Determining the water holding capacity of microbial cellulose

S.T. Schrecker & P.A. Gostomski*Department of Chemical and Process Engineering, University of Canterbury, Christchurch, New Zealand*Author for correspondence (Fax: +64-3-3642063; E-mail: peter.gostomski@canterbury.ac.nz)

Received 27 June 2005; Accepted 25 July 2005

Key words: Acetobacter xylinus, microbial cellulose, water holding capacity

Abstract

Comparing reported water holding capacities for microbial cellulose is difficult because different methodsare used. The different methods affect both the average value and the precision. In this new method, avacuum of 10 mm H2O (98 Pa) is applied to the wet cellulose to stabilize the sample prior to determiningthe wet weight. This simple method lowers the standard deviation by 50% or more over other methods.

Introduction

In recent years, microbial cellulose has receivedconsiderable attention due to its unique proper-ties especially for biomedical applications. Aceto-bacter xylinus extrudes cellulose microfibrils frompores on its cell envelope. These microfibrilsagglomerate along the side of the cell to form acellulose ribbon. The bulk membrane or pellicleis an entangled web of these ribbons. Microbialcellulose is very hydrophilic – holding over 100times it weight in water – and is far strongerthan cellulose derived from plants (Ross et al.1991, Brown Jr. 1993).

The water holding capacity of microbial cellu-lose is an important property for medical applica-tions such as wound dressing for chronic woundsand thermal burns or artificial blood vessels formicrosurgery (Klemm et al. 2001, Dalton 2004).To measure this property, the sample needs to bestabilized prior to weighing the wet sample. Pub-lished techniques include draining the cellulosepellicle on a mesh for 1 h (Serafica et al. 2002),wiping off the excess liquid (Budhiono et al. 1999)and a quick transfer from the broth to a balance(Watanabe & Yamanaka 1995). The other com-mon method is to suspend the sample and thenallow the water to drain (Schramm & Hestrin

1954, Williams & Cannon 1989). Since the meth-ods vary substantially, the average wet weight canvary significantly, making comparisons betweengroups difficult. In addition, some of these meth-ods have poor reproducibility because theydepend on the researcher’s sample handling tech-nique. To overcome these limitations, we havedeveloped a new sample stabilization method andcompared it to other published methods.

Materials and methods

Acetobacter xylinus, ICMP 15569 was grown for192 h in a rotating biological contactor, using a120 mm diameter cylinder that was 16% sub-merged in medium. The rotational speed of thecylinder was 7 rpm. The growth medium wasmodified from Serafica et al. (2002): it containedper litre: glucose 50 g, (NH4)2SO4 5 g, NaH2PO4

2.7 g, MgSO4Æ7H2O 1 g, yeast extract (Sigma)0.5 g, citric acid 1.5 g, ethanol 14 ml, trace ele-ment solution 2 ml.

Sample preparation

At harvest, the cellulose pellicle was approxi-mately 10 mm thick. The cellulose was removed

Biotechnology Letters (2005) 27: 1435–1438 � Springer 2005DOI 10.1007/s10529-005-1465-y

from the cylinder and boiled in 1 M NaOH for15 min to remove cell debris and remaining med-ium. It was rinsed twice and soaked in deionizedwater (DIW) for 48 h. Four 25 mm diameterround samples were cut for each method using awad punch. For the Vertical drain method, addi-tional samples of approximately 90� 30 mmwere prepared. Parts of the pellicle with obviousdefects such as holes were not used as samples.All samples were soaked in DIW until needed.

Wet sample stabilization

The wet weight of the microbial cellulose wasstabilized by six different methods. Some of thereported methods did not provide enough detailto reproduce exactly, therefore some assumptionswere required, e.g. sample dimensions. Four sam-ples were assayed for each method.

• Shake method: The sample was removed fromthe storage container using tweezers. Thesample was shaken twice quickly and thenweighed.

• Rapid transfer method: The sample was trans-ferred to the balance swiftly, minimizing theamount of surface water dripping off(Watanabe & Yamanaka 1995).

