effects of polyacrylamide-co-acrylic acid on cellulose production by acetobacter xylinum

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 78:964–970 (online: 2003)DOI: 10.1002/jctb.869

Effects of polyacrylamide-co-acrylic acid oncellulose production by Acetobacter xylinumGerard Joseph, Gerald E Rowe, Argyrios Margaritis and Wankei Wan∗Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario, Canada N6A 5B9

Abstract: Polyacrylamide-co-acrylic acid (PA) added to shake flask cultures of Acetobacter xylinum atconcentrations up to 3 g dm−3 resulted in increased production of bacterial cellulose. For PA concentrationsof 0–3 g dm−3, 7-day cellulose production rose monotonically from 2.7 ± 0.8 to 6.5 ± 0.5 g dm−3 at a shakerspeed of 175 rpm, and from 1.7 ± 0.01 to 3.7 ± 0.5 g dm−3 at shaker speed of 375 rpm. Addition of PAalso changed the morphology of the biomass from amorphous/stringy forms to spheroidal particles withdiameters ≤2 mm. Similarly, bioreactor cultures grown in the absence of PA formed long fibrous masseswhich deposited on the internals, while those grown in the presence of 1–2 g dm−3 PA formed smalldiscrete particles with diameters ≤0.1 mm. Tests performed with 1 and 2 g dm−3 PA, and stirrer speedsfrom 500 to 900 rpm, appeared to give the highest cellulose concentration of 5.3 ± 0.7 g dm−3 in 64–68.5 hin the presence of 2 g dm−3 PA at 700 rpm, although this value was statistically indistinguishable from thatobtained at 1 g dm−3 PA and 900 rpm. A qualitative model is proposed to describe the mechanisms by whichPA affects biomass morphology, resulting in its advantageous formation as small, dispersed, spheroidalpellets. Quantitative analysis of the results gave inverse correlations between both the fraction of fructosecarbon going to cellulose synthesis and the specific fructose consumption rate, and the maximum celluloseconcentration and the fraction of fructose carbon going to by-product formation. Since cellulose yieldwas almost universally improved by higher polyacrylamide concentration, it appears likely that increasedviscosity reduces fructose uptake rate by limiting mass transfer. 2003 Society of Chemical Industry

Keywords: bacterial cellulose; polyacrylamide; Acetobacter xylinum; thickener; flocculant

INTRODUCTIONBacterial cellulose possesses many of the sameproperties as plant cellulose but does not requireharsh chemical treatment for purification. As aresult it can be purified and still retain nativecharacteristics lost in the traditional processing ofplant cellulose. Many properties of bacterial cellulose,including promotion of wound healing,1 high tensilestrength,2 good acoustic properties,3 biocompatibility4

and a fiber size of less than 130 nm,5 have madeit a potentially important industrial and biomedicalmaterial. A significant barrier to the exploitation ofbacterial cellulose is a lack of information on large-scale production processes.

Conventional production by static culture ofAcetobacter xylinum, while moderately successful ona small scale, is not suitable to large-scale productionbecause it is very labor intensive and requires largesurface areas. Agitated culture, on the other hand, hasposed its own problems. One recurrent problem hasbeen conversion of a large part of substrate glucoseto gluconic and keto-gluconic acids, overcome either

by strain selection,6,7 or use of fructose as a carbonsource giving lower by-product formation. Optimaloxygen supply is also a difficult issue since masstransfer by bubble aeration tends to be limited bythe gelatinous nature of the product, yet sparging withoxygen-enriched air has been shown to have no effecton cellulose production rate.8 Although difficultiessuch as selection of stable bacterial strains have largelybeen overcome,9 solutions to problems such as controlof product morphology, although little discussed, haveproven to be elusive.

Evidence for the improvement of fermentationby the addition of a thickener to reduce clumpinghas been present in the literature since Cormanused agents such as gelatin and soybean flour toincrease the production of beta-carotene.10 In bacterialfermentations for cellulose production, Ben-Bassatet al11 studied a number of thickeners includingxanthan, guar gums and carboxymethylcellulose, andconcluded that polymers based on polyacrylamide(PA) worked best. Polyacrylamide, one of the mostcommonly used polymeric flocculants, is linear and

∗ Correspondence to: Wankei Wan, Department of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario,Canada N6A 5B9E-mail: [email protected]/grant sponsor: Shakti Biomedical CorpContract/grant sponsor: Natural Sciences and Engineering Research Council of Canada(Received 20 December 2002; revised version received 07 March 2003; accepted 08 April 2003)

2003 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2003/$30.00 964

Effects of polyacrylamide on bacterial cellulose production

can form strong hydrogen bonds. The versatility ofPA lies in the fact that it is available in molecularweights from 10 000 to 15 000 00012 and it can befunctionalized using the amide group. It can beobtained in co-polymerized form with acrylic acidor other co-monomers to adjust its ionic charge.

