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Biochemical Engineering Journal 52 (2010) 50–54

Contents lists available at ScienceDirect

Biochemical Engineering Journal

journa l homepage: www.e lsev ier .com/ locate /be j

heological property of self-flocculating yeast suspension

. Yu, H. Wang, L. Wang, F.W. Bai ∗

epartment of Bioscience and Bioengineering, Dalian University of Technology, Dalian 116023, China

r t i c l e i n f o

rticle history:eceived 10 April 2010eceived in revised form 30 June 2010ccepted 4 July 2010

eywords:elf-flocculating yeast suspension

a b s t r a c t

Using the online monitoring technique established for yeast flocs with the focused beam reflectancemeasurement system, the rheological property of the simulation system with self-flocculating yeast andwater was examined. By controlling yeast flocs size relatively stable at the range from 350 to 470 �m, theimpact of biomass concentration on the rheological property was first studied. Compared with regularyeast suspension, which is a Newtonian fluid in general, the suspension of yeast flocs exhibited non-Newtonian fluid behavior, from a pseudoplastic fluid with biomass concentration lower than 40 g/L to ayield pseudoplastic fluid as biomass concentration increased, since the higher the biomass concentration,

heological propertythanol fermentation

the more resistant the suspension to shear force. On the other hand, it was found that, when yeast flocs sizedecreased to about 200 �m, a linear correlation between shear rate and shear stress was established, andthe rheological property of the suspension was close to a Newton fluid, but non-Newtonian flow behaviordeveloped when the size of yeast flocs increased to above that criterion, indicating its main contributionto the non-Newtonian flow behavior of the suspension. Based on these results and rheological modelestablished with the simulation system, the rheological property of the ethanol fermentation system

yeas

with the self-flocculating

. Introduction

Ethanol fermentation with yeast cells immobilized with sup-orting materials has been studied for more than 30 years sincehe early 1970s, but unfortunately, no commercial application haseen reported up till now since a pilot plant was established andxamined by Kyowa Hakko Kogyo Co. Ltd. in the middle of the980s [1]. Ethanol, as a typical primary metabolite, its production

s tightly coupled with the growth of yeast cells, making yeast cellsmmobilized with supporting materials, particularly by gel entrap-

ent that has been studied intensively in the past and is still beingxplored continuously now, theoretically not reasonable, becausehe physical constraint of supporting materials on yeast cells seri-usly compromises their growth and ethanol production as well. Onhe other hand, extra costs, such as the preparation of immobilizedeast cells in large quantity without significant contamination, con-umption of supporting materials and contamination of supportingaterials on the quality of biomass and other byproducts that can

redit ethanol production significantly, make them economically

ot competitive [2].

When yeast cells self-flocculate and form flocs with suitableize, they can be immobilized within bioreactors without con-umption of any supporting materials, and the aforementioned

∗ Corresponding author. Tel.: +86 411 8470 6308; fax: +86 411 8470 6329.E-mail address: fwbai@dlut.edu.cn (F.W. Bai).

369-703X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2010.07.003

t was predicted.© 2010 Elsevier B.V. All rights reserved.

theoretical drawbacks and economic disadvantages of yeast cellsimmobilized with supporting materials can be overcome effec-tively. Therefore, studies have gone to ethanol fermentation withthe self-immobilized yeast cells, including their online monitor-ing and characterization technique, growth and ethanol productionkinetics, process optimization and metabolic flux analysis [3–6],which led to the establishment of a large scale fuel ethanol plantwith its production capacity of 200,000 t/y [7].

Rheological property affects hydrodynamics and heat and masstransfer performance of bioreactors as well as kinetics of cellgrowth and fermentation, making it a prerequisite for bioreactordesign and process optimization. Although rheological property ofregular yeast suspensions has been studied in the past, available lit-eratures are very limited, and experimental results are inconsistent.For example, Mancini and Moresi reported that yeast suspensionswith biomass concentration in the range of 25–200 g/L behaved asNewtonian fluids [8], while El-Temtamy et al. previously reportednon-Newtonian fluid behaviors for yeast suspensions with wetbiomass volume fraction between 0.1 and 0.6 [9].