• Vertical drain method: Samples were sus-pended vertically, held by a 32 mm wide fold-back clip for 15 min. The rectangular sampleswere gripped from the short edge. Thismethod was slightly modified from Schramm& Hestrin (1954), where the samples werehung over a peg.

• Horizontal drain method: The sample was laidhorizontally on a flat stainless steel mesh with3 mm diameter openings separated by 1 mmand allowed to drain for 60 min before weigh-ing. To avoid evaporation, the sample wascovered (Serafica et al. 2002).

• Wipe method: The cellulose was removedfrom the DIW and the surface water waswiped off the sample by hand (Budhionoet al. 1999).

• Vacuum method: Tubing connected a 47 mmdiameter filter holder (Millipore) to an openreservoir (Figure 1). The filter holder and res-ervoir were filled with DIW and a 0.22 lmmixed cellulose esters hydrophilic membrane(Millipore) was placed on the membrane

support mesh. Initially, the water level in thereservoir was set at the same height as themembrane support to eliminate air bubblestrapped below the membrane. The reservoirwater level was then lowered 10 mm belowthe level of the filter membrane. This heightdifference maintained a 98 Pa vacuum on themembrane, while maintaining continuouswater connectivity between the filter mem-brane and the reservoir. Since this pressuredifference was below the bubble point of thehydrophilic membrane (P3.52� 105 Pa) airdid not flow through the membrane. Thecellulose sample was placed on the membraneand the filter reservoir was covered. After 4 h,the sample was weighed.

Dry weight

The samples were dried at room temperature for48 h, followed by 12 h at 85 �C. They werequickly transferred to the balance for weighingto minimize possible rehydration (Figure 1).

Sample weights

All wet and dry weights were determined with abalance having 0.1 mg reproducibility.

Calculation of the water holding capacity

The water holding capacity (WHC) was the massof water removed during drying divided by thedry weight of cellulose (gwater/gdrycellulose). Thestandard deviation of the WHC was calculatedbased on four samples.

g p g

Fig. 1. Set-up for the Vacuum method: a glass filter holderconnected to a water filled reservoir.

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Results

Each method was repeated four times on cellu-lose samples from the same fermentation run.The wet weight was measured after applying thedifferent methods and combined with the dryweight, to calculate the water holding capacity(WHC). Since each method produced a differentaverage WHC, the relative standard deviationwas evaluated for comparison (Table 1).

The Rapid transfer and Shake methods gavethe highest WHC due to the limited drain timebefore the measurement. The Vertical and Hori-zon drain methods gave a lower WHC sincedraining was permitted. The Vacuum methodgave the lowest average WHC. Most of themethods gave similar variability, however theVacuum method had approximately 50% lessvariability than the other methods.

Discussion

Different methods for stabilizing wet celluloseprior to weighing were investigated to assessaverage value and variability. We found the pub-lished methods inadequate especially in the effectof technique on reported values within our owngroup and therefore comparing our results toother reports was extremely difficult.

All the methods are related by recognizingthat capillary forces hold the water in the cellu-lose pore structure and when the sample isremoved from the water, some water drains out.

The different methods stabilized the sample dif-ferently including: (1) simple gravity draining ofthe sample (Vertical, Horizontal drain), (2)impressing an additional force to acceleratedraining (Wipe, Shake, Vacuum) or (3) minimiz-ing the loss (Rapid transfer). The different meth-ods gave quite different results, thereby indicatingthe need for a clearly defined technique.

The investigated methods left varyingamounts of surface water on the pellicle. TheRapid transfer technique had the most surfacewater. Water dropped off uncontrollably duringthe transfer from the beaker to the balance,which significantly influenced the wet weight. Theamount of water lost depended on the speed ofthe transfer, the distance and the tweezer pres-sure. The Shake and Wipe methods sufferedfrom similar problems, the number and intensityof shakes/wipes affected the wet weight, and con-sistent technique was difficult.