After encountering difficulties in producing bacterialcellulose as discrete particles, we decided to testPA as a morphology control agent. While its useas a ‘protectant’ in bacterial cellulose fermentationhas been patented,11 there is no explanation in thisspecification as to how reduction of shear stress bythe polymer improves cellulose yield. Given that thisis the only published report on the effect of PA onbacterial cellulose production, the present researchaimed to systematically study, and if possible explain,such effects.

2 EXPERIMENTAL2.1 Microorganism and medium preparationThe microorganism used, Acetobacter xylinum BPR2001 (ATCC No 700178), is also known asGluconacetobacter xylinus subsp sucrofermentans. Themedium used was modified from Naritomi et al13

and consisted of the following dissolved in 1 dm3

of water: fructose 20 g, centrifuged CSL (corn steepliquor, Sigma) supernatant 80 cm3, KH2PO4 1 g,MgSO4.7H2O 0.25 g, (NH4)2SO4 3.3 g. The pHwas adjusted to 5.0 by the addition of sodiumhydroxide solution (1 mol dm−3). Polyacrylamide-co-acrylic acid (1% w/w acrylic acid, Aldrich ChemicalCompany; henceforth designated PA), having anaverage molecular weight of 5 000 000, was used invarious concentrations.

2.2 Fermentation vesselsThe shake flasks used were 500 cm3 Kimax baffledculture flasks. The vessel for all bioreactor experimentswas a Biostat MTM manufactured by B BraunBiotech. The jacketed vessel had a nominal volumeof 2 dm3, a working volume of 1.5 dm3, and adiameter of 130 mm. The impeller was a flat-blade diskturbine with four blades (diameter 47.8 mm) 9 mmbelow a gate-type impeller similar to that describedby Kouda et al.14 The reactor headplate containedports for a dissolved oxygen probe, pH electrode,thermocouple, sampling tube, a condenser and fourliquid feeds. Although measurement of dissolvedoxygen concentration throughout the fermentationswas attempted, fouling of the electrode with biomasscaused the readings to rapidly fall to zero.

2.3 Culture conditionsThe inoculum was grown in 500 cm3 baffled flasks witha working volume of 100 cm3 for 3 days at 175 rpm and28 ◦C. The culture was aseptically transferred into asterile stainless steel blending vessel and homogenizedfor 15 s at 18 000 rpm using a Waring laboratoryblender. In the case of shake flask fermentations,

each flask, containing 100 cm3 of sterile medium, wasinoculated aseptically using 1 cm3 of the homogenizedinoculum. The bacteria were cultured in a rotaryshaker/incubator at either 175 rpm or 375 rpm at 28 ◦Cfor 7 days. In the case of the bioreactor, followinghomogenization the entire contents of the inoculumflask were aseptically added to 1.2 dm3 of sterilemedium. Fermentation proceeded for approximately3 days at the selected agitation rate (400–900 rpm)and 28 ◦C. The pH was controlled at 5.0 by automaticaddition of aqueous sulfuric acid or sodium hydroxide,and air was supplied at 1 dm3 min−1.

2.4 Sample preparationDepending on the nature of the biomass morphologyin the fermentation vessel, the broth samples obtainedwere more or less representative of the state ofthe culture. Broth from shake flasks was harvestedand homogenized at 18 000 rpm for 15 s, and two5 cm3 samples were taken; homogenization was notnecessary for the samples taken from the bioreactor.Samples were centrifuged at 3000 rpm for 20 mins,washed with distilled water and centrifuged again toremove the culture broth. The washing procedurewas repeated three times. The supernatant from thefirst centrifugation was saved for the determination ofthe residual fructose concentration while the washedpellets were used to determine cell and celluloseconcentration.

2.5 Bacterial cellulose concentrationFermentation samples (prepared as described above)were treated with 10 cm3 of 1% (w/v) sodiumhydroxide at 90 ◦C for 30 min to dissolve the cells. Thebacterial cellulose obtained was filtered, washed withdistilled water, dried at 50 ◦C for 24 h, and weighed.

