Food Chemistry 120 (2010) 163–173

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Food Chemistry

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Kinetics of inulinase production by solid-state fermentation in a packed-bedbioreactor

Marcio A. Mazutti a,*, Giovani Zabot b, Gabriela Boni b, Aline Skovronski b, Débora de Oliveira b,Marco Di Luccio b, Maria Isabel Rodrigues a, Helen Treichel b,*, Francisco Maugeri a

a Department of Food Engineering, Faculty of Food Engineering, University of Campinas – UNICAMP, P.O. Box 6121, CEP 13083-862, Campinas – SP, Brazilb Department of Food Engineering, URI – Campus de Erechim, P.O. Box 743, CEP 99700-000, Erechim – RS, Brazil

a r t i c l e i n f o

Article history:Received 22 June 2009Received in revised form 14 August 2009Accepted 5 October 2009

Keywords:Solid-state fermentationInulinasePacked-bedKluyveromyces marxianus NRRL Y-7571Kinetic evaluation

0308-8146/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.foodchem.2009.10.002

* Corresponding authors. Fax: +55 54 35209090.E-mail addresses: mazutti@uricer.edu.br (M.A. Maz

Treichel).

a b s t r a c t

In this work the cell growth and inulinase production by Kluyveromyces marxianus NRRL Y-7571 wereinvestigated in a packed-bed bioreactor. For this purpose, seven experimental runs were carried out inorder to verify the influence of the inlet air temperature and volumetric air flow rate on the processdynamics. The results showed that the manipulated variables affected significantly the process perfor-mance. The best condition for enzyme production was an inlet air temperature and volumetric flow rateof 30 �C and 3 m3 h�1, respectively, reaching an activity of around 463 U gds�1. The results obtained herecan be useful in the scale-up and optimisation of packed-bed bioreactors configuration for inulinaseproduction.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

The inulinases are useful for fructoligosaccharide productionand have functional and nutritional properties for use in low-calo-rie diets, stimulation of Bifidus and as a source of dietary fibre infood preparations (Silva-Santisteban & Maugeri, 2005). Inulinasesare enzymes potentially useful on high fructose syrups (HFS) bythe enzymatic inulin hydrolysis, which have high yields of 95%(Ettalibi & Baratti, 2001). The conventional fructose productionby starch hydrolysis includes three steps: the action of a-amylase,amyloglucosidase and glucose isomerase, yielding only 45% of fruc-tose in the final product due to the thermodynamic equilibrium ofthe reaction (Mazutti, Bender, Di Luccio, & Treichel, 2006).

Solid-state fermentation (SSF) is generally defined as that inwhich microbial growth and product formation take place on a so-lid substrate in the absence of free water (Pandey, 2003). In the lasttwo decades, SSF has received increasing interest partly because, asfermentation technology, it has lower energy requirements andproduces less wastewater than submerged fermentation (Couto &Sanromán, 2006; Holker, Hofer, & Lenz, 2004). In addition, thereare increasing environmental concerns regarding the disposal ofsolid wastes, and their use as substrates for the commercial pro-

ll rights reserved.

utti), helen@uricer.edu.br (H.

duction of microbial metabolites is becoming an attractive propo-sition, mainly in Brazil that is known as one of the most importantagricultural-based economies in the world, producing coffee, sug-arcane, soybeans, cassava, fruits, etc. The industrialisation of theseproducts generates high amounts of residues that cause seriousenvironmental problems (Lu, Maddox, & Brooks, 1998; Machado,Oishi, Pandey, & Soccol, 2004). Besides these characteristics ofSSF, several works can be found in the literature employing sub-merged fermentation for inulinase production (Sguarezi et al.2008; Makino, Treichel, Mazutti, Maugeri, & Rodrigues, 2009;Treichel, Mazutti, Maugeri, & Rodrigues, 2009).

A considerable effort has been made recently employing agroin-dustrial residues as substrates to produce inulinase by SSF(Ayyachamy, Khelawan, Pillay, Permaul, & Singh, 2007; Bender,Mazutti, Di Luccio, & Treichel, 2008; Bender, Mazutti, Oliveira, DiLuccio, & Treichel, 2006; Mazutti, Ceni, Di Luccio, & Treichel, 2007;Mazutti et al. 2006; Selvakumar & Pandey, 1999; Xiong, Jinhua, &Dongsheng, 2007). This class of enzyme is important in food process-ing, with its main use as a catalyst in synthesising fructooligosaccha-rides from sucrose by transfructosilation (Santos & Maugeri, 2007).

