EFFECT OF SELECTED LIPID SUBSTRATES ON THE PROCESS OF BIOSYNTHESIS OF SURFACE-ACTIVE COMPOUNDS BY THE CANDIDA BOMBICOLA YEASTS

Małgorzata Gumienna, Maria Czarnecka, Zbigniew Czarnecki
Department of Fermentation and Biosynthesis, Institute of Food Technology of Plant Origin, The August Cieszkowski Agricultural University of Poznan, Poland

ABSTRACT

The performed investigations were concerned with the possibility of utilisation of the yeasts ATCC 22214 Candida bombicola for the biosynthesis of surface active substances on substrates containing oleic acid, sunflower oil and post-deodorising condensate as carbon sources. Glucose was the source of hydrophilic carbon in the medium, whereas nitrogen was supplied by the yeast extract. The highest yields of biosurfactants (124.5 g·dm^-3 - oleic acid, 116 g·dm^-3 - sunflower oil and 118.5 g·dm^-3 - post-deodorising condensate) were achieved using the medium in which 3 g of glucose and 0.05 g of yeast extract and 1 g of the fat substrate. The obtained biosurfactants, depending on the fat substrate applied in the culture medium, differed with respect to their appearance. The obtained preparations reduced the surface tension of water from 70 mN/m to approximately 32 mN/m.

Key words: biosurfactants, biosynthesis, Candida bombicola, fat substrate.

INTRODUCTION

In order to reduce the proportion of synthetic surfactants in food, cosmetics and pharmaceutical industries and to protect natural environment, for example, in fat recovery (Microbially-Enhanced Oil Recovery), attempts are being made to investigate possibilities of replacing them by their counterparts manufactured using microbiological synthesis, employing, for this purpose, primarily, bacteria and yeasts [3,12].

From the point of view of their properties, biosurfactants of microbiological origin are superior in comparison with their chemical counterparts, predominantly, in that they can be used in a wide range of temperatures, acidity and salt concentrations. Moreover, a greater interest in these compounds stems from the fact that biosurfactants - as "natural" products - are both biodegradable and non-toxic. Surface active agents synthesised by microorganisms belong mainly to: glycolipids, lipoproteins, phospholipids, fatty acids, neutral lipids and polymeric compounds [16, 19, 20].

Biosurfactants synthesised by microorganisms are either extracellular substances or are associated with the cell of a given organism. Their presence is observed, primarily, in the course of growth of microorganisms on media containing substrates, which are not soluble in water. Culture media used to manufacture surface-active agents require the presence of two sources of carbon: hydrophilic and hydrophobic. The most frequent sources include: glucose and plant or animal fats, their esters, fatty acids, alcohols and alkanes as well as whey, beer wort and a post-deodorizing condensate - a waste raw material from fat-processing industry and sapstok [1, 4, 7, 15].

Carbon sources (hydrocarbons, in particular) exert a strong impact on the type and composition of the manufactured biosurfactant and determine the place of its deposition [17, 21, 25]. In the case of Candida bombicola yeasts, the best source of nitrogen was a yeast extract. The substitution of yeast extract by a peptone, urea or nitrate reduces the yield of the final product. The optimal level of nitrogen for the production of surface active agents ranged from 0.2 < N < 0.6% [9, 20, 27]. However, it should be remembered that nitrogen belongs to factors securing the appropriate quantity of biomass, while the source of hydrophilic carbon determines the conversion of the available sources of carbon into biosurfactants.

Different activities are attributed to compounds obtained in the result of microbiological biosynthesis. It has been speculated that they are involved in the process of emulsifying substrates insoluble in water but they are also suspected of taking part in the cell adhesion and increasing their resistance to the action of harmful environmental factors as well as in the desorption of cells or antagonistic activities [4, 13].

The biosynthesis yield of surface active substances manufactured by yeasts ranges from some to several grams from 1 l of the medium and it depends, to a considerable degree, on the type of strain and conditions of their culture, including the medium composition.

The objective of the research project was to assess the impact of various fat substrates on the biosynthesis yield of surface-active compounds manufactured by the ATCC 22214 Candida bombicola.

MATERIALS AND METHODS

Biological material

The experimental biosurfactants were synthesised employing Candida bombicola ATCC 22214 yeasts obtained from the American Type Culture Collection.

