Enzyme and Microbial Technology 38 (2006) 101–108

The effect of agitation and aeration on the synthesis and molecularweight of gellan in batch cultures ofSphingomonas paucimobilis

Ioannis Giavasis, Linda M. Harvey, Brian McNeil∗

Strathclyde Fermentation Centre, University of Strathclyde, Department of Bioscience, 204 George street, Glasgow G1 1XW, UK

Received 20 April 2004; received in revised form 12 April 2005; accepted 5 May 2005

Abstract

The effects of agitation and aeration upon synthesis and molecular weight of the biopolymer gellan were systematically investigated inbatch fermenter cultures of the bacterium,Sphingomonas paucimobilis. High aeration rates and vigorous agitation enhanced growth ofS.paucimobilis. Although gellan formation occurred mainly in parallel with cell growth, the increase in cells able to synthesise gellan did notalways lead to high gellan production. For example, at very high agitation rates (1000 rpm) growth was stimulated at the expense of biopolymersynthesis.

Maximal gellan concentration was obtained at 500 rpm agitation and either 1 or 2 vvm aeration (12.3 and 12.4 g/l gellan, respectively). Ani r dissolvedo uction andp

lkanoates,( mayc

s (nutrient)t ear real timee avimetrica

r weight( importantf ight of theb©

K

1

teb

l(

eralith

l-ikelysuchoxy-

cesser-cessionation

0d

ncrease in aeration (from 1 to 2 vvm) enhanced gellan synthesis only at low agitation rates (250 rpm). However, high aeration oxygen was not necessary for high gellan synthesis, in fact oxygen limitation always preceded the phase of maximum gellan prodrobably enhanced polysaccharide biosynthesis.Some gellan was formed even after glucose exhaustion. This was attributed to the intracellular accumulation of polyhydroxya

such as polyxydroxybutyrate) which were found inS. paucimobilis cells indicating the existence of a carbon storage system, whichontribute to gellan biosynthesis under glucose-limiting conditions.The autolysis of the culture, which occurred at the late stages of the process, seemed to be triggered mainly by limitations in mas

ransfer, due to the highly viscous process fluid that gradually develops. Rheological measurements generally gave a very good nstimate of maximum biopolymer concentration offering the possibility of improved process control relative to time consuming grssay methods.While mechanical depolymerisation of gellan did not occur, high aeration rates (2 vvm) led to production of gellan of low molecula

at either 250 or 500 rpm). This effect of aeration rate upon gellan molecular weight is reported here for the first time, and isor the properties and applications of gellan. Mechanisms which may have led to this are discussed, but control of molecular weiopolymers is clearly an area needing further research.2005 Elsevier Inc. All rights reserved.

eywords: Gellan; Biopolymer; Agitation; Aeration; Rheology; Molecular weight

. Introduction

Gellan gum is a versatile biopolymer produced by the bac-eriumSphingomonas paucimobilis (formerlyPseudomonaslodea). It is used mainly as a gelling, thickening and sta-ilising agent in food, as well as a replacement for agar in

∗ Corresponding author. Tel.: +44 141 5484379; fax: +44 141 5534124.E-mail addresses: ioannis.giavasis@strath.ac.uk (I. Giavasis),

.m.harvey@strath.ac.uk (L.M. Harvey), b.mcneil@strath.ac.ukB. McNeil).

culture media for microbial and plant cells, and in sevother applications, either on its own, or in combination wother polysaccharides[1]. As with other biopolymers, gelan synthesis and molecular weight characteristics are lto be influenced by aeration and agitation, since in(polysaccharide) processes the efficiency of nutrient andgen transfer is affected by the high viscosity of the profluid [2]. Both agitation and aeration are involved to diffent extent in overall mass and oxygen transfer in the profluid. Agitation controls nutrient transfer and the distributof air and oxygen, while aeration determines the oxygen

141-0229/$ – see front matter © 2005 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2005.05.003

102 I. Giavasis et al. / Enzyme and Microbial Technology 38 (2006) 101–108

of the culture, but also contributes to bulk mixing of the fer-mentation fluid, especially where mechanical agitation ratesare low[3].

