Factors affecting the yield and properties of bacterial cellulose

Factors affecting the yield and properties of bacterial cellulose

(Parte 1 de 3)

Factors affecting the yield and properties of bacterial cellulose A Krystynowicz, W Czaja, A Wiktorowska-Jezierska, M Goncalves-Miskiewicz, M Turkiewicz and S Bielecki Institute of Technical Biochemistry, Technical University of Lodz, Stefanowskiego 4/10, Lodz 90-924, Poland

Acetobacter xylinum E25 has been applied in our studies in order to find optimal culture conditions for effective bacterial cellulose (BC) production. The strain displays significantly higher stability in BC production under stationary culture conditions. In contrast, intensive agitation and aeration appear to drastically reduce cellulose synthesis since such conditions induced formation of spontaneous cellulose nonproducing mutants (Cel– ), which dominated in the culture. Mutation frequency strictly depends on the medium composition in agitated cultures. Enrichment of the standard SH and Yamanaka media with 1% ethanol significantly enhanced BC production in stationary cultures. Horizontal fermentors equipped with rotating discs or rollers were successfully applied in order to improve culture conditions. Relatively slow rotation velocity (4 rpm) and large surface area enabling effective cell attachment are optimal parameters for cellulose production. Physical properties of BC samples synthesized either in stationary cultures or in a horizontal fermentor revealed that cellulose from stationary cultures demonstrated a much higher value of Young’s modulus, but a much lower value of water-holding capacity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 189–195 doi:10.1038/sj.jim.7000303

Keywords: bacterial cellulose; Acetobacter xylinum; rotating disc fermentor; stationary culture; Young’s modulus; Cel

Introduction

Bacterial cellulose (BC), a biopolymer produced by some strains of Acetobacter, has unique physical and chemical properties. These useful characteristic properties result from specific and unique BC ultrastructure, which is particularly characterized by a net of ultrafine cellulose ribbons [21,28].

Several techniques for BC production have been reported [5,6,2,28], some of which demonstrate a potential tool for economic and commercial BC production: stationary culture (in plastic trays), agitated culture (jar fermentors), cultivation in horizontal fermentors or cultivation in internal loop airlift reactors [4,14,15,23].

The choice of a cultivation technique is dependent on further biopolymer commercial destination, considering that cellulose ultrastructure and its physical and mechanical properties are strictly influenced by the culture method [1,10]. In stationary culture conditions, a thick, gelatinous membrane of BC is accumulated on the surface of a culture medium, whereas under agitated culture conditions, cellulose can be produced in the form of a fibrous suspension, irregular masses, pellets or spheres [9,21,28]. While stationary culture has been widely investigated and applied for production of some successful commercial cellulose products (nata de coco, transducer diaphragms, wound care dressing materials), agitated culture is considered more suitable for the commercial production of BC mainly due to the higher production rates that potentially can be achieved [4,17,20,30]. However, cellulose production in fermentors with continuous agitation and aeration encounters many problems, including spontaneous appearance of Cel mutants (cellulose nonproducers), which contributes to a decline in polymer synthesis [21]. Recent investigations showed that in agitated cultures, high oxygen supply and high volumetric agitation power are required for increase of BC productivity [16]. Other factors such as agitator configuration, effects of oxygen and carbon dioxide pressures on BC productivity have been investigated [15,16,32].

Efforts to apply other types of bioreactors for efficient cellulose production have been undertaken. Chao et al [6] successfully used an airlift reactor provided with oxygen-enriched gas supply to improve the oxygen transfer rate and obtain a high cellulose production rate. Static continuous culture in trays — in which the pellicle synthesized on the surface is picked up, passed through the bath in order to kill the cells and set on the winding roller — was recently reported by Sakairi et al [2]. Cellulose production in horizontal fermentors, which is considered to be a combination of stationary and agitated cultures, has also revealed promising results [5,23]. In our research, we examined the usefulness of Acetobacter xylinum E25 for efficient production of BC in selected culture conditions. Our studies started with characterization of A. xylinum

E25 with regard to its tendency to form spontaneous Cel mutants under different culture conditions. We investigated a process of BC production in stationary cultures, which were the most suitable method for cultivation of our strain. Horizontal bioreactors equipped with either rotating discs or rollers have also been successfully applied in order to improve culture conditions. Simultaneously, some characteristic physical and mechanical properties of the synthesized polymer have been determined.

Materials and methods

Microorganism

A. xylinum E25 (recently reclassified as Gluconacetobacter xylinus) [29] from the collection of the Institute of Technical Biochemistry,

Technical University of Lodz, Poland, was used.

