Scholarly article on topic 'Utilization of a waste glycerol fraction using and reusing immobilized  Gluconobacter oxydans  ATCC 621 cell extract'

Utilization of a waste glycerol fraction using and reusing immobilized Gluconobacter oxydans ATCC 621 cell extract Academic research paper on "Industrial Biotechnology"

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{Biodiesel / Biofuels / "Conversion of waste glycerol" / Dihydroxyacetone / "Fuel demand" / "Glycerol dehydrogenase" / "Glycerol waste fraction valorization" / "Immobilized cell extract" / "Renewable energy" / "Substrate consumption" / Utilization}

Abstract of research paper on Industrial Biotechnology, author of scientific article — Lidia Stasiak-Różańska, Stanisław Błażejak, Iwona Gientka, Anna Bzducha-Wróbel, Edyta Lipińska

Abstract Background Depletion of petroleum resources has enforced the search for alternative sources of renewable energy. Introduction of biofuels into the market was expected to become a solution to this disadvantageous situation. Attempts to cover fuel demand have, however, caused another severe problem—the waste glycerol generated during biodiesel production at a concentration of approximately 10% w/w. This, in turn, prompted a global search for effective methods of valorization of the waste fraction of glycerol. Results Utilization of the waste fraction at 48h with an initial glycerol concentration of 30g·L-1 and proceeding with 62% efficiency enabled the production of 9g·L-1 dihydroxyacetone at 50% substrate consumption. The re-use of the immobilized biocatalyst resulted in a similar concentration of dihydroxyacetone (8.7g·L-1) in two-fold shorter time, with an efficiency of 85% and lower substrate consumption (35%). Conclusions The method proposed in this work is based on the conversion of waste glycerol to dihydroxyacetone in a reaction catalyzed by immobilized Gluconobacter oxydans cell extract with glycerol dehydrogenase activity, and it could be an effective way to convert waste glycerol into a valuable product.

Academic research paper on topic "Utilization of a waste glycerol fraction using and reusing immobilized Gluconobacter oxydans ATCC 621 cell extract"

Accepted Manuscript

Utilization of a waste glycerol fraction using immobilized cell extract from Gluconobacter oxydans ATCC 621 and its possible re-use

Lidia Stasiak-RoZanska, Stanislaw BlaZejak, Iwona Gientka, Anna Bzducha-Wrobel, Edyta Lipinska

PII: S0717-3458(17)30011-8

DOI: doi:10.1016/j.ejbt.2017.03.003

Reference: EJBT231

To appear in:

Electronic Journal of Biotechnology

Received date: Accepted date:

4 November 2016 7 March 2017

Please cite this article as: Stasiak-Rozanska Lidia, Blazejak Stanislaw, Gientka Iwona, Bzducha-Wrobel Anna, Lipinska Edyta, Utilization of a waste glycerol fraction using immobilized cell extract from Gluconobacter oxydans ATCC 621 and its possible re-use, Electronic Journal of Biotechnology (2017), doi: 10.1016/j.ejbt.2017.03.003

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Electronic Journal of Biotechnology EJBT-D-16-00175 R1 Original Research Article Received: November 4, 2016 Accepted: March 7, 2017

Areas: Food Biotechnology; Microbial Biotechnology;

Short title: Utilization of a waste glycerol with immobilized cell extract from G. oxydans

Utilization of a waste glycerol fraction using immobilized cell extract from Gluconobacter oxydans ATCC 621 and its possible re-use

Lidia Stasiak-Rozanska*, Stanistaw Btazejak, Iwona Gientka, Anna Bzducha-Wrobel, Edyta Lipinska

Department of Biotechnology, Microbiology and Food Evaluation, Warsaw University of Life Sciences, Nowoursynowska 166 St., 02-787 Warsaw, Poland

Corresponding author: lidia_stasiak@sggw.pl

Abstract

Background: Depletion of petroleum resources has enforced the search for alternative sources of renewable energy. Introduction of biofuels onto the market was expected to become a solution to this disadvantageous situation. Attempts to cover fuel demand have, however, caused another severe problem - a waste glycerol generated during biodiesel production in the quantity of ca. 10% w/w. This, in turn, prompted a global search for effective methods of valorization of the waste fraction of glycerol. Results: The 48 h utilization of the waste fraction with the initial glycerol concentration of 30 gL-1, proceeding with 62% efficiency, enabled producing dihydroxyacetone with the concentration of 9 gL-1, at 50% substrate consumption. The re-use of the immobilized biocatalyst allowed achieving a similar concentration of dihydroxyacetone (8.7 gL-1) in two-fold shorter time at the efficiency of 85% and lower substrate consumption (35%). Conclusions: The method proposed in this work is based on conversion of the waste glycerol to dihydroxyacetone in the reaction catalyzed by an immobilized cell extract with the activity of glycerol dehydrogenase, produced from Gluconobacter oxydans bacteria and it could be an effective way to utilize the waste glycerol into a valuable product.