As for the suspensions of yeast flocs, scarcely available reporton their rheological property seems quite problematic, since onlyconcentration of yeast flocs was measured and taken into account,

while their size and its impact on the rheological property wereneglected, due to the lack of reliable online monitoring and charac-terization techniques for yeast flocs that cannot be measured offlineby taking samples from bioreactors [10]. In this article, takingadvantage of the online monitoring and characterization technique

L. Yu et al. / Biochemical Engineeri

Nomenclature

� shear stress (Pa)�0 stress yield (Pa)� shear rate (1/s)�a apparent viscosity (mPa s)K consistency coefficient (N sn m−2)n flow behavior indexX biomass concentration in the suspensions (g (dry

cell weigh)/L)C citric acid concentration (M)

o(yb

2

2

Seoibpbba(

tmtf

2

attlspfiwcmeeao

TA

Lsqr average chord length of yeast flocs (�m)R2 correlation coefficient

f yeast flocs with the focused beam reflectance measurementFBRM) system [3], the rheological property of the self-flocculatingeast suspensions was investigated, which is a prerequisite forioreactor design and process optimization.

. Materials and methods

.1. Strain, medium and pre-cultivation

The strain used in this work was the self-flocculating yeastPSC01, a fusant developed by Department of Bioscience and Bio-ngineering, Dalian University of Technology, the People’s Republicf China, from two parent strains: Saccharomyces cerevisiae K2, anndustrial strain with excellent ethanol fermentation performanceut without self-flocculating ability, and Schizosaccharomycesombe, a self-flocculating strain with good self-flocculating abilityut moderate ethanol fermentation performance. This fusant com-ines the merits of the two parent strains, which was depositedt Chinese General Microbiological Culture Collection CenterCGMCC) with a reference number of 0587.

Seed pre-culture was carried out by inoculating the stock cul-ure into a 250 mL Erlenmeyer flask containing 100 mL sterilized

edium composed of glucose 30 g/L, yeast extract 4 g/L and pep-one 3 g/L. The flask culture was carried out at 30 ◦C and 150 rpmor 24 h.

.2. Preparation of the self-flocculating yeast suspensions

The flask culture was vacuum-filtrated by the Buchner funnelnd washed by deionized water for three times. Since the fermenta-ion broth without yeast flocs was previously studied and identifiedo be a Newtonian fluid with low viscosity of 2–3 mPa s [11], muchower than apparent viscosity of the self-flocculating yeast suspen-ions, series dilution with water was applied to the yeast flocs torepare a simulation system with different biomass concentrations,tting the ethanol fermentation system that is normally operatedith biomass concentration from 30 to 60 g/L. Given low biomass

oncentration of the fermentation system from inoculation to nor-

al fermentation, biomass concentration below 30 g/L was also

xamined. The concentration of yeast flocs in the suspensions wasxperimentally measured to be 2.3, 14.6, 23.7, 31.3, 40.7, 51.2, 62.1nd 78.2 g/L, respectively. Furthermore, the rheological propertyf the ethanol fermentation system was measured and compared

able 1verage chord length of yeast flocs in the suspensions.

X (g L−1) 2.3 14.6 18.9 27.5Lsqr (�m) 343.16 352.71 380.78 430.2

ng Journal 52 (2010) 50–54 51

with that predicted by the rheological model established with thesimulation system.

The average chord length of yeast flocs detected by FBRM wasused to characterize their size, and its impact on the rheologicalproperty was investigated by de-flocculating yeast flocs with citricacid, a moderate chelator for Ca2+ and Mg2+ that bridge yeast cellsfor their flocculation [12]. The self-flocculating yeast suspensionswith biomass concentrations of 14.9, 34.7 and 72.9 g/L were appliedand deflocculated with citric acid at the concentrations of 0, 0.02,0.05, 0.10 and 0.15 M to create yeast flocs with different sizes.

2.3. Analytical methods

The culture of yeast flocs with a volume of 200 mL was trans-ferred into a 500 mL beaker that was stirred with a magnetic stirbar (30 mm × 8 mm) at the speed of 250 rpm to make yeast flocssuspended homogeneously, which simulated the hydrodynamicconditions for yeast flocs to be suspended homogeneously withinthe suspended-bed bioreactor [7].

The FBRM that was inserted into the beaker measured theaverage chord length of the yeast flocs, following the protocol pre-viously established by Ge et al. [3]. The rheological property ofthe self-flocculating yeast suspensions was measured by the rota-tional viscometer NDJ-1, which was equipped with a stator wheresamples were positioned and five rotors with their rotating speedsrange from 6 to 60 rpm for the measurement of the apparent viscos-ity. All biomass concentrations were expressed in dry cell weight(DCW) per volume suspension [3].