Culture media

Nine culture media containing oleic acid (90% technical), sunflower oil and a post-deodorizing condensate (99.7% fat substances) as hydrophobic sources of carbon, glucose as the source of hydrophilic carbon and a yeast extract as a source of nitrogen were used in the performed investigations. Initial quantities of these constituents in each medium are shown in Table 1.The initial pH value of experimental media was brought to the level of 5.8 by adding 0.1 M solution of HCl. All media were sterilised at the temperature of 121°C for 20 min.

Table 1. Composition of culture media

Medium constituents [g · dm-3]	Culture
	with oleic acid Pp  Pw1  Pw2			with sunflower oil Pp  Pw1  Pw2			with post-deodorizing condensate Pp  Pw1  Pw2
Glucose	100	100	100	100	100	100	100	100	100
Yeast extract	5	5	5	5	5	5	5	5	5
Oleic acid	100	100	100	-	-	-	-	-	-
Sunflower oil	-	-	-	100	100	100	-	-	-
Post-deodorizing condensate	-	-	-	-	-	-	100	100	100

Pp - control media. Pw - media supplemented: 1 in the course of culture, the substrate was supplemented with glucose in the amount of 14.5 g·dm-3/day, beginning with day 3. 2 substrate was enriched with glucose in the amount of 25 g · dm-3/day, beginning with day 3.

The course of culturing

Depending on the carbon raw material applied in the medium, the duration of the performed experiments ranged from 14 to 21 days, while the temperature was maintained at 30°C. The experiment was conducted in shaken flasks of 300 cm³ volume in which, initially, there was 100 cm³ medium in each flask. The medium was inoculated with the volume of 10 cm³ inoculum, which contained from 2.8 x 10⁹ to 3.5 x 10⁹ cells of Candida bombicola yeasts.

The media with the basic composition were enriched from day three with glucose in the amount ranging from 1.45 to 2.5 g/100 ml of the medium (glucose introduced earlier was assimilated by yeasts). The reactivity of the experimental media was maintained at the same level of 3.5 throughout the experiment using, for this purpose, 5M NaOH. The conditions of running batch cultures were selected on the basis of investigations by Zhou and Kosaric [27] with the author´s own modifications [15].

Analytical methods

Throughout the experiment, the following parameters were checked every 48 h: biomass yield [27], concentration of reducing sugars [23], pH value and the level of biosurfactants [27,15].

In order to document the course of the multiplication of yeast cells and the process of biosynthesis, pictures were taken under the scanning electron microscope (SEM). Samples for microscopic observations (1 cm³) were taken during the process of culture. The inoculum constituted the initial material for the comparison. Yeasts preparations were prepared in accordance with the modified methodology described by Naperała-Filipiak et al. [24] by fixing them with glutaraldehyde buffered with sodium catodylate. Later the preparation was additionally fixed using the osmium gaudroxide buffered with sodium cacodylate, dehydrated with a series of alcohols of concentrations ranging from 30% to 90% and acetone (90% to 100%), dried in an apparatus designed to dry biological objects in a critical point (SPD-030 apparatus manufactured by the Balzes company) and sprayed with gold in an ion sprayer SCD-050 manufactured by the Balzes company. The experimental objects were examined in a scanning electron microscope SEM 515 of the Philips Company equipped in a special attachment for digital picture processing DISS (Digital Image Scanning System).

Measurements of the surface tension were performed using a Sigma 70 tensiometer manufactured by KSV Ltd. Company from Finland, which employs a platinum ring as a measuring system [6]. Aqueous biosurfactant solutions from culture were prepared in seven concentrations ranging from 0.0001% to 0.1%.

Statistical analysis

A two factorial analysis of variance as well as a significance test were carried out. Calculations were carried out using Statistica 5.0 program.

RESULTS AND DISCUSSION

Characterisation of the process of biosynthesis of surface-active compounds

The Candida bombicola yeasts multiplied using the method of batch culture synthesised surface-active compounds with different efficiency in relation to the fat substrates contained in the medium as well as to the amount and method of the addition of glucose (Fig. 1a, b, c). According to Asmer et al. [2], these compounds belong to glycolipids and are extracellular metabolites secreted to the culture medium.

The media and their modifications applied in this study allowed to obtain the efficiency of biosurfactants ranging from 116 g·dm^-3 to 124.5 g·dm^-3 (Fig. 1a, b, c).