2.6 Cell dry weight concentrationThe remaining sample was suspended in 10 cm3 ofa solution of cellulase from Penicillum funiculosum(Sigma C-0901). The cellulase solution was preparedas a 2.5% (w/v) suspension of crude cellulase in citratebuffer (pH 5), mixed for 10 min, then centrifuged toremove suspended solids. The mixture of cellulaseand biomass was kept at 37 ◦C for 2 h to hydrolyze thecellulose. The sample was then washed, filtered, driedat 50 ◦C for 24 h, and weighed.

2.7 Fructose concentrationThe fructose concentration in the sample’s super-natant was analyzed using the dinitrosalicylic acid(DNS) method.15 The sample was diluted, then 2 cm3

was mixed with 3 cm3 of DNS reagent and placed ina boiling water bath for 5 mins. After cooling to roomtemperature, the absorbance was read at 540 nm.

3 RESULTS3.1 Effects of polyacrylamide in shake flasksAs a prelude to growing A xylinum in a stirredbioreactor, shake flask experiments were performed

J Chem Technol Biotechnol 78:964–970 (online: 2003) 965

G Joseph et al

without polyacrylamide (PA) to determine how thebacteria would behave in an agitated environment.A fructose medium concentration of 20 g dm−3

was used to shorten fermentation time, and reducethe deleterious effects of high bacterial celluloseconcentration on mixing and mass transfer. Thefermentations produced an average of 3.4 g dm−3

of cells and 2.7 g dm−3 of cellulose with significantvariability in triplicate experiments. Cellulose wasproduced in a range of forms, from long water-swollenfibrous strings to small particles having an estimateddiameter of less that 1 mm.

To test the effect of PA on cell growth andcellulose production, baffled flasks were preparedwith 100 cm3 of CSL–fructose medium containing8% (v/v) CSL and 2% (w/v) fructose along withother components, as described in Section 2. PAwas added in the concentration range of 0 g dm−3

to 3 g dm−3, and quadruplicate flasks were shakenat 175 rpm for 7 days. The results given in Table 1show a trend toward higher cellulose productionwith increasing PA concentration, concomitant withdecreasing fructose consumption. The morphology ofthe biomass produced ranged from stringy, amorphousparticles at 0 g dm−3 of PA to distinct spheroidalparticles at 3 g dm−3 of PA; most of the latter haddiameters of less than 2 mm.

In order to test whether improving oxygen transferby increased agitation would raise fructose consump-tion and cellulose yield, the experiment was repeatedin duplicate flasks with shaking at 375 rpm. As thedata given in Table 2 show, less cellulose was pro-duced, although its concentration was again positivelycorrelated with the amount of PA added.

Table 1. Cell and cellulose production, and fructose consumption,

versus polyacrylamide (PA) concentration in flasks shaken at 175 rpm

for 7 days. All values are in g dm−3, and represent the average ± 1

standard deviation for four replicate experiments

PAconcentration

Cell dry matterconcentration

Celluloseconcentration

Fructoseconsumption

0 3.7 ± 0.5 2.7 ± 0.8 18.3 ± 0.41 2.3 ± 1.2 4.6 ± 1.1 17.4 ± 0.92 2.4 ± 0.04 4.8 ± 1.0 16.2 ± 1.53 3.7 ± 1.7 6.5 ± 0.5 15.8 ± 1.5

Table 2. Cell and cellulose production, and fructose consumption,

versus PA concentration in flasks shaken at 375 rpm for 7 days. All

values are in g dm−3, and represent the average ± 1 standard

deviation for duplicate experiments

PAconcentration


Celluloseconcentration

Fructoseconsumption

0 2.4 ± 0.2 1.7 ± 0.01 15.9 ± 1.61 3.4 ± 0.1 2.1 ± 0.4 18.7 ± 0.012 3.9 ± 0.8 2.3 ± 0.3 18.7 ± 0.03 3.4 ± 0.3 3.7 ± 0.5 16.3 ± 0.5

3.2 Effects of polyacrylamide on bioreactorperformanceBioreactor runs without PA were performed using 2%(w/v) fructose and 8% (w/v) CSL at agitation ratesfrom 400 to 900 rpm (results not shown). Cellulosewas produced, not as discrete particles, but as longfibrous bodies that became entangled in the impellers,on the sparger, and on the dissolved oxygen and pHprobes. This led us to experiment with polyacrylamideaddition to try to improve product morphology.