The Vertical drain method allowed a largeamount of the water drain off. However, due tothe vertical drying position, the dimensions ofthe sample influenced the wet weight. The twosample sizes yielded statistically different waterholding capacities (>99% probability at 95%confidence). The amount of water retained by thecellulose matrix varied along the length, with thebottom portion remaining essentially saturatedwhere water dripped off, a common feature whenporous material drains (Selker et al. 1999). Grav-ity draining should have caused the longer rect-angular samples to have equal or lower bulkwater content than the shorter round samples.However, the clip caused additional localizeddrainage. Since the round cellulose samples weresmaller, the clip compression affected a greaterpercent of the round samples. Another contribut-ing effect was the more focused point of drainingin the round samples, meaning a smaller portionof the sample remained saturated. Therefore, thesample size, shape and support technique possi-bly influence the Vertical drain technique.

The Horizontal drain method eliminatedmany of the influencing factors of the Verticaldrain method, since the vertical dimension wasthe sample thickness and the whole sample wassupported evenly. The drain time was longerthan the Vertical drain, which is the most likelycause for the lower average water content.However, this method does not rapidly reach

Table 1. A comparison of water holding capacity (WHC)using different methods for stabilizing the wet cellulose priorto weighing.

Method Average WHC

(gwater/gdry cellulose)

Relative standard

deviation

(r/WHC) (%)

Vacuum 148 5.5

Vertical drain

(rectangular sample)

285 9.88

Rapid transfer 309 11.2

Horizontal drain 204 11.4

Wipe 249 13.1

Vertical drain

(round sample)

209 13.5

Shake 291 15.3

Values are for four repeats of each method.

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equilibrium (Serafica et al. 2002). Additionally,the draining process depended heavily on theaperture size and width of the mesh. Smallerholes would not let the water pass though easily,mostly due to surface tension. The majority ofthe water drained from the side of the pellicle,not from the bottom.

Jamye & Rothamel (1948), developed a cen-trifugation method that was used by Klemmet al. (2001), and Krystynowicz et al. (2002) formicrobial cellulose. This method was not repro-duced in our work. The recommended 800� gcentrifugal force would drain significantly morethan just the surface water from the pellicle.Accelerations between 2 and 5 g would generatesimilar forces to the Vacuum method, but mostcommercial centrifuges do not spin this slowly,thus requiring a custom centrifuge apparatus.The centrifugation method at appropriate speedsshould give similar results to the Vacuum meth-od, but would not reach equilibrium signifi-cantly faster.

The Vacuum method was designed to counterthe problems identified with other methods. Dur-ing its development, we decided to use readilyavailable laboratory components. The key vari-ables for the method included the magnitude andduration of the vacuum applied to the sample.Previous studies have shown that the waterrelease rate from samples can be quite variable(Serafica et al. 2002). Steady state was normallyachieved in 1–2 h (data not shown), so 4 h waschosen to account for sample differences.

The 10 mm hanging water column or 98 Pavacuum removed free water from the surface ofthe cellulose thereby minimizing dripping duringtransfer to the balance. The applied vacuumeliminated edge/surface saturation in the samplecompared to the Vertical or Horizontal drainmethods. However, even at this low vacuum,the sample thickness decreased slightly as waterdrained from the pores and the cellulose struc-ture partially collapsed. The sample thicknessprobably decreased in the other methods butwas not observed because of less water loss.The Vacuum method yielded the lowest relativestandard deviation of all the methods, becausethe technique was more repeatable and longerequilibration times. Four hours of equilibrationwas longer than applied to the Horizontal or

Vertical drain methods and the variability ofthose methods may have improved if the sam-ples were equilibrated longer. However, we con-cluded that the Vacuum method was much lesssusceptible to technique, sample size or appara-tus influence, thereby allowing better compari-son of WHC between research groups.

Acknowledgements

The authors gratefully acknowledge the supportprovided by technical staff in the Department ofChemical and Process Engineering and School ofBiological Sciences at the University of Canter-bury.

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