.... Cellulose 11: 403 411, 2004. " I[) 2004 KlulVer Academic Publishers. Printed in /he Ne/her/ands. 403

Structural investigations of microbial cellulose produced in stationary and agitated culture

Wojciech Czaja1,2, Dwight Romanovicz' and R. Malcolm Brown, 1r. 1,*

ISection of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78713, [/SI1; 2Author is currently affiliated with the Institute of Technical Biochemistry, Technical University of Lodz, Stefanowskiego 4, 10, Lodz 90-924, Poland; *AUlhor for correspondence (e-mail: rmbrown(c~mail.utexas.edu)

Received 24 November 2003; accepted in revised form 6 April 2004

Key words: Acetobac/er, Agitated culture, ATCC 53582, Bacterial cellulose, Fermentation, FT-IR, Micro­bial cellulose

Abstract

Structural characteristics of microbial cellulose synthesized by two different methods have been compared using FT-IR and X-ray diffraction techniques. Cellulose synthesized by Acetobacter xylinum NQ-5 strain from agitated culture conditions is characterized by a lower Ia mass fraction than cellulose that was produced statically. Such a decrease was in good correlation with smaller crystallite sizes of microfibrils produced in agitated culture. Formation of characteristic cellulose spheres during agitation has been investigated by various electron and light microscopic methods. On this basis, a hypothetical mechanism of sphere formation and cell arrangement in the agitated culture has been proposed. During agitation, cells are stacked together in organized groups around the outer surface of the cellulose sphere.

Introduction cellulose include: an acoustic transducer dia­phragm made of dried cellulose sheet (Nishi

Cellulose is the most abundant biopolymer on et al. 1990), wound dressing material (artificial earth and is produced by a variety of organ­ skin) made of wet and purified cellulosic mem­isms, ranging from vascular plants to algae and brane (Fontana et al. 1990), or Nata de Coco, a prokaryotic organisms such as cyanobacteria traditional Philippine fermented dessert, which (Jonas and Farah 1998; Nobles et al. 2001). In became very popular in Japan a few years ago addition, there are some strains of the (Yoshinaga et al. 1997). In recent years, an prokaryotic, non-photosynthetic organism, Ace­ interest has developed in producing bacterial tobacter, which have the ability to synthesize cellulose on a large commercial scale. Some at­high-quality cellulose organized as twisting rib­ tempts have been made in the area of optimi­bons of microfibril bundles (Brown et al. 1992). zation of culture conditions (Kouda et al. 1997a, Bacterial cellulose demonstrates unique proper­ b), medium composition (Matsuoka et al. 1996), ties including high mechanical strength, high strain improvement (Ishikawa et al. 1995; crystallinity, high water holding capacity and Vandamme et al. 1998), or the scaling-up pro­high porosity, which make it a very useful bio­ cess, but few production yield enhancements material in many different industrial processes have been reported so far. (Brown 1998; Iguchi et al. 2000). So far, the There are two methods to produce bacterial best-known commercial applications of bacterial cellulose: (a) stationary culture, which results in

I

404

the accumulation of a gelatinous membrane of cellulose on the surface of the medium; and (b) agitated culture, where cellulose is synthesized in deep media in the form of fibrous suspensions, pellets, or irregular masses (Watanabe et at. 1998; Chao et at. 2000). While stationary culture con­ditions have been quite successfully investigated and described, agitated culture of Acetobacter strains causes many problems, among which strain instability, non-Newtonian behavior during mixing of bacterial cellulose, or proper oxygen supply are the most common (Kouda et at. 1996, 1997a, b). Despite those problems, some researchers have suggested that agitated culture might be the most suitable technique for eco­nomical scale production (Ross et at. 1991; Yoshinaga et at. 1997).

Detailed structural characteristics carried out using electron diffraction analyses (Sugiyama et at. 1991) and (CPMAS) l3C NMR (VanderHart and Atalla 1984; Yamamoto and Horii 1993) revealed that native cellulose is a composite of two different crystalline phases called la and Ifl. Normally, Acetobacter xylinum cellulose displays character­istics of highly crystalline, I",-rich cellulose (Van­derHart and Atalla 1984).