Keywords: biodiesel; biofuels; conversion of waste glycerol; dihydroxyacetone; fuel demand; glycerol dehydrogenase; glycerol waste fraction valorization; immobilized cell extract; renewable energy; substrate consumption; utilization.

1. Introduction

Fuel demand has been intensively growing worldwide in recent years. At the same time, increasing attention has been paid to the protection of natural environment [1,2]. It was, therefore, necessary to find alternative sources of renewable energy. The replacement of petroleum with biofuel was expected to become a successful solution to this situation. Instead, it has contributed to the generation of waste glycerol which today poses a

severe environmental problem [1]. It is estimated that with each year the global production of biodiesel will increase by 42% compared to the preceding year. Experts of the fuel market have estimated that by 2016 the level of biodiesel production would reach 140 millions of tons, which means generation of ca. 14 millions of tons of the waste glycerol [3].

The development of biodiesel production methods with reduced generation of the waste glycerol is currently at the laboratory stage and many years would pass till their application on the industrial scale [4,5]. It is, therefore, advisable to search for effective and safe to the environment methods for the utilization of waste glycerol. Many of the so far proposed methods were based on the widely understood metabolic potential of microorganisms that converted the waste glycerol into valuable and industrially-desirable chemical compounds [6].

Some papers have appeared recently [1,3] that present new possibilities of the microbiological utilization of waste glycerol including, e.g., the method for 1,3-propanediol production by Citrobacter freundii strain in the culture medium with waste glycerol [7]. Similar investigations were successfully conducted with bacteria of the species Klebsiella pneumoniae and Clostridium butyricum, with proven capability to produce 1,3-propanediol from waste glycerol [8,9,10]. Attempts were also undertaken to convert glycerol into ethanol [11], citric acid [12] and polyhydroxyalkanates [13].

Another interesting idea of waste glycerol management may be its transformation into dihydroxyacetone (DHA), namely a compound with vast industrial applicability. Today, DHA is applied in, e.g., food, cosmetic and pharmaceutical industries and in medicine, but its application possibilities are still being extended [14,15,16,17]. The annual production of DHA reaches ca. 2000 tons [18]. The most common method of its industrial production involves glycerol biotransformation with the use of free cells of acetic acid bacteria of Gluconobacter oxydans species. This method has, however, some technological drawbacks that are difficult to eliminate [18]. The main problems of biotechnological production of DHA include susceptibility of G. oxydans bacteria to a high concentration of glycerol and inhibition of this compound oxidation by increasing DHA concentration in the culture medium [19].

The G. oxydans ATCC 621 strain of acetic acid bacteria oxidizes glycerol to DHA in the reaction catalyzed by glycerol dehydrogenase (GlyDH, EC 1.1.99.22). This enzyme is strictly connected with the cytoplasmic membrane of G. oxydans [20,21], and its action depends on the presence of PQQ cofactor [22]. Due to a strongly hydrophobic character and low stability of the purified fraction of GlyDH it is difficult to determine the spatial structure of this enzyme [23,24,25]. The optimal temperature of GlyDH action ranges from 23 to 25°C, and the optimal pH from 7.0 to 7.5 [23,24,26].

Our previous study [27] demonstrated the feasibility of applying an immobilized cell extract with the activity of glycerol dehydrogenase from G. oxydans for biotransformation of a glycerol solution into DHA. This work reports on the attempt to utilize the waste fraction of glycerol after biodiesel production for DHA generation in the reaction catalyzed by an immobilized cell extract with the activity of GlyDH from G. oxydans.

Another objective of this study was to check whether the cell extract used in one utilization cycle would exhibit enzymatic activity in the successive cycle.

2. Materials and methods

2.1. Biological materials

The study was conducted with acetic acid bacteria strain G. oxydans ATCC 621 (Manassas, NY).