The time courses of cell and cellulose concentrationsfor experiments with PA concentrations of 1 or 2 gdm−3 and stirrer speeds from 500 to 900 rpm areshown in Figs 1–5. At 500 rpm with 1 g dm−3 PA,production of both cells and cellulose ended at lowlevels although substantial fructose was still present(Fig 1). At 700 rpm a lag period of 15–25 h wasobserved before growth and polymer formation beganat a significant rate (Figs 2 and 3); the reason for thislag is unclear. In the presence of 2 g dm−3 PA, both celland cellulose formation were generally prolonged overa greater period of time than at 1 g dm−3, ultimatelyreaching higher concentrations. At 900 rpm the rateof cell and polymer formation over time was rathermore consistent than at 700 rpm (Figs 4 and 5). Whilecell concentration generally reached the same level aspreviously, product formation was not as high as thatobserved with 2 g dm−3 PA at 700 rpm.

Table 3 summarizes the bioreactor results obtainedas a function of polyacrylamide concentration andstirrer speed. In the table the cell and fructoseconcentrations given are those which correspond to thepoint of maximum cellulose concentration. Althoughit is clear that the error bars would often overlap dueto the sampling problem described, highest productformation appeared to be realized at 2 g dm−3 PA and700 rpm, although the average cellulose concentrationwas statistically indistinguishable from that at 1 g dm−3

PA and 900 rpm at the 67% confidence level.

Figure 1. Bioreactor microbial (A xylinum) cell, cellulose and fructoseconcentrations versus fermentation time with stirring at 500 rpm and1 g dm−3 polyacrylamide.

966 J Chem Technol Biotechnol 78:964–970 (online: 2003)


Figure 2. Bioreactor microbial (A xylinum) cell and celluloseconcentrations versus fermentation time with stirring at 700 rpm and1 g dm−3 polyacrylamide.


3.3 Biomass morphologyObservations of biomass morphology made over timein shake flasks and the bioreactor are summarized here.In the minutes following inoculation of either typeof vessel, a cloudy suspension containing irregularlyshaped particles barely visible to the naked eye wasobserved. After about 3 h, the liquid phase cleared asthe visible particles increased in size while remainingirregular in shape, evidencing flocculation.

In the shake flasks without PA, the biomass particleswere a mixture of irregularly shaped particles andsoft, spheroidal bodies with a great variety of physical



dimensions. In the shake flasks with PA, the solidparticles eventually formed spheroidal pellets whosesize appeared to be inversely proportional to shakerspeed, and directly related to PA concentration. Thepellets had an opaque core and a translucent outercoating, and for a given set of conditions thereappeared to be a high degree of uniformity in particlesize. In the case of the bioreactor without PA, all ofthe biomass particles became lodged on the internals,leaving the fermentation broth clear. The biomassremained attached to the internals as solid masseswhich increased slowly over time. In the case of thebioreactor in the presence of PA, biomass particles in


G Joseph et al

Table 3. Maximum cellulose concentration, and corresponding cell

concentration and fructose consumption, versus PA concentration

and bioreactor stirrer speed. All values are in g dm−3, and represent

the average ± 1 standard deviation; the experiments were performed

in triplicate, except for the single run at 500 rpm

PA concentration(g dm−3), stirrerspeed (rpm)


Maximumcellulose

concentrationFructose

consumption

1, 500 2.88 2.08 18.21, 700 2.51 ± 0.33 3.56 ± 0.50 16.8 ± 0.92, 700 3.49 ± 0.82 5.30 ± 0.74 17.6 ± 1.51, 900 4.33 ± 0.11 4.41 ± 0.90 18.4 ± 0.12, 900 3.43 ± 0.70 3.37 ± 0.89 18.8 ± 0.4

suspension increased in quantity, with a small fractiongradually accumulating on the internals.

4 DISCUSSION4.1 Biomass morphologyBen-Bassat et al11 described the function of polyacry-lamide as that of a ‘protectant’ from the adverse effectof high agitation rates on cellulose yield, but said noth-ing about its effect on the morphology of biomass (iecombined cellulose and cells). The observations sum-marized in Section 3.3 indicate that PA has a profoundeffect on biomass morphology in agitated systems andwhile the influence of PA-based flocculants on cel-lulose in the pulp and paper industry has been welldocumented,16 the A xylinum fermentation has theadditional complication of being a living system. Thuscellulose fibers are constantly produced and entanglewith one another, such that a cellulose particle wouldstill grow if other particles were not being added to itthrough flocculation. In the following we attempt tobriefly describe some of the mechanisms which mightbe responsible for the phenomena observed in thiscomplex system.