In our investigations, we have studied the synthesis and structural characteristics of bacte­rial cellulose produced in stationary and agitated culture by A. xylinum NQ5 strain (ATCC 53582). This particular strain is characterized by a periodic series of reversals in the direction of cellulose ribbon synthesis and produces an agar colony which contains cellulose synthesized in tunnels (Thompson et at. 1988). It is also unique for an uncharacteristic absence of spontaneous mutation during the agitation process (Brown and Lin 1990: Saxena et at. 1990). X-ray dif­fraction was used to characterize the effects of agitation on the crystalline arrangement of glu­can chains within microfibrils. Furthermore, the effect of different rotational speeds of the agita­tion on the structure of the cellulose was inves­tigated. Estimation of la and Ip cellulose fractions in ceJlulose samples from different cul­ture conditions was carried out using FT-IR spectroscopy. Light and electron microscopic techniques were used to examine the formation of cellulose spheres that are characteristic for this particular strain when grown in agitated culture.

Materials and methods

Microorganisms

Acetobacter xylinum NQ5 (ATeC 53582) strain from the collection of Section of Molecular Genetics and Microbiology, Cniversity of Texas at Austin, was used in this study.

Culture medium

Schramm Hestrin medium (Hestrin and Schramm 1954) without pH adjustment was used in all experiments unless otherwise specified.

Culture conditions

The cells for the inoculum were cultured in flasks either statically or on a rotary shaker with addition of O. J% cellulase enzyme (Celluclast 1.5L™ from Trichoderma resei, Novo Nordisk Bioindustrials, Inc., Denmark) for 3 days at 28°C. In the first case, a thick gelatinous mem­brane was squeezed aseptically to remove cells em bedded inside the pellicle, and the cell sus­pension was transferred as the inoculum for the main culture. In the second case, cells were har­vested by centrifugation, then resuspended in the fresh culture medium. The main cultures were grown in the flasks either statically or on a rotary shaker (Lab-Line Instruments, Inc. USA) oper­ating at different rotational speeds (in the range 90- 250 rpm), for 7 days at 28°C. The synthe­sized cellulose was separated from the medium by filtration. The quantity of cellulose produced was measured as dry mass of polymer after washing with 2% sodium hydroxide (overnight) followed by three changes of distilled water in order to remove cells and medium embedded in the cel­lulose material.

FT-IR spectroscopy

Each cellulose sample was air-dried on a glass slide in the form of a thin film, which was then placed across a hole in a magnetic holder. FT-IR spectra were obtained using a Perkin-Elmer spectrometer (Spectrum 2000). All spectra were

recon resoll' 400 Cl

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X-ray

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405

12) strain vIolecular fTexas at

Schramm ~d in all

I

. in flasks ker with =elluclast Nordisk days at us mem­ave cells cell sus­

t1 for the ;vere har­ed in the Ires were a rotary

A) oper­he range : synthe­~dium by llced was washing followed order to the cel­

a glass ;vas then ". FT-IR in-Elmer tra were

recorded with the accumulation of 32 scans, a resolution of 2 cm- I in the range from 4000 to 400 cm- I, normalized using the band at 2900 cm I due to the COC stretching vibration. The!, fraction of the samples was calculated by the following equation (Yamamoto et aL 1996): f~ = 2.55f;R - 0.32, where f;R of cellulose can be calculated as A 1 '(A~ + All) and A~ and Ap are absorbencies at 750 and 710 cm- l

, respectively.

X-ray diffraction

Cellulose samples in the form of sheets dried on glass slides were placed in the X-ray holder. X-ray diffraction spectra were recorded using Ni-filtered Cu-KIX radiation (Il 0.15418 nm) produced by either a Rigaku RINT 2200 X-ray generator equipped with a Position Sensitive Proportional Counter (PSPC) as the detector or a Philips PW 1720 X-ray generator operating at 35 kV and 25 mA, equipped with a Philips vertical scanning diffractometer and a diffracted beam monoch­rometer. Scans were perfolmed over the 5 40 28 range using steps of either 0.05° or 0.01° in width. The data were analyzed using the WinFit software program (Krumm 1997) or the Jade 5 XRD soft­ware program. The crystallite size was estimated by substituting the full-width at half-maximum (FWHM) into the Scherrer equation (Nieduszyn­ski and Preston 1970; Alexander 1979).