Waste glycerol from biodiesel production originated from BIOAGRA-OIL S.A. plant (Tychy, Poland). Characteristic of waste glycerol which was used in our experiments: 24°Blg, pH 6.12, concentration of glycerol 659,5 gL-1, dry substance content 69,76%, medium content of individual elements [mg» g-1]: Ca (0,33), K (0,07), Mg (0,04), Na (19,06), P (0,08).

2.2. Culture media

Culture medium for storage of G. oxydans strain [gL-1]: yeast extract 5, peptone 3, mannitol 25, agar 15, 48 h, 28°C.

Inoculation medium [gL-1]: yeast extract 30, ethanol 20, pH 5.0, 24 h, 28°C.

Culture medium for GlyDH activation [gL-1]: yeast extract 5, glycerol 20, (NH4)2SO4 5, pH 5.0, 48 h, 28°C [28].

Reagents: Avantor Performance Materials Poland. The culture media were sterilized at a temperature of 121°C for 20 min.

2.3. Preparing of cell extract

After G. oxydans culture in the activation medium (the end of stationary phase), the resultant biomass was centrifuged and rinsed. Wet biomass (0.44 g) was suspended in 60 cm3 of sterile distilled water and subjected to ultrasonic disintegration (210 W, 18 kHz, 4°C, 5 min) in an Omni Ruptor 4000 apparatus with Titanium 3/8 DiaSolid tip [29,30].

2.4. Immobilization

The disintegrated cell extract was mixed (1:1, v/v) with sodium alginate (40 gL-1, Fluka) and added to a 0.2 M solution of CaCl2 (Avantor Performance Materials Poland) using a syringe with a needle 0.9 mm in diameter. The immobilized cell extract was incubated in CaCl2 at 4°C for 3 h [31].

2.5. Biotransformation of the waste fraction of glycerol to DHA

The immobilized cell extract was transferred into 150 cm3 of the waste glycerol fraction, diluted with distilled water to glycerol content of 30 gL-1, pH 7.5. The experiment was carried out in 500 cm3 flasks, on a reciprocating shaker (200 rpm) at 23°C for 168 h. Afterwards, the immobilized cell extract was separated, rinsed with sterile distilled water and immediately transferred to flasks containing the fresh waste fraction of glycerol.

2.6. Determination of glycerol concentration

Glycerol concentration was determined with the use of Free Glycerol Reagent (FGR, Sigma-Aldrich F6428). The FGR (0.80 cm3) was mixed with 0.01 cm3 of the analyzed sample and the mixture was incubated for 5 min at 37°C. Blank and standard samples, instead of the exact experimental sample, contained 0.010 cm3 of water or Glycerol Standard Solution (Sigma-Aldrich, G7793), respectively. Absorbance (A) of the exact sample, blank sample and standard was read out at the wavelength of 540 nm. Glycerol concentration [g/100 cm3] was computed from the formula: (Asample-Ablank)/(Astandard-Ablank)x0.26.

2.7. Determination of DHA concentration

2 cm3 of 3,5-dinitrosalicylic acid (Fluka) were added to 2 cm3 of the analyzed sample. The mixture was incubated at 100°C for 10 min, then cooled and transferred quantitatively into 20 cm3 of water. Absorbance of the sample was measured at the wavelength of A = 550 nm against control which contained water instead of the sample. DHA concentration was determined based on the regression equation of absorbance dependency on the concentration of standard solutions [32].

2.8. Reaction efficiency

The efficiency of reaction was calculated based on the stoichiometric equation of a chemical reaction indicating that 90 g mol-1 DHA was produced from 92 g mol-1 of glycerol.

Biotransformation was conducted in three independent series. Each determination was carried out in three replications.

2.9. Statistical analysis

Standard deviation was calculated for all experimental results. 3. Results and discussion

3.1. Utilization of the waste fraction of glycerol with the use of an immobilized cell extract with the activity of glycerol dehydrogenase from G. oxydans ATCC 621

Changes in DHA and glycerol concentration, substrate consumption and efficiency of the utilization of the waste glycerol fraction with the use of an immobilized cell extract with the activity of glycerol dehydrogenase from G. oxydans were presented in Table 1.