It has been shown earlier that the viscous, pseudoplasticfermentation fluids typical of microbial biopolymer produc-tion processes require high agitation to promote good masstransfer and achieve high product concentrations[2]. Con-versely, an increase in agitation means that more energyis consumed for the process (increasing production cost),and very high agitation has been shown to lead to mechan-ical damage to polysaccharides[4]. Similarly, oxygenationis important for cell growth for an aerobic species such asS. paucimobilis, and gellan production has been reported toalmost completely cease when the dissolved oxygen in thebioreactor is controlled at zero (DO = 0%) throughout theprocess[5].

The effect of aeration on gellan synthesis has not beenclearly established and the impact of both aeration andagitation on gellan molecular weight has not been stud-ied. Research into the relationship between the environmentwithin the bioreactor vessel and gellan molecular weight isimportant, since the molecular weight of a biopolymer affectsits quality and applications.

In the present study the effect of agitation and aeration onthe physiology of the micro-organism (growth, substrate con-sumption), gellan synthesis, the rheology of the fermentationfl ares ofp

2

m edb ter-n e.

0a 1 or2 houtt n of3 thep rode( mm,I

H),3N g/lo 82).T maltg act( ”,S four5 tionm sksw r-

mentation medium (inoculum size 10%). The optical density(OD) of the inoculum prior to addition in the fermenter was1.1–1.3 at 500 nm.

Gellan and biomass concentration were determined bymeans of centrifugation, precipitation and oven-drying toconstant weight, as described by Manna et al.[6]. The averagevalues of triplicate measurements are reported here.

Glucose concentration (in cell and gellan free centrifu-gates) was assayed with a glucose enzymatic kit (SIGMA,Glucose 500 Trinder, Kit No. 315-500). Ammonium concen-tration was measured with an ammonia electrode connectedto a digital display unit (Corning, model 240). Glucose andammonium concentration was again analysed in triplicatesamples, from which the average value is reported.

The detection of intracellular polyhydroxyalkanoates wasbased on the Sudan staining method, as described by BollesLee[7]. A Sudan IV stain was used (BDH, Cat. No. 26105).To an aliquot ofS. paucimobilis culture fluid, an equal vol-ume of an alcoholic solution of the dye was added. Afterincubation of this solution at room temperature for 30 min,the culture was examined under the microscope. Cells con-taining polyhydroxyalkanoates appeared to have large, darkviolet intracellular inclusions.

Rheological measurements were carried out with a RM180 Rheomat (Mettler-Toledo AG, Schwerzenbach), using acylinder-type, cup-and-bob measuring head to measure brothv1 ev f dif-f 9 sw er tog n thefi xingo

t tog-r byp .9,1 K).)v ar-m 5 cmd td.,D asu frac-t ies,M 00,H iono th0 hee ml/h,m aciaB oomt lateda sc )f pH

uid, and the molecular weight of the biopolymer itselfystematically investigated in batch fermenter culturesS.aucimobilis ATCC 31461.

. Materials and methods

The production of gellan gum bySphingomonas pauci-obilis from ATCC 31461 was studied in a 15 L stirrioreactor (B. Braun Biostat, ED ES10, Braun Biotech Inational, Meslungen, Germany) with 10 L working volum

The process set points were: pH, 7.0; temperature, 3◦C;gitation rate, 250, or 500, or 1000 rpm; aeration rate,vvm. Both agitation and aeration were constant throug

he process. The pH was controlled by automatic additioM NaOH and 3 M HCl. The dissolved oxygen (DO) inrocess fluid was monitored on-line by an oxygen electBroadley James Co., model D400 Oxyprobe, width 25rvine, USA).