Sectioning for light and electron microscopy

The microbial cellulose material was fixed in 4% glutaraldehyde, washed in cacodylate buffer, and then fixed again in 2% osmium tetroxide (OS04). After washing in distilled water, the cellulose was dehydrated in stepwise concentrations of ethanol followed by absolute acetone, then infiltrated with resin -acetone solutions. Cellulose was embedded in epoxy resin (EPON; Electron Microscopy Sci­ences, USA) and allowed to polymerize at 60 ~C

for 24 h. Both thick (about 1 ,LIm) and thin sections were cut on a Reichert OM2 ultramicrotome, using either a glass or diamond knife, respectively. Thick sections were stained with 1% bromo toluidine and observed with a Zeiss Universal Light Microscope. Ultra-thin sections were gently placed on the grids, stained with lead citrate, washed in 0.02 N NaOH

and boiled distilled water and finally post-stained with 2% uranyl acetate. Grids were then examined with a Philips 420 transmission electron mIcro­scope (TEM) operating at 100 kV.

Scanning electron microscope (SEM) observations

Cellulose samples were fixed in 4% glutaraldehyde followed by 2% osmium tetroxide and dehydrated using the same procedure as for sectioning. Sam­ples were either freeze-dried or critical point dried (Samdri-790, Tousimis Research Corp.) and then coated with gold (30~OO, Ladd Research Indus­tries, Inc.). A Hitachi S-4500 field emission scan­ning electron microscope operating at 10 or 15 kV was used for examination of the samples.

Results and discussion

Most of the Acetobacter xy/inul11 strains used worldwide in research synthesize cellulose stati­cally in the form of a gelatinous membrane. When these cultures are grown in agitated conditions the results often give a poor yield (Yoshinaga et aL 1997). Considering the properties of our Aceto­bacter NQ5 strain, especially its great genetic sta­bility, it might be one of the best available strains to apply using large-scale, agitated and aerated fermentation systems. A time course of cellulose synthesis both in stationary and agitated culture shown in Figure 1 indicates that after 7 days of culture almost the same quantity of cellulose was produced.

However, after 16 days we did not notice any further significant increase in cellulose synthesis under agitated culture, whereas the pellicle grown in stationary culture reached a dry mass value of 10 gil (data not shown). One possible explanation may focus on the different ways of oxygen distri­bution and substrate penetration during growth.

While the mechanism of pellicle formation and its oxygen profile have been recently investigated (Verschuren et aL 2000), not much attention has been paid to cellulose accumulation in agitated culture conditions. The NQ5 strain in agitated culture produces cellulose in the form of charac­teristic spheres, as shown in Figure 2.

Thick sections cut across the fixed and embedded cellulose spheres revealed a specific

406

3.5 ____ stationary

3 ----.- agitated

25 <:: ~ 2 Q) III o :i 1.5 Qj u

0.5

o._==--.::r---,----,----,----,----,---------,,----------, o 24 48 72 96 120 144 168 192

cultivation time [hours]

Figure I. Time course of bacterial cellulose synthesis in stationary and agitated culture.

Figure 2. Cellulose spheres formed in the agitated culture conditions; scale bar = 5 nlnl.

localization of Acetobacter cells. Sections pre­sented in Figure 3 show that most of the cells are distributed at the surface of the sphere and only a few of them are randomly scattered inside.

Such a particular arrangement might be ex­plained by the following: cells which are intro­duced into the fresh medium become attached around the surface of air bubbles existing in the agitated liquid; cells start to reproduce and syn­thesize cellulose ribbons forming eventually a more compact structure shown in Figures 2 and 3. In this hypothesis the surface distribution of cells

would suggest that the cellulose is synthesized only at the surface and that the cells fail, somehow, to become entrapped in the pellicle as in static cul­ture. Perhaps shear forces during agitation cause cells to become separated from the surface of the sphere.

Alternatively, cells may have a periodic ribbon synthesis phase whereby the cells actually have a cycle of synthesis, separation from the sphere, rejoining the sphere and initiating ribbon synthe­sis. Such a scenario could explain why the center of the sphere is solid and has only cellulose ribbons present. Such a specific surface distribution of cells has been also proved by SEM observations (Fig­ure 4b) and TEM observations of thin sections taken from the same specimen (Figure 3c, d).