After 24 h of the process, the mean concentration of produced DHA reached 6.9 ± 0.07 gL-1, the mean concentration of glycerol in the solution reached 22 ± 0.06 gL-1, and the reaction proceeded with the mean efficiency of 90%, which was the highest noted efficiency in the first utilization cycle (Table 1). After this time, substrate consumption reached 27% (Table 1). After 48 h of biotransformation, mean DHA concentration increased compared to the previous measured value and reached 8.9 ± 0.03 gL-1 (Table 1). After this time, the content of glycerol remaining in the waste fraction decreased by half compared to the initial concentration. After 48 h, the mean efficiency of utilization decreased by ca. 28% and reached the value of 62% (Table 1). Successive reaction did not cause an increase in DHA concentration, but its slight reduction (Table 1). The mean glycerol content in the waste fraction after 72-h valorization accounted for 11 ± 0.10 gL-1 (Table 1). The greatest glycerol consumption (63%) was determined after 72 h of utilization (Table 1).

Results obtained demonstrate that GlyDH present in the cell extract exhibited the highest activity in the first 24 h of the process. It is consistent with literature data [18]. Glycerol dehydrogenase is an enzyme which is bound with the cytoplasmic membrane of G. oxydans bacteria, and whose active center is in the periplasmic space. Glycerol is directly oxidized to DHA which, in turn, is released outside the cell [18]. The purified GlyDH fraction was proved to be less stable and to lose its activity more rapidly than the fraction whose enzyme remains bound with the cytoplasmic membrane [33]. It was also demonstrated that the purified enzyme lost 70% of its activity in the third day of storage [24]. Owing to low stability of the purified enzyme, it has not been thoroughly characterized so far [34]. The activity of GlyDH depends on active acidity of the medium. It was determined that GlyDH (isolated from the strain Gluconobacter sp. 33 and cleaned of membranes) exhibited the highest activity in a pH range of 7.0-7.5, whereas stability at pH 8.5-9.5 [24]. In the present study, the initial pH of the reaction was pH 7.5. After 168 h of utilization, the active acidity of the waste glycerol decreased to 4.0. Acetic acid bacteria oxidize glycerol to DHA. A glycerol aldehyde is, simultaneously, formed in this reaction that is then converted into glyceric acid which probably contributed to medium pH decrease to the value of 4.0 [18]. This change of active acidity during utilization could have an immediate effect upon GlyDH activity decrease and, consequently, upon lesser increases of DHA concentration. In future studies, utilization ought to be conducted under conditions that would ensure stable pH of the waste fraction optimal for the activity of GlyDH (e.g. in a biofermenter). No studies have been published so far on the characteristics of GlyDH from the strain used in our study (G. oxydans ATCC 621). Therefore, it may be assumed that optimal parameters of this enzyme activity may differ from those reported in research works for GlyDH isolated from strains of acetic acid bacteria [25,33].

The efficiency of biotransformation after 72 h reached 44%, and in the successive measuring intervals it did not exceed 28% (Table 1). After 100 h, no glycerol was found in the waste fraction (Table 1). Similar results were obtained by Celik et al. [35], who demonstrated that G. oxydans NBRC12528 strain oxidized glycerol to DHA only in the first 24 h of the reaction. Further biotransformation did not cause any increase in DHA concentration, but only transformation of the remaining glycerol into glyceric acid [35,36].

Apart from the predominating glycerol, the waste glycerol usually contains also many impurities including residues of methanol, NaOH, fats, oils, esters, small quantities of sulfur compounds, proteins and minerals [35]. The composition of the waste glycerol fraction depends, among other things, on the type of catalyst applied for biodiesel production, efficiency of transesterification, efficiency of biodiesel and catalyst recovery, and also on conditions of separation of the polar and non-polar fraction [1,37]. Some contaminants of the waste fraction, particularly free fatty acids, may inhibit many processes of bacterial fermentation, e.g. production of 1,3-propanodiol in cells of C. butyricum [10,38,39], and may negatively affect many bacterial metabolic processes

In the present study, although DHA concentration did not increase after 48 h of utilization, some part of glycerol was consumed (Table 1). Apart from GlyDH, the immobilized cell extract contained also other enzymes, including enzymes bound with the cytoplasmic membrane, which (despite the application of conditions optimal for the activity of GlyDH) could exhibit some activity against glycerol and catalyze some oxidation reactions, e.g. of alcohols and polyols, in a stereo- or regioselective manner

[41]. Cells of acetic acid bacteria contain at least eight characterized and two so far not characterized dehydrogenases bound with the cytoplasmic membrane [20]. These include, among others, alcohol dehydrogenase [42,43], inositol dehydrogenase [44], aldehyde dehydrogenase [20,45], sorbitol dehydrogenase [46,47], D-glucone dehydrogenase [48], glucose dehydrogenase [49], lactate dehydrogenase [50], and glycerol dehydrogenase [25,51]. It cannot be excluded, therefore, that the mentioned dehydrogenases were active during waste glycerol biotransformation to DHA. The presence of these enzymes in the immobilized cell extract could, to some extent, affect the reactions proceeding during utilization of the glycerol waste (glycerol could be transformed into other compounds than DHA), and by this means also process efficiency.