The fermentation medium contained (g/l): glucose (BD0; K2HPO4 (Aldrich), 0.5; MgSO4 7H2O (BDH), 0.1;H4NO3, (Fishon’s) 0.9; yeast extract (Oxoid), 0.5 and 1f a complex salt solution, defined by Kang et al. (19he stock culture was grown and maintained in yeastellan (YMG) medium, which contained (g/l): yeast extrOxoid), 10; malt extract (Oxoid), 10; gellan (“Gelriteigma), 10. The inoculum was prepared by inoculating00 ml shake flasks containing 250 ml of sterile fermentaedium (of pH 7) with one loopful of culture each. The flaere incubated for 48 h at 30◦C and then added to 9 L of fe

iscosity at shear rates ranging from 65 to 1291 s−1 (65, 99,52, 233, 357, 549, 841, 1291 s−1). For comparison of thiscosity (average values of triplicate measurements) oerent fermentation fluids, a constant shear rate of 54−1

as chosen. This (high) shear rate was chosen in ordive measurable viscosity values in thin process fluids (irst hours of the fermentation), and to ensure good mif the viscous fermentation medium.

The peak molecular weight (MWp) and the MWp distribu-ion of gellan were studied using gel permeation chromaaphy (GPC). A MWp calibration curve was producedlotting the known MWp values of dextran standards (6682, 509 and 1000 kDa Sigma Chemicals Ltd., Dorset, Uersus elution time. A plastic column (XK 16 model, Phacia Biotech, Uppsala, Sweden) of 60 cm length and 1.iameter packed with Fractogel EMD BioSEC (Merck Lorset, UK), with a fractionation range of 1–1000 kDa wsed. The column was connected to a differential re

ive index (RI) detector (model 1755, Bio-Rad Laboratorunich, Germany) and a chart recorder (model YT 10owe & Co. Ltd., London, UK). The eluent was a solutf 75 mM tetra-methyl-ammonium-chloride (TMACl), wi.05% sodium azide (NaN3) added as a preservative. Tluent flow rate through the measurement cell was 25aintained by a peristaltic pump (model P-1, Pharmiotech, Uppsala, Sweden). Elutions were carried out at r

emperature. All samples studied by GPC were deacetys described by Manna et al.[6]. Briefly, deacetylation waarried out by heating the samples to 100◦C (in water bathor 15 min, cooling to 85◦C, subsequent alkalisation to a

I. Giavasis et al. / Enzyme and Microbial Technology 38 (2006) 101–108 103

of 10, and final neutralisation of sample pH. Eight milligramof deacetylated gellan sample were dissolved in 2 ml of elu-ent, to give a final concentration of 4 mg/ml. The samplesolution was filtered before injection with Whatman filters(GF/C, pore size 1.2�m) to remove any residual cells andany gellan aggregates which were not dissolved in the eluent.The injection volume was 0.2 ml. For the MWp distributionof gellan the samples of the highest MWp for each processwere compared. The MWp distribution of gellan was studiedfrom the elution profiles of each sample.

3. Results and discussion

3.1. Cell growth

The growth of S. paucimobilis was closely linked tothe agitation rate. The highest biomass (4.5 g/l) occurredat 1000 rpm, while it did not exceed 3.4 g/l in the processoperated at 500 rpm and 1 vvm (Fig. 1). When agitation wasreduced further (250 rpm, 1 vvm) biomass accumulation wasalso reduced (Fig. 1). An increase in the aeration rate from 1to 2 vvm also led to an improvement in cell growth, especiallywhen the agitation rate was low (250 rpm) (Fig. 1). This isprobably due mainly to the improved oxygen supply to thec

-i asst oseua thani teu iallyc 0 rpma agi-t d bym

Fu

Fig. 2. Glucose consumption in batch cultures ofS. paucimobilis ATCC31461 under different agitation and aeration conditions.