At this point, it is important to mention that all the above men-tioned works reporting the inulinase production by SSF were car-ried out in tray bioreactors using only a few grams of substrate.Besides, this technology suffers from its limited capacity and pooraeration control. Thus, other bioreactor configurations have beeninvestigated, including the packed-bed and rotating drum bioreac-tor (Lu et al. 1998).

164 M.A. Mazutti et al. / Food Chemistry 120 (2010) 163–173

The packed-bed reactor seems to be a promising bioreactor con-figuration, since it can be used in two ways: (i) to analyse empiri-cally the global evolution of a process and determine theenvironmental parameters for controlling the temperature andmoisture of the solid medium, (ii) to study mass and heat transferphenomena and oxygen diffusion (Durand, 2003). Besides, the yeastKluyveromyces marxianus is sensitive to shear stress and agitation,as previously reported by Silva-Santisteban and Maugeri (2005).

One important step in the development of the SSF process is theproper description of biological activity. Results of these descrip-tions can be used for modelling, optimisation and scale-up. Theimpossibility of separating the biomass from the substrate andthe heterogeneous characteristics of SSF processes are the principaldifficulties found when kinetics accomplishment is attained. Thesefacts impede the acquirement of representative samples and areparticularly acute in the case of microbial growth. Even with thedifficulties that are encountered in the SSF, the kinetic procedurecan not be substituted by simple and overall process description(Machado et al., 2004). Measuring O2 uptake or CO2 production rateis a commonly employed methodology for estimating cell growth inthe aerated fermentation process (Terebiznik & Pilosof, 1999).

In this context, the objective of the present work was to evalu-ate the kinetics of cell growth and inulinase production by K.marxianus NRRL Y-7571 in a packed-bed bioreactor. A stoichiome-try correlation between CO2, ethanol, metabolic water, O2 and totalreducing sugar was determined. Besides, the metabolic heat pro-duction was estimated by a proper energy balance in the inletand outlet air stream leading to better knowledge of the behaviourof the system for future work in scaling-up.

2. Materials and methods

2.1. Agroindustrial residues

The medium composition was optimised in a previous work byMazutti et al. (2006) as following: 2 kg of sugarcane bagasse sup-plemented with pre-treated cane molasses 15% (w/w), corn sleepliquor (CSL) 30% (w/w), and soybean bran 20% (w/w). Sugarcanebagasse was obtained from COTREL Ltd. (Erechim, RS, Brazil), CSLfrom Corn Products Brazil (Mogi Guaçu, SP, Brazil), soybean branfrom Olfar SA (Erechim, RS, Brazil), and cane molasses from ÉsterRefinery (Campinas, SP, Brazil).

The cane molasses was pre-treated before its use following themethodologies described by Sguarezi et al. (2008). The pH of canemolasses 200 g l�1 solution was adjusted with sulphuric acid from1 N to 5.0. The solution was set to rest at 24 �C for 24 h. The med-ium was then centrifuged at 5000g for 15 min, and the final pH wasadjusted to 4.0 with NaOH 1.0 M.

2.2. Microorganism and medium

The strain of K. marxianus NRRL Y-7571 used for inulinase pro-duction was maintained on YM agar medium (g l�1): yeast extract

Table 1Stoichiometry and yielding coefficients obtained for the inulinase production by SSF in pa

Runs Volumetric flow rate(m3 h�1)

Inlet air temperature(�C)

Stoichiometry Coeffi(C-mol compound/C

a b

1 2.0 27.0 3.59 0.172 2.0 30.0 3.59 0.173 2.0 33.0 3.24 0.174 3.0 27.0 3.29 0.175 3.0 30.0 3.93 0.176 3.0 33.0 3.11 0.177 2.4 30.0 3.63 0.17

* The unit of the YX/S, YX/O and RQ is g/g and the unit of YQ/X is KJ/g.

3.0, malt extract 3.0, peptone 5.0, glucose 10.0, agar 20.0, and sub-cultured every 3 weeks.

Cell production for pre-inoculum was carried out in 50 ml testtubes with 10 ml of liquid YM medium. This was inoculated with aloopful of the stock culture and incubated at 30 �C for 24 h. Each testtube (10 ml) with YM medium was transferred to a 500 ml Erlen-meyer flask with 100 ml of pre-inoculum medium and incubatedin a rotatory shaker at 30 �C and 150 rpm for 24 h. The mediumwas composed by (g l�1): sucrose 20.0, yeast extract 5.0, K2HPO4

5.0, NH4Cl 1.5, KCl 1.15, and MgSO4�7H2O 0.65. The cell mass in thepre-inoculum was determined by a direct weight measurement be-fore its use in the fermentation (Mazutti et al., 2007).