The homogenized inoculum consists of a suspensionof free cells and biomass particles (ie masses ofcells enmeshed in cellulose). With time the celluloseparticles increase in size through growth, and likelyflocculation. The main evidence for the latter processis the transformation of the broth from a cloudysuspension to a clear solution containing particleswithin 24 h after inoculation. The size of the particlesand their morphology depends on whether thefermentation is carried out in a stirred bioreactor or inshake flasks. In shake flasks the majority of particleshave diameters larger than about 0.5 mm, and arespheroidal in shape; the remainder of the biomasstends to exist as fibrous clumps. The presence of PAdrives the system towards the former morphology.The spheroidal pellets appear to be made up oftwo layers, as observed by Chao et al17 in an air-lift bioreactor, namely an opaque core and a moretranslucent outer layer. In the bioreactor, probablybecause of the higher shear caused by the impellers,polyacrylamide has a more profound effect than inshake flasks due to its shear reduction properties. In

the absence of PA, biomass accumulates in large, solidclumps on the internals while the broth remains freeof particles. In the presence of PA, a suspension ofbiomass forms, and, while there is still some solidsdeposition on the internals, this consists largely ofdiscrete particles which eventually collapse into thebroth under gravity. Thus polyacrylamide appearsto affect biomass morphology both by acting as aflocculant, and by a mechanism(s) involving reductionof shear effects.

4.2 Cell and cellulose yieldsBefore discussing cell and product yields it is necessaryto consider the available substrates. Given that80 cm3 dm−3 of corn steep liquor (CSL) was present inthe medium in addition to 20 g dm−3 of fructose, theformer would be expected to contribute substantialamounts of both lactic acid and amino acids aspotential carbon sources. The medium is estimatedto contain approximately 14 g dm−3 of lactic acid and8.9 g dm−3 of amino acids, based on the followingtypical parameters for CSL:18 specific gravity, 1.25;54% (w/v) total solids; 26% (w/w) lactic acid (DMbasis); 35% (w/w) free amino acids (based on crudeprotein); 47% (w/w) crude protein (DM basis).

Lactic acid has been shown to promote rapid earlygrowth of the same and closely related strains ofA xylinum as studied herein by serving as an energysource for catabolism in the TCA cycle, but it isnot used as a substrate for cellulose formation,19 nordoes it increase ultimate cell concentration.20 As foramino acids, it was shown that 0.05 g dm−3 methio-nine added to fructose basal medium produced thesame cell and cellulose concentrations as a mixturecontaining a total concentration of 1.85 g dm−3 of 14amino acids.20 It has also been found that, with glu-cose as carbon source, as much cellulose is producedwith ammonium sulfate as nitrogen source as withcasein hydrolyzate or peptone.21 To a good approxi-mation, then, we are justified in considering fructoseas the only substrate contributing a significant amountof carbon to biomass formation.

In shake flasks containing PA at 1–2 g dm−3, YX/S

at 375 rpm was in the range 0.18–0.21 g g−1 com-pared with 0.13–0.15 g g−1 at 175 rpm. Similarlyin the bioreactor with 1 g dm−3 PA, YX/S (biomassyield based on fructose consumed, g dry matter g−1)increased steadily from 0.09 to 0.17–0.23 g g−1 asstirrer speed was increased from 500 to 700–900 rpm.Since in both types of vessel greater agitation wouldbe expected to increase oxygen transfer into thebroth, these results suggest that the dissolved oxy-gen concentration experienced by the cells embed-ded in the cellulose matrix was the principal factordetermining growth yield. In the bioreactor with2 g dm−3 PA, however, YX/S was similar at 700 and900 rpm (0.18–0.20 g g−1). Under these latter con-ditions of high broth viscosity, oxygen transfer mayhave been limited by bubble size, largely unaffected byimpeller speed.



Figure 6 shows cellulose yield based on fructoseconsumed, YP/S (cellulose yield based on fructose con-sumed, g g−1), plotted as a function of polyacrylamideconcentration, calculated for the 13 fermentationsfrom the tabulated data. In shake flasks YP/S wasconsistently greater at 175 rpm than at 375 rpm, andincreased with PA concentration. In the bioreactor at700 rpm but not at 900 rpm, YP/S followed the samepattern. Similarly Tables 1–3 show that cellulose pro-duction increased with PA concentration in all casesexcept at 900 rpm in the bioreactor. We believe thatthis results from preferential synthesis of cellulose rel-ative to by-products when the cells experience a lowfructose concentration. Our reasoning, justified below,goes as follows: all else being equal, the fructose con-centration at the surface of the cells surrounded bya gel-like cellulose network is controlled by its rateof diffusion through the gel; the latter is inverselyproportional to PA concentration through its effecton liquid-phase viscosity; and relatively high fruc-tose concentration accelerates its rate of consumption,causing the cells to divert a greater amount of substratecarbon from formation of cell mass and cellulose to by-products, including organic acids, acetan and carbondioxide. Thus, in general, higher PA concentration istranslated into an increase in cellulose production.