In most processes a rapid decay in biomass was observedsoon after maximum biomass was reached. This must be dueto an autolytic process triggered by the combined effect ofexhaustion of the nitrogen (N) source, as indicated by theearly exhaustion of ammonium (Fig. 3), and limitations inmass transfer caused by the increased viscosity of the pro-cess fluid. As seen inFig. 3in these processes the ammoniumion is consumed at a high rate in the first 9–13 h and is rapidlyexhausted (apart from the process with 250 rpm and 1 vvm)and, as reported previously by Lobas et al.[5] (using a processmedium similar to that in the present study) the culture thengrows on nitrate and later on an organic N source (e.g. yeastextract). It was found by Lobas et al.[5] that cell growthwas limited by the nitrogen rather than the carbon source,when initial glucose concentration was 30 g/l. Notably, thepresent results indicate that the high apparent viscosity ofthe process fluid may cause mass transfer problems, limitingnutrient and especially glucose availability to the cells. More

F3

ells at 2 vvm aeration rate.From the above, it is clear that forS. paucimobilis, increas

ng agitation enhances cell growth, by enhancing overall mransfer. Indeed, when agitation was low (250 rpm) glucptake was much slower than in the other processes (Fig. 2)nd similarly ammonium depletion occurred much later

n better agitated processes (Fig. 3), indicating poor substraptake. Increasing the aeration rate from 1 to 2 vvm partompensates for the poor mixing and mass transfer at 25lthough it makes less contribution to mixing at higher

ation rates (500 rpm), where mass transfer is dominateechanical agitation.

ig. 1. Biomass formation in batch cultures ofS. paucimobilis ATCC 31461nder different agitation and aeration conditions.

ig. 3. Ammonium concentration in batch cultures ofS. paucimobilis ATCC1461 under different agitation and aeration conditions.

104 I. Giavasis et al. / Enzyme and Microbial Technology 38 (2006) 101–108

Fig. 4. (a) Pseudoplastic behaviour of a typical gellan process fluid: as shearrate increases, apparent viscosity falls. The sample with the highest apparentviscosity in the process operated at 500 rpm and 1 vvm was used. (b) Appar-ent viscosity of the gellan process fluid in batch cultures ofS. paucimobilisunder different agitation and aeration conditions. Rheological measurementswere carried out at a constant shear rate of 509 s−1.

specifically, the most rapid decline in biomass observed inthe processes with 500 or 1000 rpm agitation rate and 1 vvmaeration rate coincides with the highest apparent viscosityvalues of the process fluid (especially at 500 rpm where mix-ing is less effective) (Fig. 4b). Conversely, the processes with500 rpm–2 vvm and 250 rpm–1 vvm, where autolysis occursmore slowly and to a lesser extend, are characterised by lowapparent viscosity of the process fluid and, consequently,better mixing and nutrient availability. Thus, the potentialglucose limitation imposed by poor mass transfer in the vis-cous fluids must also be considered for the explanation ofextensive autolysis in those processes.

Also, in the present study, a late increase in ammoniumconcentration in the fermentation fluid (Fig. 3) is observablein some processes, which may be closely associated withautolysis. Autolysis in microbial cultures is usually charac-terised as an active process[8] which consumes energy, andin the present study, this seems to be the case, with re-uptakeof the released ammonium ions in the 1000 rpm, 1 vvm pro-cess showing that a process analogous to the cryptic growth

Fig. 5. Gellan formation in batch cultures ofS. paucimobilis ATCC 31461under different agitation and aeration conditions.

noted by White et al.[8] may occur in these bacterial culturesalso (Fig. 3).

3.2. Gellan production

The gellan concentration was highest at an agitation rateof 500 rpm, regardless of the aeration level (at either 1 or2 vvm). At 1 and 2 vvm aeration rates, gellan reached 12.3and 12.4 g/l, respectively (Fig. 5). At 1000 rpm–1 vvm, gel-lan concentration was less than 9.0 g/l, despite the improvedcell growth, while at 250 rpm and 1 vvm aeration rate, themaximum gellan concentration dropped to 4.1 g/l. Underlow agitation conditions (250 rpm) an increase in aerationenhanced gellan formation (as well as growth).

