24].

Table 1. Medium sets used to evaluate hydrocarbon decomposition

Medium	Synthetic Medium (SM) supplemented with:
SM1	a definitive yeast inoculum (105 cells/ml)
SM2	a 0.4 mg petroleum oil
SM3	a definitive yeast inoculum (105 cells/ml) and a 0.4 mg petroleum oil
SM4	C. lipolytica cells immobilized in sodium alginate and a 0.4 mg petroleum oil
SM5	C. lipolytica cells immobilized in sodium alginate and chitosane and a 0.4 mg petroleum oil

Evaluation of the biomass outgrowth in cultures
Stains of yeasts - C. lipolytica (P-115/HB), R. mucilaginosa (P-153/BF), Geotrichum sp. (P-1003/HB) and T. mucoides (R-104/HF) in cultures conducted in SM1, SM3, and SM4 were enumerated by standard method of plating appropriate sample dilutions on Sabouraud agar (Oxoid, England) at weekly intervals for 5 weeks.

Statistical analysis
Results served to calculate the means, standard deviations and significance of variances (Scheffe’s test). Correlation between a culture type and decomposition of petroleum hydrocarbons was evaluated by Kruskall-Wallis test. Linear diagrams were evened by the weighted least square fitting. Statistical analysis were performed using Statistica 6.0 PL software.

RESULTS

Evaluation of strain hydrophobicity
Only C. lipolytica and Geotrichum sp. expressed CSH to hexadecane and petroleum oil (Figure 1) whereas R. mucilaginosa revealed affinity to hexadecane exclusively. T. mucoides adhered neither to hexadecane nor to petroleum oil (Figure 1).

Figure 1. Hydrophobicity of strains to hexadecane and petroleum oil

Survival of yeast strains in prepared cultures
Statistical analysis of the biomass outgrowth in particular cultures were conducted using Scheffe’s test at the confidence level p<0.05. Geotrichum sp. and T. mucoides did not grow in cultures described as SM1 and SM3. No significant variances in the number of cfu mL^-1 for SM1 and SM3 cultures of R. mucilaginosa were found. Only in C. lipolytica cultures supplemented with a mixture of hydrocarbons more intensive rate of the outgrowth was observed contrary to the cultures without supplementation. In both experiments values describing the biomass outgrowth did not reveal statistically significant differences (Figure 2). Cultures including immobilized cells (SM4 and SM5) contained statistically significant numbers of C. lipolytica free cells on 21^st day of the experiment (Figure 2).

Figure 2. Comparison of the intensity of growth of different strains on petroleum oil hydrocarbons

Decomposition of petroleum hydrocarbons
Statistical analysis conducted using Kruskall-Wallis test at p<0.05 revealed correlation between the type of culture and decomposition of petroleum hydrocarbons (Figure 3). The loss of hydrocarbon mass in medium without yeasts inoculum during the period of experiment was not statistically significant (Figure 3) and evaluated at the level of 10% of initial mass on the last day of experiment (Figure 4). Similar amounts of petroleum oil were observed also for R. mucilaginosa cultures (Figure 3) with a final 72.5% of the preliminary hydrocarbon content (Figure 4). The most intensive biodegradation processes occurred in C. lipolytica cultures (Figure 3). It was confirmed statistically that the intensive elimination of introduced hydrocarbons happened in cultures containing free (SM3) and immobilized cells (SM4, SM5) and reached 94.1%, 96.5% and 96.1%, respectively. The analysis of rate of hydrocarbons decomposition revealed that in both cases it reached statistically significant level as early as on 7^th day of the experiment in case of non-biodegraded petroleum oil (Figure 4). Analysis performed using Scheffe‘s test at 0.01 enabled to infer that processes taking place in cultures containing immobilized cells were significantly more intensive (Figure 4). It was learnt that the amount of hydrocarbons remained in this culture on 7^th day of the experiment was comparable to their content in the culture with free cells, however, not earlier than on 21^st day, when the elimination levels of 5.1%, 5.4% and 4.9% were obtained for cultures SM4, SM5 and SM3, respectively. Further progress of biodegradation run at this level (Figure 4).