A constant temperature of 30 ◦C was applied to all measure-ments, since ethanol fermentation with yeast is normally carriedout at this temperature with small variations, which exerts negli-gible impact on the rheological property of the suspensions.

3. Results and discussion

3.1. The rheological property of the self-flocculating yeastsuspension

The average chord length of yeast flocs was measured by FBRM,and the results are documented in Table 1. As can be seen, it wasrelatively constant, ranging from 350 to 470 �m, which would notaffect the rheological property of the suspension significantly.

The impact of shear rate on shear stress was further examinedfor the suspension. As illustrated in Fig. 1, shear stress increasedwith the increase of shear rate and the concentration of yeast flocsas well. Under low biomass concentration of 2.3 g/L, a linear rela-tionship between shear rate and shear stress was observed, and thesuspension was close to a Newtonian fluid, due to the main contri-bution to the rheological property coming from the liquid phase.However, as biomass concentration increased, non-linear relation-ship between shear stress and shear rate was developed, indicatingthat the rheological property of the suspension converted to a non-Newtonian fluid.

Classification of non-Newtonian fluids depends on the rela-

tionship between the shear stress developed and the shear rateimposed. Since the data illustrated in Fig. 1 clearly indicate thatyield stress developed for the self-flocculating yeast suspensionas the biomass concentration increased, the constitutive equation� = �0 + K�n was thus adopted, and the values of the fluid consis-

40.7 51.2 62.1 78.25 463.83 470.82 448.53 348.77

52 L. Yu et al. / Biochemical Engineering Journal 52 (2010) 50–54

FbT

tas

mttcdo

capo

TCs

Table 3Average chord length of yeast flocs deflocculated by citric acid.

Lsqr (�m) C (M)

0 0.02 0.05 0.10 0.15

ig. 1. Rheological property of the self-flocculating yeast suspensions with differentiomass concentrations. The average chord length of the yeast flocs is illustrated inable 1.

ency coefficient K and flow behavior index n were estimated withpolynomial regression with a confident interval of 95%, which are

ummarized in Table 2.Since the entanglement of yeast flocs and the interaction of

acromolecules such as surface proteins that are responsible forhe self-flocculation of yeast cells were enhanced as the concentra-ion of yeast flocs increased, the consistency coefficient increased,orrespondingly. And in the meantime, the flow behavior indexecreased, indicating that more deviation from a Newtonian fluidccurred.

From the constitutive equation, we can see when biomass con-

entration was lower than 40 g/L, no stress yield was developed,nd the flow behavior of the suspension was characterized by theseudoplastic fluid pattern. With the increase of the concentrationf yeast flocs, stress yield was established, and the flow behavior of

able 2onsistency coefficient, flow index and constitutive equation of yeast flocsuspensions.

X (g L−1) K n Constitutive equation R2

2.3 0.003 0.85 � = 0.003�0.85 0.998714.6 0.018 0.80 � = 0.018�0.80 0.998018.9 0.028 0.75 � = 0.028�0.75 0.999127.5 0.036 0.64 � = 0.036�0.64 0.989340.7 0.073 0.58 � = 0.01 + 0.073�0.58 0.978751.2 0.087 0.43 � = 0.02 + 0.087�0.43 0.982862.1 0.110 0.39 � = 0.03 + 0.110�0.39 0.981078.2 0.130 0.24 � = 0.06 + 0.130�0.24 0.9583

X (g/L) 14.9 427.42 334.61 258.82 160.92 73.8234.7 366.02 360.61 320.05 163.50 94.2672.9 326.00 316.96 280.66 151.73 87.09

the suspension converted to the yield pseudoplastic fluid pattern,which seems reasonable since the higher the concentration of yeastflocs, the more resistant the suspension to shear force exerted.

3.2. Impact of yeast flocs size on the rheological behavior of thesuspension

Previous research on the rheological behavior of regular yeastsuspensions indicated Newtonian fluids when biomass concentra-tion is not extremely high [10]. The difference between yeast flocsand regular yeast lies in the fact that yeast flocs are much larger insize, and on the other hand, when yeast cells self-flocculate, theirinteraction is enhanced, which could affect the rheological propertyof their suspension.