Correspondence: Dr Stanislaw Bielecki, Institute of Technical Biochemistry, Technical University of Lodz, Stefanowskiego 4/10, Lodz 90-924, Poland Received 1 March 2002; accepted 18 July 2002 w.nature.com/jim

Culture medium The Schramm and Hestrin medium [1] with initial pH adjusted to 5.7 or its modifications, as well as Yamanaka medium [30] or its modifications (described in the text), were used unless otherwise specified.

Culture conditions Preinoculum for all experiments was prepared by transferring a single Acetobacter Cel + colony grown on SH agar medium into a 50-ml Erlenmeyer flask filled with liquid SH medium. After 48– 64 h of cultivation at 308C, the cellulose pellicle formed on the surface of the culture broth was either squeezed or vigorously shaken in order to remove active cells embedded in the cellulose membrane. Ten milliliters of the cell suspension was introduced into a 500-ml Erlenmeyer flask containing 100 ml of a fresh SH medium. The culture was carried out statically for 48 h and the cell suspension derived from the synthesized cellulose pellicle was used as the inoculum for further cultures.

All cultures were grown at 308C. The stationary cultures in either plastic trays (0.25 0.17 0.08 m) or in Erlenmeyer flasks filled with different volumes of the medium lasted for 7 days. Cultures in horizontal bioreactors equipped with discs or a roller were carried out in two fermentors having different volumes: 1 and

2l, respectively, for 7 days. Agitated cultures were carried out in 500-ml flasks on a rotary shaker at 90 rpm. The synthesized cellulose was harvested, purified by boiling it in 1% NaOH for 2 h, treated with 5% acetic acid and finally thoroughly washed in tap water until the product became transparent.

Stability studies of A. xylinum E25 The stability of the strain has been studied using a modified method described by Ben-Bassat [1], involving serial transfers in stationary and agitated cultures. Under stationary conditions, the cultures were incubated for 48 h after which a cell suspension from the vigorously shaken cellulose pellicle was used to inoculate fresh medium in the successive static culture. This process has been repeated for four subsequent transfers. The cell suspension from each transfer was diluted, spread on SH agar and incubated for 6 days at 308C. After that time, the appearance of Acetobacter colonies was examined at a magnification of 12 in order to select and count Cel mutants. The same procedure has been applied for agitated cultures. Based on the morphological differences, the stability of the strain was determined.

Determination of water-holding capacity (WHC) WHC was determined by the modified method described elsewhere [28]. Cellulose samples were centrifuged for 15 min at various centrifugal forces. The volume of remaining liquid was measured. WHC was described as the ratio of the liquid volume to the cellulose dry weight.

Figure 1 Morphology of A. xylinum E25 colonies grown on SH agar. Thick arrows indicate colonies of cellulose producers (Cel + ) and thin arrows indicate colonies of cellulose nonproducers (Cel ).

I transferII transferIII transfer

Cel mutants[%] glucose glucose +1% of ethanol fructose fructose + 1% of ethanol

Figure 2 Effect of medium composition on A. xylinum E25 stability.

Table 1 The effect of culture medium composition on the synthesis of BC

Yamanaka medium; YE — Yamanaka medium with 1% ethanol (vol/vol); YM — mathematically optimized Yamanaka medium; YME — mathematically optimized Yamanaka medium with 1% ethanol (vol/vol).

Figure 3 Dynamics of cellulose synthesis by A. xylinum E25 under stationary culture conditions.

Factors affecting the yield and properties of bacterial cellulose A Krystynowicz et al

Mechanical testing Mechanical properties (tensile strength, Young’s modulus) of dried cellulose samples have been measured using a ZWICK 1435-type mechanical tester.

TEM and SEM Fixed and dehydrated samples were either freeze-dried or critical point-dried (Samdri-790; Tousimis Research) and then coated with gold (30800; Ladd Research Industries). A Hitachi S-4500 field emission scanning electron microscope at 10 or 15 kV was used for sample examination. TEM observations were performed using a Philips 420 transmission electron microscope at 100 kV.

X-ray diffraction X-ray diffraction spectra were recorded using an HZG-4 diffractometer at 30 V and 25 mA. Scans were performed over the 5–408 2Q range using step 0.18 in width. The analysis of the diffractogram was carried out using the method of Hindeleh and Johnson [12]. The crystallinity index was estimated by dividing the area of the resolved crystalline peaks by the total area of the diffraction profile for 5–408.

Results and discussion

Stability studies of A. xylinum under different culture conditions The major obstacle encountered in agitated and highly aerated cultures of A. xylinum is the tendency of cellulose-producing strains to revert to noncellulose-producing mutants (Cel mutants), which contributes to a decline in BC production [2,9,21].