Fig. 1a, b, c. Effect of the supplementation glucose on biosurfactants production in the culture media containing: (1a) oleic acid, (1b) sunflower oil, (1c) post-doeodorizing condensate

a)

b)

c)

All media applied in our experiments had their pH value regulated and maintained at 3.5 beginning from the third day. The regulation was introduced following suggestions of Davila et al. [11], Rau et al. [26] and McCaffrey and Cooper [22] who reported that the pH interval 2.5 to 3.5 was the optimal level for the biosynthesis of surface-active compounds. This was also confirmed by experiments conducted by Gumienna et al. [15] on media supplemented with oleic acid.

The fat substrates used in these studies differed with regard to their composition and, hence, with regard to their availability to yeasts. The results obtained in earlier experiments by Gumienna et al. [15] indicated the need to supplement the culture medium with a carbon source. The application of media supplementation with two carbon sources increased the efficiency of the isolated compounds by 30% in relation to the control medium. In the described experiment, in order to enhance the effectiveness of the process of biosynthesis and to reduce the costs of the process, the author enriched the control media with the amount of glucose ranging from 1.45 g to 2.5 g ·100 cm^-3 every 24 h beginning from the third day of culture.

The introduction of 2.5 g ·100 cm^-3 glucose into the medium, in which oleic acid was used as the fat substrate, allowed to reach on the 14th day of culture the yield of biosurfactants of 124.5 g·dm^-3. This output was by 60% higher than the yield achieved in the case of media which were not enriched by glucose. On the other hand, in comparison with the culture in which the addition of glucose was 1.45 g ·100 cm^-3, the biosynthesis process increased by 15% (p < 0.05) (Fig. 1a). A similar trend was observed in the case of experiments in which the sunflower oil and post-deodorising condensate (the most complex substrate) were used as carbon sources. Respective outputs of 116.5 g·dm^-3 and 118.5 g·dm^-3 of biosurfactants were obtained on day 14 (substrate with sunflower oil) and on day 12 (substrate with the post-culturing deodorising condensate) of culture on the medium enriched every 24 h with the additional amount of glucose - 2.5% (p < 0.05) (Fig. 1b, c).

Therefore, it can be said that the supplementation of the experimental media with 2.5 g ·100 cm^-3 glucose resulted in a significant (p < 0.05) increase in the levels of synthesised biosurfactants.

The level of the glucose utilised from the media was influenced by the type of the applied fat substrate as well as by the method of culture (Fig. 2a, b, c). A 90% loss of glucose already on the 3rd day of culture was recorded in the media in which oleic acid and sunflower oil were used as hydrophobic substrates. On the other hand, in the case of media in which 2.5 g glucose per 100 cm³ of the medium was applied, its level after the third day of culture reached about 20 g·dm^-3- the medium with the sunflower oil and 40 g·dm^-3 - the medium with the oleic acid (Fig. 2a, b). However, the glucose from the culture medium was utilised by the yeasts most effectively in the case of the medium in which the post-culturing deodorising condensate was applied as its concentration on the third day, irrespectively of the method of culture, was at the level of 1 g·dm^-3 (Fig. 2c).

Fig. 2a, b, c. Changes of glucose level in the course culture on media containing: (2a) oleic acid, (2b) sunflower oil, (2c) post-doeodorizing condensate

a)

b)

c)

When analysing the yield of the cell biomass of the experimental cultures, it turned out that it depended not only on the quantity of the added glucose but also on the source of hydrophobic carbon applied in the media. The most intensive production of the cell biomass was observed on the medium with the oleic acid supplemented with 2.5 g ·100 cm^-3 of the medium (30 g·dm^-3 - 10th day of culture) (Fig. 3a). It is quite probable that this considerable increase of the biomass in the case of the medium supplemented with the oleic acid in which the concentration of glucose was increased threefold can indicate that the Candida bombicola yeasts were also capable of utilising, for energy purposes, the oleic acid as an "easily" available substrate and, by doing so, prolong the logarithmic phase of the cell growth and extend the duration of culture. In addition, this can also explain the increased concentration of glucose in this medium during the final phase of culture.

Fig. 3a, b, c. Effect of the supplementation glucose on biomass production in the culture media containing: (3a) oleic acid, (3b) sunflower oil, (3c) post-doeodorizing condensate

a)

b)

c)

Studies carried out by Lee and Kim [21] showed that a surplus of "easy" carbon source (glucose) for synthesis was desirable in the growing medium because during the batch fermentation, 50% of carbon was utilised as energetic material, 37% - for biosurfactant synthesis and the remaining 13% - for the growth of yeasts cells.