These results show that increased agitation (i.e. from 250to 500 rpm) favours gellan synthesis by improving oxygentransfer and substrate uptake, which is crucial in a viscouspolysaccharide process fluid. The enhancement of polysac-charide synthesis by agitation was also observed in experi-ments on pullulan[9], and xanthan[10] production, wherebiopolymer accumulation was a direct function of stirringspeed (within an agitation range of 200–800 rpm). However,in the present study there seems to be a limit to the pos-itive effect of agitation on gellan production since at veryhigh agitation rates (1000 rpm), gellan concentration fell. Itis clear that under such conditions cell growth was stimulatedaa ita-t tionp rgelys fore,a ins ento

theo ra-t of

t the expense of gellan synthesis (Figs. 1 and 5). In addition,lthough good bulk mixing is important, an increase in ag

ion from 500 to 1000 rpm did not increase gellan formarobably because the fermentation medium became latagnant after approximately 30 h, in both cases. Theres indicated by Nienow[11] an increased agitation rateuch a fluid would not lead to any significant improvemf mixing out with the immediate impeller zone.

A moderate agitation rate of 500 rpm appears to beptimal for gellan production, with either 1 or 2 vvm ae

ion rate (Fig. 5). This is in some variance with the results

I. Giavasis et al. / Enzyme and Microbial Technology 38 (2006) 101–108 105

Fig. 6. Dissolved oxygen (DO) concentration (as percentage of air satura-tion) in the process fluid, in batch cultures ofS. paucimobilis, under differentagitation and aeration conditions.

Dreveton et al.[2] who concluded that the highest gellan pro-duction is achieved under the best oxygen transfer condition(however, in their study lower agitation rates and differentagitation systems were generally applied). Interestingly, herethe increase in aeration from 1 to 2 vvm caused a delay inreaching the peak in gellan concentration, thus modest agi-tation and modest aeration (500 rpm, 1 vvm) give a shorterprocess (Fig. 5). This delay at high aeration implies a possibleadverse effect of aeration on the rate of gellan synthesis, incontrast to the enhancement of cell growth noted above. Thedissolved oxygen profiles (DO%) showed that at 1 vvm aer-ation DO limitation occurs sooner in all cases (around 15 h),compared to the processes with 2 vvm aeration (Fig. 6). Inother words, it seems that there is a link between early oxygenlimitation and early peak in gellan concentration, and that thephase of maximum gellan synthesis requires a prior stage ofoxygen limitation.

The role of oxygen limitation in polysaccharide biosyn-thesis is worth further investigation for the gellan process.Contradictory results on this matter have been reported forother microbial polysaccharides. Many support the idea thatoxygen enhances biopolymer formation and production rate.For instance this has been observed for xanthan[10,12], curd-lan [13] and pullulan[14]. Nevertheless, contrary reports onthe effect of oxygen exist, especially in glucan production.Specifically, other reports on pullulan[15,16]and scleroglu-c ndi-t to y ber

raryo ee evi-o wass DOc on

process is not vulnerable to short reductions in oxygen sup-ply and less sensitive to reduced oxygen levels than otherpolysaccharide processes (e.g. xanthan,[12]).

In the present study gellan synthesis occurred mainly dur-ing the growth phase, regardless of the agitation and aerationrate, indicating that gellan formation was growth-associatedin these cultures to a degree. This is in agreement withthe reports of Lobas et al.[5] and Manna et al.[6]. How-ever, despite the concomitant increase with biomass in thefirst 25–30 h of the process, some gellan synthesis contin-ued in some of these processes during the stationary phase,or even during periods where biomass levels were falling(Figs. 1 and 5). From a process viewpoint, the formation ofsignificant quantities of biopolymer during the growth phaseoffers a potential advantage, since overall process duration isreduced relative to those biopolymer processes where prod-uct is formed only or mainly after cessation of growth, suchas the fungal biopolymers discussed above. It also opens thepossibility of continuous production processes.

3.3. Carbon storage

In the control process (500 rpm, 1 vvm) most gellan isproduced after 20 h, when only 5–10 g/l of glucose remainsin the process medium (Figs. 3 and 5). In addition, in thepresent study, and at variance with the results of Lobas eta edi austedf e oft 0 h)i to beu

beenrE -d -d cessc fpdgn -c senceo ts ofS el-l pya ragem imet ellanp

3

ts seu-d ity

an[17] showed optimum biopolymer synthesis under coions of low dissolved oxygen. Rau et al.[17] postulated thaxygen-induced repression of biosynthetic enzymes maesponsible for limiting polysaccharide production rate.