Another interesting point is a microscopic comparison of cellulose microfibril structure, syn­thesized in stationary and agitated culture condi­tions. Figure 4c, d demonstrates clearly the differences between both cellulose samples. Gen­erally, a fine net built of entangled cellulose rib­bons represents both of them. A close observation revealed that mostly uniaxially oriented ribbons characterize cellulose formed in the stationary culture, whereas cellulose synthesized under agi­tated conditions demonstrated a structure of dis­orderly, curved, overlapping ribbons. Such a disordered microstructure could be a result of constant motion forces occurring during agitation. The thickness of the cellulose microfibrils also seems to differ between those two samples, with

Fi d 10 sr

tf sl

If

Cl

c; X d p

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Sl

a ti fl

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407

lesized only )mehow, to 1 static cul­ation cause rface of the

)dic ribbon ally have a the sphere, )on synthe­he center of )se ribbons tion of cells ltions (Fig­in sections 3c, d).

nicroscopic Icture, syn­ture condi­:learly the lples. Gen­llulose rib­,bservation ed ribbons stationary under agi­me of dis­s. Such a

result of ~ agitation. fibrils also nples, with

Figure 3. Thick (a, b) and thin (c, d) sections of cellulose sphere produced in agitated culture; the characteristic ring of cells is situated close to the surface of the ball (see a); () small groups of entangled ceJls - initial stage of sphere formation; ( ,) some of the cells localized inside the sphere formed an ordered layer; we can probably assume that these overlapping layers might be another stage of sphere formation; scale bars: (a) 25 1,m, (b) 60 11m, (c) lOa nm, (d) 100 nm.

the one from agitated culture distinguished by the slightly thinner microfibrils.

In order to compare the microstructural changes in cellulose samples from both differenL culture conditions and especially to estimate if the shaking causes any disturbance in the crystallization process, X-ray diffraction was used. Figure 5 presents X-ray diffraction patterns taken for both cellulose sam­ples, which represent a typical profile of cell ulose 1.

Quantitive analysis of the reflections corre­sponding to all three peaks in those X-ray profiles revealed that they are shifted to wider angles in the cellulose sample from agitated culture. Compari­son of 28 angle values revealed also that in case of agitation, the (110) and (110) reflections are posi­tioned closer together than in the cellulose profile from cellulose grown statically.

These changes in the d-spacings appear to rep­resent a different proportion of Ie< and If! cellulose allomorphs as reported previously (Yamamoto

et a1. 1989; Watanabe et a1. 1998). The crystallite sizes calculated for peaks 1, 2 and 3 using the Scherrer formula (Nieduszynski and Preston 1970) are shown in Table I. They clearly demonstrate existence of smaller crystallite sizes in cellulose from agitated culture.

The conditions of stress occurring during agi­tation appear to interfere strongly in the process of nascent microfibrils crystallization, favoring the formation of smaller size microfibrils and in­creased 1/3, the more stable allomorph. Such a hypothesis is in a good agreement with previous reports (Yamamoto et a1. 1996; Hirai et a1. 1998).

To determine the exact values of mass fractions of cellulose Ie< and Iii, FT-IR spectroscopy was applied. The enlarged regions of the FT-IR spectra shown in Figure 6 present peaks assigned to Ie,

I(750 cm- ) and If! (710 cm- I ) fractions. Careful observations of those peaks reveal that

the Ix contribution in cellulose synthesized in

I

408

Figure 4. SEM miC!"ographs of the bacterial cellulose produced under different cultme conditions: (a) surface of a sphere formed in the agitated culture, (b) bacteria seen close to the surface of a cracked cellulose sphere, (c) structure of cellulose produced statically, (d) structure of cellulose produced during agitation.

1400 d3 (200)

1200 -­stationary --agitated

5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 2 theta [degree]

Figure 5. X-ray diffraction patterns obtained from bacterial cellulose samples synthesized in stationary and agitated culture condi­tions. Three typical diffraction peaks occurring in the region of 10- 25° are labeled as d), d" and d.1.

agitated culture is lower in comparison with sharp fraction is lower in the cellulose sample from agi­and well defined peaks in the same spectrum of tated culture conditions, confirming our X-ray stationary produced cellulose. The numerical data results. from those spectra are presented in Table 2. Besides the external, environmental stresses on According to the formula proposed previously Acetabaeter during agitation, another possible (Yamamoto et al. 1996) the estimated Ta mass explanation of such I'l. mass fraction decrease may

Table togra

Cellu

Static Agita

also (eM alan 199~

Abs

Abs

Figw enlar

1000 'iii' Co ~ 800 ~ 'iii c:: 600 'E

400

200

0

Ql

d, (110)

409

'armed in the ,tatica lIy , (d)

.Iture condi­

rom agi­ur X-ray

.I·esses on possible

'ease may

Table I. d-spacings, crystallite sizes and percent crystallinity of different bacterial cellulose samples determined from X-ray diA'rac­tograms.