A significant impact of glucose on the production of the cell biomass was also recorded in the case of the medium in which the sunflower oil was applied (p < 0.05) (Fig. 3b). On the other hand, in the case of the culture with the post-deodorising condensate, the supplementation of the medium with glucose in the amount of 2.5 g ·100 cm^-3 of the medium did not have a significant influence (p > 0.05) on the yield of the biomass (Fig. 3c).

The obtained results allowed concluding that the most favourable glucose addition to the culture medium, which assured the increase of the biosynthesis of surface-active compounds by the Candida bombicola yeasts, occurred when the medium was supplemented with 2.5 g ·100 cm^-3 glucose every 24 h. Similar results were reported by Asmer et al. [2] and Klekner et al. [18], who found that the best conditions for the biosurfactant biosynthesis were achieved when the weight ratio of the hydrophilic to hydrophobic substrate in the medium was 3:1. However, the obtained results also showed that the fat substrate used in the culture media also exerted a significant effect (p < 0.05) on the final yield of the product.

Morphological characterisation of yeast cells during culturing

Throughout the experiment, yeast cells were examined under the scanning electron microscope with the aim to determine their morphological changes and the impact of these changes on the biosynthesis process. Yeast cells were examined during their growth in the inoculum (yeasts before the introduction to the culture) as well as on the 1st, 10th and 12th days of culture when the post-deodorising condensate was applied as the fat substrate (Photo 1a, b, c, d).

Photo. 1. Photographs were taken under scanning electron microscope (SEM) yeasts cell Candida bombicola individuals days of culture: a) inokulum, b) day 10 of culture, c) day 12 of culture, d) structure of biosurfactants were observed of culture. Extension 512 x 512 x 256

a) inokulum yeasts	b) inokulum

c) yeasts - 10 day of culture	d) yeasts - 10 day - cell elongated

e) yeasts - 12 day of culture	f) biosurfactants

Although different hydrophobic substrates were applied in the established experiments, the synthesis dynamics of the biosurfactants was similar. Regardless of the applied fat substrate and the quantity of the glucose added to the culture media, the intensive increment of the biomass of surface-active compounds was initiated when the number of live yeast cells in the medium declined. Daniel et al. [10] maintain that it is the cell autolysis that is responsible for the phase of dying of yeast cells and, consequently, for the drop in their concentration in the culture medium. Before they were introduced into the culture media (from the inoculum), the Candida bombicola yeast cells did not show morphological differences. They were oval-shaped and revealed scars after budding (Photo 1a, b). However, on the day of the maximum production of the cell biomass (day 10 of culture), the initiation of the cell degradation (cells partially over-digested) was observed. Simultaneously, some of the examined cells changed their shapes (Photo 1c, d) and became more elongated. Most probably, daughter cells after budding failed to separate and formed one cell. Bonin and Wzorek [5] observed a similar behaviour of cells during fermentation. They suggested that the observed changes in the shape of cells could have been caused by their adaptation to the low glucose concentration in the medium. In their opinion, increased cell dimensions allowed better contact with nutrients. The above-mentioned thesis was confirmed in this study because glucose was assimilated by yeasts almost completely already after day 3 and, regardless of its applied concentration in the medium, the quantity of glucose in the medium on the 10th day of culture did not exceed 1 g^.dm^-3 (Fig. 2c). On the other hand, on day 12 of culture, when the level of biomass dropped in all media, the increase of the biosurfactant production was observed. Most probably, the intracellular structure in aging yeast cells underwent degradation during the process of autolysis and this, in turn, caused the outflow of cytoplasm from cells (Photo 1c, d, e). Enzymes produced during the logarithmic phase responsible for the process of synthesis were released into the culture medium together with the cytoplasm. Once in the medium, these enzymes took part in the biosynthesis and, hence, enhanced the output of surface-active compounds [10]. In addition, on days 10 and 12 of culture, yeast cells surrounded by some kind of "secretion" were observed. Microscope examination showed that the "secretions" were formed by crystals of the biosurfactant most probably secreted into the medium (Photo 1f).

On the basis of microscopic observations, it was concluded that the process of yeast cell autolysis may have a direst impact on the observed increase of the surfactant production because their level always increased when there was a considerable decline in the amount of the biomass in the medium. In addition, changes in the yeast cell morphology (development of elongated cells, cell autolysis) observed during culture most probably depended, to a significant degree, on the conditions existing in the culture media (expiration of the easy source of carbon - glucose from the medium, limited quantity of nitrogen).