Also, in the present study, a short period of tempoxygen limitation (DO around zero) (Fig. 6), had no adversffect on polysaccharide production, although in a prus study gellan production stopped when the cultureubjected to continuous prolonged oxygen limitation (ontrolled at 0%)[5]. This shows that the gellan formati

l., [5] and Manna et al.[6], gellan accumulation continun some cases even after glucose appeared to be exhrom the process medium. This might indicate that somhe glucose that is utilised by the culture (e.g. in the first 2s stored inside the cells as an energy reserve, in ordersed at a later stage when residual glucose is very low.

Accumulation of energy storage compounds haseported for many bacteria, includingRhizobium spp. [18],. coli [19] and Azotobacter vinelandii – an alginate proucing organism –[19] all of which can also form polyhyroxyalkanoates, especially under conditions where exarbon source is readily available[19]. The existence oolyhydroxybutyrate as an energy storage compound inPseu-omonas paucimobilis (the former designation ofSphin-omonas paucimobilis) has been reported[20], but to dateo one had examinedSphingomonas in a fermentation proess for the presence of this storage compound. The pref this storage material was examined in culture aliquo. paucimobilis, and the characteristic dark violet intrac

ular inclusions were readily visualized by light microscofter staining, confirming the presence of intracellular stoaterials, i.e. polyhydroxyalkanoates. This is the first t

hat the relevance of intracellular carbon storage to the grocess has been considered.

.4. Rheology of the process fluid

In agreement with previous studies[2,21], in the presentudy the process fluid exhibited a marked degree of poplasticity (Fig. 4a). Examination of the apparent viscos

106 I. Giavasis et al. / Enzyme and Microbial Technology 38 (2006) 101–108

(µ�) of the process fluid under different agitation and aerationconditions showed that the maximum viscosity (173 MPa s−1

at shear rate of 549 s−1) was achieved at 500 rpm and 1 vvm(Fig. 4a), where gellan concentration was also maximal. Con-sideration ofFig. 4b andFig. 5 shows that in all processesconducted at 1 vvm aeration the time profile of apparent vis-cosity was closely correlated with gellan concentration in theprocess fluid. Conversely, the relatively high gellan concen-tration at 500 rpm and 2 vvm (12.4 g/l) was accompanied by asignificantly lowerµ� than might have been expected for thisconcentration of gellan, and a similar effect was noticed at250 rpm and 2 vvm, where apparent viscosity did not increasein proportion with polysaccharide concentration (Fig. 4b). Inother words, there was no simple correlation in these pro-cesses between the concentration of biopolymer formed andthe apparent viscosity of the process fluid. Since the rheologi-cal characteristics of crude gellan solutions (i.e. whole broth)are dominated by the polymer, the most probable explanationfor this is that alteration of the process conditions leads, inturn, to changes in the molecular weight of the biopolymer,thus influencing rheology.

However, for any one set of process conditions, apparentviscosity was a good indicator of when maximal biopolymerconcentration was achieved, and may well have relevanceto control of these processes, since it potentially gives nearreal time information about the process whilst the standardg

3

eenr dif-f ucedi earlyt esti-m tog-r ry).T thiss ative( llan.H gel-l ylatedg te ins nal-y ride

Fig. 7. Peak molecular weight (MWp) of gellan from batch cultures ofS.paucimobilis ATCC 31461 under different agitation and aeration conditions.

(TMACl), which is supposed to prevent aggregation[21]. Inaddition, deacetylation helps remove any residual cells andimpurities from gellan, which might be present in native gel-lan and contribute to inaccurate measurements.

In the present study the peak MW (MWp) values estimatedshowed that the highest MW was achieved in the processoperated at 500 rpm and 1 vvm, ranging from 650 to 850 kDa(Fig. 7). Towards the end of this process the MW of thepolysaccharide is reduced significantly (Fig. 7), accompaniedby a reduction in gellan concentration (Fig. 5) and conse-quently, broth apparent viscosity (Fig. 4b). This may be dueto the action of gellan lyases, possibly as a result of the glu-cose depletion occurring at that time (around 39 h) (Fig. 2),since it has been reported that gellan can be degraded enzy-matically byS. paucimobilis to produce glucose, rhamnoseand glucuronyl monomers[25]. However, under other agita-tion and aeration conditions such a severe drop in the MWwas not observed, despite the fact that glucose exhaustionoccurred in all processes.