Figure 3. Degradation of petroleum oil hydrocarbons

Figure 4. Differences in degradation of petroleum oil hydrocarbons

DISCUSSION

Hydrocarbons of petroleum oil head the list of the most deleterious environmental pollutants. Their decomposition in nature is slow and depends on ecological balance. The course of such processes is more frequently disturbed by increasing amounts of wastes introduced into the environment as a consequence of civilization development and widespread industrialization. Generally, all kinds of petroleum-derived substances are susceptible to microbiological decomposition but the factor which influences decomposition deeply is a hydrocarbon structure. Breakdown of hydrocarbon mixture in homogenic cultures may result in inhibition of decomposition and destroy microorganism cells. The most frequently it is the effect of initial enzymatic reactions whose products are either toxic for or not assimilated by microorganisms [1]. Such a theory may explain the presence of Geotrichum sp. in samples during the first part of the experiment and its subsequent vanishing (Figure 2). Then, it is recommended to use heterogenic cultures focused at mutual supplementation, especially that not all petroleum components are degraded at the same level [2,29].

To function properly each biological ecosystem needs not only the qualitative and quantitative cooperation of microorganisms but also the presence of all components responsible for transformation rate. Our studies did not demonstrate the intensive multiplication of microorganisms breaking up petroleum hydrocarbons if compared to cultures without the substratum (Figure 4). Partial reactions characterized by a biomass transmission and not by its significant increase are of crucial importance for heterogenic systems [10,26]. Galas et al. [9] suggested that the presence of dehydrogenases indicated the ability of microorganisms to rapid hydrocarbon decomposition and demonstrated that particular enzymatic activity of Bacillus sp. and Micrococcus sp. increased parallel to substratum concentration and correlated with application of petroleum oil. In case of cultures inoculated with C. lipolytica a slight biomass increment may be a direct result of utilization of a cell-produced liposane. This protein-polysaccharide substance consists of 83% of carbohydrates and 17% of proteins which creates a very good source of carbon and energy [6].

Natural processes of decomposition involving microflora develop in areas where water connects with petrol. Its presence in the contaminated environment is a result of elimination and selection of specialized forms. To efficiently start the process not only the definite range of enzymes is needed but also finding and attaching to a petroleum molecule. According to our results only C. lipolytica demonstrated affinity to petroleum oil (Figure 1) and decomposed it efficiently (Figure 3). As a hydrophobic form it eliminated 94.1% of hydrocarbons in medium whereas a hydrophilic R. mucilaginosa decomposed only 27.5% (Figure 3). The importance of CSH in the substratum breakdown was emphasized also by Arino et al.[2]. To evaluate the hydrocarbon biodegradation possibilities of P. aeruginosa the authors demonstrated correlation between its CSH and a biosurfactant production. They stated that the ability to secretion of rhamnolipids as surface active agents increased parallel to reduction of CSH. Surface active agents increase a natural cell hydrophobicity facilitating its connection with a substratum insoluble in water in cultures containing hydrocarbons. It is of crucial importance as hydrophobicity of yeasts and yeast-like organisms is reduced when the culture enters the stationary phase of growth [8]. Physiological ability of C. lipolytica to synthesize an extracellular soluble-in-water emulgator [6] becomes a factor of a permanent substratum emulgation and initiates the process of petroleum decomposition. Surfactants are responsible for the process of substratum dissolution facilitating its transportation to microorganism cells. Biosurfactant characteristics indicates that they are not specific [2,7] but their presence is promoted by a particular conditions of a conducted culture.