Cellulose sample d-Spacings (A) Difference in 28 angle Crystallite sizes (nm) Percent crystallinity (%)

d l dz d) (Peak I - peak 2) CI', Cl'z CI', C

Sta tionary 602 5.23 3.85 223 7.9 8.6 6.7 89 Agitated 59 5.17 383 2.15 7.9 6.6 6.4 84

also be connected with {J-I,4-endogluconase reported that this enzyme plays an essential role in (CMCax) synthesis, which occurs in the medium the cellulose formation process (Tonouchi et al. along with the cellulose production (Tahara et al. 1995; Koo et al. 1998). It has also been reported 1997; Koo el al. 1998). Generally, it has been that the CMCase activity at the end of the agitated

1.1 (a)

f Abs 400!0.5

J/

~ ~ooo 3000 2000 1000 400

Wavenumber[cm-1)

0.9 r-------------------T"'il"n---,r-----------------, (I))

0.8

0.6

Abs

0.4 400 I

\I

~J 3000 2000 1000

Wavenumber[cm-l)

Figure 6. FT-IR spectra of bacterial cellulose from stationary (a) and agitated (b) culture conditions (part of the graph has been I Ienlarged to highlight the peaks assigned to cellulose) Ix (750 cm- ) and II' (710 cm· ).

400

1

261

410

Table 2. Cellulose I" and I, content (%) and crystallinity index of bacterial cellulose from different culture conditions deter­mined by FT-IR measurements.

Cellulose I, IR crystallinity index It sample (abs. at 1427(895 cm- I

)

Stationary 76 24 4.84 Agitated 71 29 4.48

culture was more than 10 times higher than in the stationary culture (Watanabe et al. 1998). The same report (Watanabe et al. 1998) showed that this higher enzyme activity in the agitated culture was a reason for lower D?w (weight-average de­gree of polymerization) fractions in that culture. Considering this fact, we can suggest that such a high CMCase activity in the agitated process might also have an influence on I~ and Iii contri­bution in the cellulose. This mechanism should be understood in more detail.

'he crystallinity index calculated for our sam­ples based on the F'T-IR spectra revealed also a reduction in crystallinity for samples of agitated cellulose. "he decrease in crystallinity is in good agreement with data determined based on the X­ray profiles analysis. jJ1 that case an estimrrted percent crystallinity for cellulose grown statically was also slightly higher than for cellulose synthe­sized in agitated culture.

Cur studies have focused on the structural investigation of cellulose formed in agitated cul­ture conditions and on an interesting Aeetobaete,. behavior and its product accumulation during agitation. A. xylinum 1,rQ5 has been found to dfectively synthesize cellulose in agitated culture in the form of unique, large spheres. :v.any other strC1ins of Aeetobaete,. undergo mutation to non­cellulose producing cells under agitation, and this often is a problem with maintenance of active cellulose-producing strains; however, with the NQ5 strain, mutations to the non-cellulose state do not occur. :.n fact, no mutations to the non­cellulose producing state have been observed in more than 20 years growing this strain. This fact might huve a great impact on its further applica­tion in a large-scale fermentation process with such strains as NQ5. ~n several studies up to now, cellulose production in stationary culture has been investigated, but a quite low productivity and high prod uction costs were often the limiting factors (Matsuoka el al. 1996;~' oshinaga et al. 1997). In

addition, while the static technique does not offer many optimization alternatives, an efficient bac­terial cellulose synthesis in agitated and aerated conditions might be a cost-effective technological system (Ross et al. 1991). It has been reported that microbial cellulose production and aerobic Aee­tobaete,. cell growth are strictly related processes (1\.1arx-Figini and Pion 1974). For improvement of cellulose productivity, a high oxygen supply in agitated ami aerated culture is required to increase the total cell density and consequently to achieve high production rates (Kouda et al. I997b, 1998; Yoshinaga et al. 1997). Besides a good production yield, the novel properties of such a NQ5 cellulose synthesized under agitated conditions might have many different advantages useful in industrial applications.

Acknowledgements

f,mong many colleagues from the lab, authors are especially grateful to Dr Krystyna Kudlicka for her advice and fruitful discussion during this re­search and to Mr Richard Santos for his technical assistance. We thank Dr Tetsuo Kondo for use of FT-IR and X-ray diffraction instruments. This work was supported in part by a Grant to R.M.B. from the Welch Foundation (F-1217) and the Johnson & Johnson Centennial Chair Fund.Spe­cial thanks go to the Polish-American Fulbright Commission for a grant awarded to Dr Wojciech Czaja.

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