The most direct evidence for this hypothesis isthe inverse relationship between cellulose yield fromfructose, and specific fructose consumption rate,derived from the data in Tables 1–3, and shown inFig 7. From this it is evident that, for each shake-flaskagitation rate as well as in the bioreactor, the fraction offructose carbon used for cellulose synthesis increasedas the specific rate of fructose consumption decreased.More indirect evidence comes from analyzing thesame data for by-product formation. Figure 8 showsmaximum cellulose concentration in the variousexperiments as a function of the fraction of fructose

Figure 6. Cellulose yield by A xylinum based on fructose consumedversus polyacrylamide concentration.

Figure 7. Cellulose synthesis by A xylinum. Fraction of fructosecarbon going to cellulose synthesis versus specific fructoseconsumption rate in shake-flask and bioreactor experiments.

Figure 8. Cellulose synthesis by A xylinum. Maximum celluloseconcentration versus fraction of fructose carbon metabolized toby-products.

carbon going to by-product formation, defined asanything other than cell or cellulose synthesis, andcalculated assuming cell carbon content of 48%(w/w).22 As expected, an overall inverse relationshipof product and by-product formation is observed(correlation coefficient of 0.87). What could notbe predicted is that for each shake-flask agitationrate by-product formation decreased and celluloseformation rose as PA concentration was increasedfrom 0 to 3 g dm−3. Moreover in the bioreactor thehighest extent of substrate conversion to by-productsoccurred at 500 rpm with 1 g dm−3 PA present.


G Joseph et al

Under these conditions both cellulose production andgrowth were poor (Table 3), the latter at least dueapparently to oxygen limitation since fructose waspresent in abundance (Fig 1). This high substrateavailability per unit cell mass resulted in over three-quarters of the 18.2 g dm−3 of fructose consumedbeing metabolized to form by-products. With bettermass transfer at 700 rpm oxygen availability increased,cellulose formation rose and by-product formationfell as PA concentration was increased from 1 to2 g dm−3 (Fig 8); both YX/S and YP/S increased as well(Fig 6). Here the higher viscosity due to increasedPA concentration apparently reduced the fructoseconcentration experienced by the cells. However at900 rpm the exact opposite was observed, ie celluloseformation decreased and by-product formation rose asPA concentration was increased from 1 to 2 g dm−3;both YX/S and YP/S also fell. Here the higher agitationrate increased mass transfer of fructose both in theliquid phase and, possibly, also within the cellulosegel. However with 2 g dm−3 PA present, averagecell concentration at peak cellulose level was only3.43 g dm−3 compared with 4.33 g dm−3 at 1 g dm−3

PA (Table 3), presumably due to oxygen limitation;and the increased fructose availability per cell resultedin preferential formation of by-products.

Finally, indirect evidence that polyacrylamideimproves cellulose yield by restricting fructose avail-ability is provided by the fact that both of the processesdesigned for large-scale bacterial cellulose productionlimited the broth concentration of substrate by usingfed-batch fermentation.23 For example the processdeveloped by Byrom for Imperial Chemical Industrieslimited carbon source (generally glucose) concentra-tion to no more than 0.5 g dm−3.24 Although thiswould make it unnecessary to add PA for control-ling substrate availability, it might still be indicated todirect product morphology into the desirable particu-late morphology.

Clearly further work is needed to fully understandboth the morphological and physiological effectsof polyacrylamide on bacterial cellulose production.Whether or not such additives are used in large-scale fermentation processes, an understanding of themechanisms whereby they increase cellulose yield canonly further our ability to economically produce thisuseful polymer.

ACKNOWLEDGEMENTSWe thank Shakti Biomedical Corp for partial financialsupport of this work; and the Natural Sciencesand Engineering Research Council of Canada forindividual research grants awarded to A Margaritis andWK Wan, and a graduate scholarship to G Joseph.

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effects of polyacrylamide-co-acrylic acid on cellulose production by acetobacter xylinum

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