Nowadays, the role of factors accelerating the biodegradation processes is cell or enzyme encapsulation. The capsule structure imitates biological membranes throughout a cell may naturally contact with the external environment. Application of immobilized cells of C. lipolytica in our studies caused the acceleration of hydrocarbon biodegradation. It was statistically confirmed that after 7 days of incubation, the amount of hydrocarbons in samples with immobilized cells was significantly lower (16.0% and 20.0%) than in cultures containing free cells (43.6%) (Figure 4). Comparable amounts of substratum residues were detected not earlier than on 21^st day of incubation (Figure 4). The increase in efficacy of biological processes within entrapped cells is a result of higher concentration of enzymatic proteins confined in a small srate and the lack of necessity of dew drop colonization by individual cells. In case of C. lipolytica cultures the efficacy is independently accelerated by produced liposane. This substance acting as an extracellular emulgator translocate freely throughout capsule layers and enhances decomposition. Although C. lipolytica is characterized by a natural ability to immobilize cells by autoaggregation and accumulation them around a substratum, it undoubtedly causes the elongation of the total speed of biodegradation. Immobilized cells in comparison with free cells, display significantly higher resistance to infections, external factors, and accidental changes during conducted processes which may exert negative effects on their metabolism [20]. Acceleration of biodegradation at increasing levels of the substratum and cell protection were the main reasons of immobilization trials of microorganisms capable of decomposition of environmental pollutants.

Natural, easy-to-degradate products such as alginate or karageniane are the immobilizing carriers most frequently applied in natural systems. Our studies attracted the attention to the possibility of capsule overgrowing by immobilized microorganisms. It caused the occurrence of free cells in cultures on 14^th day of the experiment whose amount increased during a conducted experiment (Figure 2). To reduce a release of C. lipolytica cells from alginate capsules they were coated with chitosane. Chitosane is a cationic polyelectrolyte which creates an outer membrane on the external surface of alginate capsule as the effect of reaction with polyanionic alginate. Unfortunately, the addition of polymer did not help. As in the cultures containing alginate-immobilized cells also in that case the presence of free yeast cells was observed (Figure 2). In both cases the occurrence of free forms did not caused a reduction of petroleum hydrocarbon residues. It was evaluated that in all variants of experiment concerning C. lipolytica cells, the efficient biodegradation process covered on average 95% of hydrocarbon mixture (Figure 4). Results obtained by Prieto et al. [15,21] indicate the significant differences at the level of substratum elimination depending on the type of conducted culture. Immobilization of Pseudomonas sp. and Trichosporon sp. caused the complete utilization of naphthalene by applied bacteria [21] and phenol by yeasts [15]. In case of cultures containing free cells introduced substrata were degraded up to 60.0% and 66.7% of their initial amount, respectively. Our studies did not reveal such significant differences in final elimination of included hydrocarbons, although the substance was characterized by a more complex chemical structure. In all cultures (SM3, SM4, SM5) the approximately 5% of traced hydrocarbons were remained (Figure 4). It is highly probable that capsule-released C. lipolytica cells utilized liposane and alginate incorporated in the medium as the source of carbon and energy and due to it their biomass increased (Figure 2).

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Elżbieta Bogusławska-Wąs
Department of Food Microbiology,
Agricultural University of Szczecin, Poland
Papieża Pawa VI 3, Szczecin, Poland
email: ewas@tz.ar.szcecin.pl

Katarzyna Czeszejko
Department of Food Microbiology,
Agricultural University of Szczecin, Poland
Papieża Pawa VI 3, Szczecin, Poland

Artur Bartkowiak
Department of Packaging and Polymers,
Agricultural University of Szczecin, Poland


Waldemar Dąbrowski
Department of Food Microbiology,
Agricultural University of Szczecin, Poland
Papieża Pawa VI 3, Szczecin, Poland

Agnieszka Michniewicz
Department of Food Microbiology,
Agricultural University of Szczecin, Poland
Papieża Pawa VI 3, Szczecin, Poland

Katarzyna Szameto
Department of Food Microbiology,
Agricultural University of Szczecin, Poland
Papieża Pawa VI 3, Szczecin, Poland

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