The process of biotransformation was continued for 168 h to make sure that DHA content was not decreasing along with time, e.g. as a result of further transformations. From the 96th h, DHA concentration accounted for 7.7 ± 0.01 gL-1, and in the last measuring period, i.e. after 168 h, for 7.4 ± 0.30 gL-1 (Table 1). These results demonstrate stability of the produced dihydroxyacetone. Earlier study [52,53] indicated that DHA was the most stable at pH 4.0, and such a value of active acidity was determined in our study after completed utilization.

3.2. Utilization of the waste glycerol fraction with re-used immobilized cell extract with the activity of glycerol dehydrogenase from G. oxydans ATCC 621

The second part of the study was conducted in order to verify the feasibility of re-using the catalytic activity of GlyDH in the immobilized cell extract from G. oxydans for utilization of the waste fraction of glycerol. Changes in concentrations of DHA and glycerol, substrate consumption and efficiency of utilization conducted with the re-applied cell extract were summarized in Table 2.

After 24 h of utilization with the re-used cell extract, the mean concentration of DHA

-1 -1 reached 8.7 ± 0.06 gL (Table 2). This value was higher by 1.8 gL from the value

determined after the same time during utilization with the cell extract applied for the first

time (Table 1). Glycerol consumption during 24 h of the second utilization cycle (with re-

applied cell extract) reached 35% and was higher by 8% compared to the first cycle of

utilization (Table 1, Table 2). Utilization efficiency after 24 h reached 85% (Table 2). It is

speculated that the results obtained could be influenced by the earlier activation of

GlyDH induced by a low glycerol concentration in the culture medium [54] and by

transferring the cell extract from lower pH (the mean pH after 168 h reached 4.0) to pH

7.5 (pH value of the freshly prepared glycerol waste). After 48 h of the second utilization

cycle, the medium concentration of DHA accounted for 7.6 ± 0.06 gL-1, glycerol content

in the waste - for 19 ± 0.14 gL-1 (at 36% substrate consumption), whereas process

efficiency reached 73% (Table 2) and was higher by 11% from the efficiency determined

after 48 h of the first utilization cycle (Table 1). After 48 h of the reaction with the re-

applied cell extract, no increase was observed in DHA concentration in the post-reaction

mixture (likewise during the first cycle). The above results suggest that waste glycerol

may be effectively utilized during the first 48 h of the process. It is an important clue for

the future development and improvement of waste valorization method for DHA

production. An attempt should be undertaken in future studies to conduct utilization in

the semi-continuous mode with fresh portion of the waste fed every 48 h and with

collection of the resultant product under optimal conditions for GlyDH activity

(temperature 23°C and pH 7.5).

In contrast to the first cycle of utilization, the re-used cell extract did not cause further transformation of glycerol, which was indicated by glycerol concentration (19 ± 0.14 gL-1) maintaining at the same level till the end of experiment (Table 2). Glycerol that was completely transformed during biotransformation with immobilized cell extract (Table 1), was present in the mixture media at a concentration of about 11 gL-1 during the biotransformation with re-application of the immobilized cell extract from G. oxydans (Table 2). It is believed that the immobilized cell extract used in the first reaction contains enzymes that have sufficient concentration of cofactors (GlyDH is PQQ-dependent enzyme) and therefore to be fully active and converted to glycerol. Probably reuse of immobilized cell extract without cofactor supplementation could reduce the activity of enzymes involved in the biotransformation of glycerol or cause complete inactivation of them. Small changes in DHA concentration in the successive hours of utilization cycle (ranging from 0.68 ± 0.05 gL-1 in the 72nd h to 6.5 ± 0.01 gL-1 in the 168th h) obviously influenced changes in process efficiency despite unchanging concentration of the product (Table 2).