A potential explanation of this may lie in the rate of gellanformation. As shown inTable 2, the mean gellan forma-tion rate at 500 rpm and 1 vvm is much higher during theperiod preceding glucose exhaustion (0–39 h) compared tothe other processes, and falls rapidly after glucose exhaus-tion. This implies a sudden transition (shock) for these cul-tures from glucose excess to absence of glucose at a timew osec ther

TE rature

S ype

[ ted, su[ lated,[ lated, c[ lated, c[ lated, c[ lated,P tylated y (GPC)

ravimetric measurements can take hours.

.5. Gellan molecular weight

Widely different values of molecular weight have beported for gellan to date. To an extent this is due to theerences in source of the gellan (commercial or prodn a research lab, acetylated or deacetylated), but clhe analytical methods used influence the final valuesated for the molecular weights (gel permeation chroma

aphy/light scattering (LS)/intrinsic viscosity/osmometable 1 shows these values, including the results oftudy. A clear difference exists between the values for nacetylated) gellan, and deacetylated or commercial geowever, it is possible the very high values of acetylated

an reported may represent an artefact, since the acetellan (in contrast to deacetylated) is known to aggregaolutions and cause filter clogging when performing LS asis, even in the present of tertramethylammonium chlo

able 1stimated values for gellan molecular weight (MW) reported in the lite

ource MW (kDa) Gellan t

22] 1600–2200 Acetyla22] 880–960 Deacety23] 434 Deacety24] 238 Deacety24] 55 Deacety21] 130–620 Deacetyresent study 200–850 Deace

hen metabolism is highly active and the rate of gluconsumption is correspondingly high, whereas, in the o

and in the present study

Method

pplied by industry Light scattering (LS)commercial Light scattering (LS)ommercial Light scattering (LS)ommercial Light scattering (LS)ommercial Osmometryproduced in lab Intrinsic viscosity, produced in lab Gel permeation chromatograph

I. Giavasis et al. / Enzyme and Microbial Technology 38 (2006) 101–108 107

Table 2Mean gellan formation rates from 0 to 39 h (pre-glucose exhaustion period),and from 39 to 58 h (post-glucose exhaustion) in batch cultures ofS. pauci-mobilis, grown under different agitation and aeration conditions. A negativeprefix in gellan formation rate indicates s reduction in gellan concentration

Agitation(rpm)

Aeration(vvm)

Mean gellanformation rate (g/l/h)0–39 h

Mean gellanformation rate (g/l/h)39–58 h

1000 1 0.202 −0.011500 1 0.304 −0.218250 1 0.090 −0.016500 2 0.232 0.078250 2 0.194 0.053

processes, the more gradual approach of carbon limitationand the moderate rates of glucose consumption may per-mit time to adapt cell metabolism to the evolving conditions.Thus, the activation of gellan lyases may be related to highglucose consumption rate and the concomitant sudden glu-cose depletion. Sutherland and Kennedy[25] found thatS.paucimobilis cannot grow on gellan as a sole carbon source,so they suggested that the main role of gellan lyases mightbe in gellan biosynthesis. However, in a balanced nutrientmedium (and after sufficient cell growth), these enzymes maydegrade the polysaccharide, under intense and rapid deple-tion of the carbon source, in order to serve the high energydemand of the cells. The late re-appearance of glucose aftercarbon exhaustion in this process is consistent with this rolefor gellan lyases (Fig. 2), as is the net disappearance of gellan(Table 2) in the processes at 1 vvm.

This has clear implications for the control of such pro-cesses, since under these conditions (500 rpm and 1 vvm)although the gellan concentration and MW achieved are thehighest in all these processes, very close control of the processis required to detect the optimal point for gellan concentra-tion and molecular weight in order to harvest. All the otherprocesses would be easier to control since they do not exhibitsuch severe changes.