Table 3 documented the size of yeast flocs deflocculated withcitric acid at different concentrations. As can be seen, when cit-ric concentration increased to 0.15 M, the average chord length ofyeast flocs in the suspension decreased to below 100 �m, a sig-nificant de-flocculation enough to make the self-immobilization ofyeast flocs failed within bioreactors, since yeast flocs at this sizecannot be separated from the effluent effectively by their self-sedimentation under continuous operation conditions, and thuswill be washed out of bioreactors quickly [8].

The impact of the size of yeast flocs on the rheological behaviorof the suspension was thus investigated, and the results are illus-trated in Fig. 2. As can be seen, when the average chord lengthof yeast flocs decreased to about 200 �m, nearly linear relation-ship between shear rate and shear stress was observed, therefore,the rheological behavior was close to a Newtonian fluid, like reg-ular yeast suspension. However, significant non-Newtonian flowbehavior developed when the average chord length of yeast flocswas above that criterion.

The consistency coefficient and flow index of the suspensionare documented in Table 4. It is worth noticing that under thesame biomass concentration conditions, the consistency coefficientdid not change significantly as yeast flocs were deflocculated bycitric acid and their average chord length decreased, correspond-ingly, but the flow behavior index was affected more. And on theother hand, under different biomass concentration, when the aver-age chord length of yeast flocs was controlled at the same level,the flow behavior index was affected slightly, but the consistency

coefficient changed much more. For example, when the suspen-sions with the biomass concentrations of 14.9, 34.7 and 72.9 g/Lwere deflocculated by 0.02, 0.05 and 0 M citric acid, their averagechord length was measured to be 334.60, 320.05 and 326.00 �m,

Table 4Impact of yeast flocs sizes on the rheological behaviors of the suspensions.

C (M)

0 0.02 0.05 0.10 0.15

X (g/L) 14.9 K 0.013 0.008 0.006 0.003 0.002n 0.41 0.49 0.50 0.76 0.80

34.7 K 0.034 0.020 0.014 0.004 0.003n 0.33 0.36 0.37 0.71 0.83

72.9 K 0.110 0.079 0.055 0.039 0.028n 0.39 0.64 0.68 0.70 0.71

L. Yu et al. / Biochemical Engineering Journal 52 (2010) 50–54 53

F east fll

atsc

3

flas

Fweyw

ig. 2. Impact of yeast flocs sizes on the rheological behaviors of the suspensions. Yength of the yeast flocs is illustrated in Table 3.

nd flow behavior index n was 0.49, 0.37 and 0.39, indicating thathe main contribution on the non-Newtonian flow behavior of theuspensions might be from their size and interaction among yeastells due to their self-flocculation and close contact.

.3. Rheological property of the ethanol fermentation system

Based on the study of the rheological property of the self-occulating yeast suspension with the simulation system, thepparent viscosity of the ethanol fermentation system was mea-ured experimentally and compared with that predicted by the

ig. 3. Comparison of the apparent viscosity predicted by the rheological modelith experimentally measured for the ethanol fermentation system with 12.0% (v/v)

thanol, less than 5 g/L residual glucose, and 2.8, 11.3, 16.1, 27.5, 35.3 and 39.2 g/Least flocs (�a increased, correspondingly), while the average size of the yeast flocsas adjusted between 323.6 and 403.5 �m.

ocs were deflocculated by citric acid at different concentrations. The average chord

rheological model. As can be seen in Fig. 3, the R-squared valueis 0.9906, indicating that the rheological model can be used to pre-dict the rheological property of the real fermentation system withnegligible errors.

4. Conclusions

Compared with regular yeast cell suspension, the self-flocculating yeast suspension presents non-Newtonian flowbehavior, due to the unique morphology of yeast flocs, which aremuch larger in size, and the enhanced interaction of yeast cellsthat aggregate together. Only at low biomass concentration thatis experienced at the early stage after inoculation, can Newtonianflow behavior be observed for the suspension. With the increase ofyeast flocs concentration, non-Newtonian flow behavior will corre-spondingly develop for the suspension, from pseudoplastic fluid toyield pseudoplastic one, and the main reason for this phenomenonis contributed to the self-flocculation of yeast cells.

Acknowledgements

The authors appreciate the financial support from Natural Sci-ence Foundation of China (No. 20576017) and High-Tech Researchand Development Program of China (No. 2007AA10Z358).

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