Characterisation of surface properties of the isolated compounds

The performed experiments allowed to conclude that the output of the biosurfactant synthesis by the Candida bombicola yeast cells was significantly affected (p < 0.05) by the source of hydrophobic carbon and its ratio to the carbon characterised by hydrophilic properties. The Candida bombicola yeast cells secreted into the culture media compounds, which, depending on the applied fat substrate, differed with respect to their appearance (Photo 2a, b, c). Compounds isolated from cultures containing oleic acid looked like white powder irrespectively of the applied media composition and supplementation of the carbon source (Photo 2a). A similar consistency - powder of creamy-beige shade, was also observed in the case of preparations obtained from the media in which the post-deodorising condensate was employed (Photo 2c). On the other hand, in the case of media containing the sunflower oil, the obtained product had the form of gold greasy paste (Photo 2b). The observed variations in the physical appearance of the products isolated from the experimental media, which contained different fat substrates, indicate their diversification with regard to their structural makeup and, consequently, imply different surface and biological properties of the obtained compounds. Some of them precipitated in the course of culture and produced sediment. According to Rau et al. [26], these are lactone forms, which are characterised by poor water solubility. On the other hand, acid forms are primarily concentrated in the culture medium. The solubility of these compounds depends strongly on the pH value. Glycolipids exhibit the best solubility at pH above 6 [9,14].

Photo. 2a. Compounds isolated from cultures containing oleic acid - look like white powder

Photo. 2b. Compounds isolated from cultures containing sunflower oil - look like gold greasy paste

Photo. 2c. Compounds isolated from cultures containing the post-deodorising condensate - look like powder of creamy-beige shade

In addition, it turned out that the carbon source affected not only the output of the produced compounds but also the properties of the obtained biosurfactants. According to Cooper and Paddock [9], Otto et al. [25]; Adamczak and Bednarski [1]; Hu and Ju [17] as well as Cavalero and Cooper [8], the capability to reduce surface tension can result from the structure of biosurfactants and this, in turn, depends on the chemical structure of the carbon source and the mechanism of biosynthesis of surface-active compounds.

The dependence was confirmed while investigating the capability to reduce surface tension. It turned out that the biosurfactants synthesised on the media containing oleic acid and the post-deodorising condensate reduced water surface tension from 72 mN·m^-1 to, respectively, 34 mN·m^-1 and 35 mN·m^-1 at the biosurfactant concentration in water solution of 0.1%. On the other hand, compounds isolated from the media containing the sunflower oil reached the value of water surface tension of 35 mN·m^-1 already at the biosurfactant concentration in water solution of 0.05%. The final value of the surface tension was 32 mN·m^-1 at the biosurfactant concentration in the solution of 0.1% (Fig. 4).

Fig. 4. Effect of concentration biosurfactants on changes of surface tension of aqueous solutions

CONCLUSIONS

1. Media containing glucose (10%), fat substrate (10%) and yeast extract (0.5%) turned out to be the most effective for the biosynthesis of surface-active compounds. However, it was also necessary to enrich the media every 24 h with the amount of 2.5 g·100 cm^-3 of the medium from day 3 of the culture.

2. The performed experiments confirmed the possibility of utilising the post-deodorising concentrate - a by-product from the fat-processing industry - for the biosynthesis of surface-active substances. The post-deodorising concentrate as the fat substrate applied in the culturing medium provided a valuable source of carbon of hydrophobic properties similar to that provided by oleic acid and sunflower oil.

3. The composition of the isolated mixture and, consequently, the form of the obtained final product were both affected, primarily, by the source of carbon of hydrophobic properties. The obtained products reduced water surface tension from 70 mN·m^-1 to about 32 mN·m^-1 at 0.1% concentration in the solution of biosurfactants isolated from the medium with the sunflower oil.

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Małgorzata Gumienna
Department of Fermentation and Biosynthesis,
Institute of Food Technology of Plant Origin,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 31, 60-624 Poznan, Poland
ph: (+4861) 848 72 67
email: e-mail:gumienna@au.poznan.pl

Maria Czarnecka
Department of Fermentation and Biosynthesis,
Institute of Food Technology of Plant Origin,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 31, 60-624 Poznan, Poland

Zbigniew Czarnecki
Department of Fermentation and Biosynthesis,
Institute of Food Technology of Plant Origin,
The August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 31, 60-624 Poznan, Poland
email: zbyczar@au.poznan.pl

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