The concentration of DHA obtained in the second cycle of utilization could be affected by PQQ content in the reaction medium. It is assumed that during the first biotransformation process the concentration of PQQ was sufficient to maintain high activity of GlyDH in the immobilized cell extract. During the second cycle of utilization (conducted with the use of the same immobilized extract), the content of PQQ could be too low to ensure the proper functioning of GlyDH. Glycerol dehydrogenase is an enzyme whose activity depends on the presence of PQQ cofactor [24]. The proposed method for utilization of waste glycerol should be modified in the future considering the appropriate concentration of PQQ in the

medium of action of the immobilized cell extract with the activity of GlyDH. The efficiency of biotransformation could also be influenced by the selection of the carrier and by method of cell extract immobilization. Partial or complete saturation of the active sites of GlyDH with the substrate could occur during oxidation of glycerol, which might impair the course of the reaction. The release of DHA to the culture medium could be impaired by the resistance of the alginate carrier which had to be overcome by diffusing molecules of the substrate and the product. Some limitations resulting from insufficient aeration of the reaction medium could not be excluded as well. Other Authors [55] provided an experiment with better success of re-use immobilized enzyme. They adsorbed GlyDH into magnetically-separable mesoporous silica with 38 nm mesocellular pores connected via 18 nm window mesopores (ADS), and further crosslinked via a simple glutaraldehyde treatment to prepare nanoscale enzyme reactors of GDH (NER). The residual activities of the free GDH and ADS could no longer be detected after 8 days and 22 d, respectively, while the NER maintained 64% of its initial activity even after 24-day incubation. The time-dependent conversion of glycerol to DHA was measured for both ADS and NER not only by ^analyzing the generation of NADH spectrophotometrically but also via the HPLC analysis measuring the increase of the concentration of DHA. Magnetically-separable NER maintained 39% of its initial activity after seven cycles of re-application, while the residual activity of ADS dropped to 13% of its initial activity after only two re-applications [55].

Oxidation of glycerol to dihydroxyacetone catalyzed by immobilized cell extract requires further optimization because efficient of DHA is not sufficient. In traditional method using whole-cell G. oxydans, glycerol and DHA can inhibit the metabolic activity of G. oxydans and consequently - inhibit the production of DHA. Traditional method requires cell proliferation every time when reaction starts. Furthermore supplementation of culture media is needed for create optimal condition for G. oxydans growth, for example magnesium or some amino acids [56]. Crystalization of final product from media is dificult and the first step is separation of bacteria cells. Obtaining DHA with immobilized cell extract involves the use of the enzyme. In the same time G. oxydans cells are inactivated after desintegration. Therefore supplementation of media by growth components are not necessary as well as earlier activation of GlyDH (which shortens the preparation stage of the process). Immobilization allows easy separation of the biocatalyst from the reaction mixture. In this study also showed that the immobilized cell extract does not lose GlyDH activity and it can be re-used in the next cycle of glycerol oxidation, which is an advantage of the proposed method, because in the future (after optimization these method) it can shorten the process and lower the cost effectiveness.

Conflict of interest

The authors declare that they have no conflict of interest. References

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Table. 1. Changes in concentration of DHA and glycerol (arithmetic mean of free independent series ± standard deviation), substrate consumption and utilization efficiency of the waste glycerol fraction with the use of an immobilized cell extract from G. oxydans.

Time [h] 0 24 48 72 96 120 144 168

DHA [gL-1] 0 ± 0.00 6.9 ± 0.07 8.9 ± 0.03 8.3 ± 0.0 7.7 ± 0.01 7.5 ± 0.06 7.2 ± 0.08 7.4 ± 0.03

Glycerol [gL-1] 30 ± 0.00 22 ± 0.06 15 ± 0.02 11 ± 0.10 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00 0.0 ± 0.00

Substrate

consumption 0 27 50 63 100 100 100 100

Reaction efficiency [%] 0 90 62 44 28 26 25 24

Table. 2. Changes in concentration of DHA and glycerol (arithmetic mean of three independent series ± standard deviation), substrate consumption and efficiency of utilization of the waste fraction with reapplication of the immobilized cell extract from G. oxydans.

Time [h] 0 24 48 72 96 120 144 168

DHA [gL-1] 0.0 ± 0.00 8.7 ± 0.06 7.6 ± 0.06 6.8 ± 0.05 6.9 ± 0.03 6.6 ± 0.03 6.6 ± 0.02 6.5 ± 0.01

-1 Glycerol [gL-1] 30 ± 0.00 19 ± 0.16 19 ± 0.14 19 ± 0.14 19 ± 0.14 19 ± 0.14 19 ± 0.14 19 ± 0.14

Substrate consumption [%] 0 35 36 36 36 36 36 36

Reaction efficiency [%] 0 85 73 65 66 63 63 62