In contrast to other biopolymers, such as scleroglucan,which are subject to time dependent mechanical degradation[ byh ald o thev closea rma-t tes)[

plesta vvm)r redt gard-l at1 eryl vma reasa the

gellan process, because it means that the physicochemicalproperties of gellan can be readily manipulated, by selectingdifferent process conditions (i.e. aeration). Thus, a polysac-charide can be produced which forms gels of either highstrength and brittleness (high MW gellan), or more elasticand soft ones (low MW gellan), according to the desirablegellan application.

This significant effect of aeration on gellan MW hasnot been reported to date, but it is in agreement with sim-ilar findings concerning other biopolymers. For instance,Quagliano and Miyaazaki[27] found that high MW poly-�-hydroxybutyrate was produced at low aeration rates(1100 kDa at 0.5 vvm), but this value was greatly dimin-ished at a high aeration rate (111 kDa at 2.5 vvm), althoughan explanation for these results was not given.

It is possible that gellan lyases could be the cause of thelower MW in these processes. However, based on the presentdata, the action of gellan lyases alone at high aeration ratesdoes not satisfactorily explain these observations, becausethe low MW of gellan in the processes with high aerationis consistently observed throughout these processes (fromthe early stages till the end) (Fig. 7), while generally gellanlyases have been shown to be induced only in the late stagesof the fermentation, when the culture is in the stationary orthe death phase, and exogenous carbon sources are at verylow or limiting levels [28]. Since in these processes MWi factora ar tob therg ilitys rtheri

4

rs int tionb uc-t att vm)l itiver y, at ro-d vm)g MWi max-i byd asesf ump aera-t atedc usb ner-a t ofm

4], the MW of gellan did not appear to be influencedigh agitation (1000 rpm) (Table 2). The absence of physicegradation with increased stirring rates may be due tiscous and locally stagnant process fluid, and also thedherence of gellan molecules to the cell membrane (fo

ion of tight network of cell and polysaccharide aggrega26].

Generally, despite some small variations among samaken at different process times, it is evident fromFig. 7thatll processes carried out under moderate aeration (1esulted in the production of gellan of higher MW compao the processes where 2 vvm aeration was applied, reess of the agitation rate (high MW gellan was formedvvm even at low agitation rates of 250 rpm, where v

ittle biopolymer was produced). More specifically, at 1 veration a polysaccharide of 560–850 kDa is formed, whet 2 vvm, MW falls to 200–320 kDa. This is important for

s consistently lower than in less aerated processes acting continuously throughout the process would appee the likely cause of the reduced polymer MW. Thus, anoellan-degrading mechanism related to oxygen availabhould be considered for these conditions. This merits fu

nvestigation, which is underway.

. Conclusions

Both aeration and agitation rate are key parametehe production of gellan. High aeration and high agitaoth favour cell growth, but not necessarily gellan prod

ion. High agitation (1000 rpm) stimulates cell growthhe expense of gellan synthesis. Also, high aeration (2 veads to a reduced gellan formation rate indicating a posole of oxygen limitation to gellan biosynthesis. Certainlemporary oxygen limitation does not hinder biopolymer puction. Moderate agitation and aeration (500 rpm, 1 vives highest gellan concentrations (12.3 g/l), and gellan

s also highest. Under these conditions, achievement ofmal gellan concentration and MW is rapidly followedecreases in both, probably due to the action of gellan ly

ollowing glucose exhaustion. Similar levels of maximolysaccharide concentration are obtained with higher

ion (2 vvm), but the gellan produced under highly aeronditions is of consistently lower MW. Gellan MW can the manipulated by variation of process conditions. Gelly, apparent viscosity could be used to identify the poinaximal polysaccharide concentration.

108 I. Giavasis et al. / Enzyme and Microbial Technology 38 (2006) 101–108

The presence of polyhydroxyalkanoates inS. paucimobilisunder conditions of glucose excess may explain the continuedsynthesis of gellan despite glucose exhaustion.

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