There are some hypotheses which state that one of the possible reasons responsible for the inactivation of the gene coding cellulose synthase is transposable elements capable of moving to a new site in the genome [7]. Different culture conditions are the selective factor that affects formation of these Cel mutants. As early as in 1954, Hestrin and Schramm [1] reported and described in detail a spontaneous occurrence of Cel mutants in agitated cultures. They isolated morphologically different types of Acetobacter colonies, which displayed different abilities to synthesize cellulose [29]: Cel +, fully able to synthesize cellulose (round, gelatinous and markedly convex colonies); Cel , cellulose-nonproducing mutants (generally flat and dull colonies) with or without the ability for reversion to Cel + forms.

As reported by a number of researchers, under stationary culture conditions that do not allow for a uniform medium aeration, Cel + colonies dominate. This phenomenon can be explained in terms of Acetobacter cells’ behavior upon culturing under different conditions: in static conditions, cellulose-synthesizing cells move towards the oxygen-rich medium–air interface, where they form a gelatinous membrane that limits access of oxygen into the lower parts of the culture. In agitated cultures that provide a sufficient and uniform aeration, intensive cell growth is preferred instead of polymer synthesis, which results in Cel domination in the whole culture (Figure 1). We examined the influence of different medium compositions on occurrence of A. xylinum E25 mutants both in stationary and agitated culture conditions.

We have shown that in stationary cultures, the type of carbon source and presence or absence of ethanol do not have any significant impact on Cel mutants’ occurrence. In the whole stationary culture, Cel forms were not observed. In contrast, in agitated cultures, Cel mutants occurred immediately after the first culture transfer and their numbers increased continuously during subsequent transfers, reaching a maximum value of 80% after the third transfer.

Based on the results from agitated cultures, a general conclusion can be drawn that mutation frequency strictly depends on the culture medium composition. Figure 2 clearly shows that the

Medium volume, V [ml]

Thickness of

Wet membrane mass [g]

Dry membrane mass [g]

BC yield [g /l]

Figure 4 Biosynthesis of BC in the RDF. (a) Cultivation in RDF (b) BC attached to the discs after 7 days of culture.

Factors affecting the yield and properties of bacterial cellulose A Krystynowicz et al

maximum amount of Cel mutants was observed in the original SH medium, without ethanol. A much smaller number of Cel mutants appeared in the medium containing fructose as carbon source (about 35–40% after the second transfer) and in the medium containing glucose enriched with ethanol (15% after the second transfer). Furthermore, in the medium containing glucose without ethanol, the number of Cel mutants reached the largest value of almost 80% of the Acetobacter population.

In order to determine the capability of Acetobacter Cel mutants for spontaneous reversion into Cel + forms, we examined the cell suspension from each of the different types of cultures. Single characteristic Cel colonies have been isolated from SH agar medium, transferred into fresh liquid SH medium and allowed to grow statically for 7 days. In the case of a successful reversion, the cellulose pellicle has been observed on the surface of a culture broth after a few days. The cellulose membrane was not formed in the case of nonreverting Cel mutants. About 20% of all Cel colonies displayed the ability for reversion to active cellulose producers; however, an increase in a number of transfers coincided with an enhancement of a number of Cel mutants that could not revert to Cel + forms.

Stationary culture

Our stability studies proved that A. xylinum E25 is suitable for BC production in stationary cultures, whereas agitated cultures promote formation of Cel mutants and result in a significant reduction of BC synthesis. These findings confirmed our preliminary studies focused on BC synthesis in stirred and aerated bioreactors using A. xylinum E25 (data not shown), which still have to be investigated in order to find the optimal conditions. Thus, our further efforts have been aimed at aspects of BC production in stationary cultures.

The composition of the nutrient medium as well as the strain activity are the fundamental factors affecting BC production and the profitability of the biotechnological process. The model SH medium [1], containing glucose as a carbon source and yeast extract and bactopeptone as nitrogen sources, is most commonly used in studies on BC synthesis by Acetobacter strains. In several studies on factors affecting BC production yield, not only glucose but also other carbon sources — like numerous mono-, di- and polysaccharides, as well as some organic compounds (i.e., glycerol) metabolized via gluconeogenesis pathway — were used. It is an ability to produce a required hydrolase by the bacterial strain which enables it to use oligo- and polysaccharides as carbon sources. Otherwise, enzymatic hydrolyzates of these substrates are applied [24].

We used a medium based on that developed by Hestrin and

(Parte 1 de 3)

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