2.3. Solid-state fermentations

The packed-bed bioreactor consists of a cylindrical stainless bedconnected to an air humidifier, which supplies air at a relativehumidity of 95–100%. The saturated air at the operation tempera-ture enters in the bottom of the bioreactor and passes through thebed, until the exit located at the top of it. The bioreactor was filledwith 2 kg of dry sugarcane bagasse, supplemented as describedabove. The moisture content was corrected to 65% (w/w) and auto-claved at 121 �C for 20 min. The fermentation runs were startedwith the inoculation of an optimised volume corresponding to acell mass of 14 g (data not shown). All experiments were carriedout by 24 h. A total of seven experiments, described in Table 1,were performed at different inlet air temperatures (27.0, 30.0and 33.0 �C) and volumetric flow rate (2.0, 2.4 and 3.0 m3 h�1).Experiments were carried out in triplicate runs and all standarddeviations were lower than 10%.

2.4. Experimental and calculated data

The total reducing sugar concentration and inulinase titres weremonitored at 0, 2, 4, 8, 12, 18 and 24 h of fermentation. The sugarwas extracted from the medium (10 g) by adding 100 ml of bi-dis-tillated water in 20 g of moist fermented medium (Mazutti et al.,2007), following incubation at 40 �C and 150 rpm for 60 min. TRSwere measured by the 3,5-dinitrosalicylic acid method (Miller,1959). After fermentation, the enzyme was extracted from the sug-arcane bagasse by adding sodium acetate buffer 0.1 mol l�1 pH 4.8in a solid/liquid ratio of 1:10, following incubation at 50 �C and150 rpm for 30 min. Inulinase activity was assayed in the superna-tant after filtration. An aliquot of 0.5 ml of the appropriately di-luted enzyme source was incubated with 4.5 ml of 2% (w/v)sucrose solution in sodium acetate buffer (0.1 mol l�1 pH 4.8) at50 �C (Mazutti et al., 2007). Reducing sugars released weremeasured by the 3,5-dinitrosalicylic acid method (Miller, 1959).A separate blank reaction was run for each sample to correct thenon-enzymatic release of sugars. One unit of inulinase is definedas the amount of enzyme that released 1 lmol of reducing sugarsper minute under the standard assay conditions. The results were

cked-bed bioreactor.

cients-mol biomass)

Yielding Coefficients(g/g or *kJ/g)

k c d r YX/S YX/O RQ *YQ/X

0.43 1.10 0.74 1.48 0.27 2.06 3.53 16.650.70 1.28 1.00 1.30 0.26 1.27 2.52 12.490.77 1.21 1.07 1.03 0.29 1.16 2.17 13.360.58 1.11 0.82 1.19 0.29 1.54 2.62 10.490.82 1.48 1.13 1.45 0.24 1.08 2.48 10.410.70 1.12 1.00 0.99 0.31 1.27 2.21 11.950.61 1.24 1.24 1.39 0.26 1.45 2.80 15.23

M.A. Mazutti et al. / Food Chemistry 120 (2010) 163–173 165

expressed in terms of inulinase activity per gram of dry solids(U gds�1). Following the same described above, the inulinase activ-ity using inulin as a substrate was also determined at the opti-mised experimental condition. The moisture content wasdetermined by the method of desiccation in an oven with air circu-lation (model FANEM 320-SE) at 105 �C. The sugar concentration,moisture content and inulinase activity were evaluated at four dif-ferent bed weights, with the samples collected at 0–10, 10–20, 20–30 and 30–40 cm.

The inlet and outlet air temperatures were continuously moni-tored by a temperature probe PT100 (NOVUS, Brazil). In addition,the temperature of the moist substrate was monitored at 10, 20and 30 cm inside the bioreactor. The respiratory metabolism ofthe microorganism was evaluated by determining the CO2 produc-tion. The outlet air from the bioreactor was analysed by a CARBO-CAP GMT220 sensor (VAISALA, Inc., Finland), which is silicon basedand its operation is based on the NDIR Single-Beam dual-wave-length principle. The temperature probes and CO2 sensor were con-nected to an acquisition board (FIELDLOGGER NOVUS, Brazil) withan interval of 30 s among each acquisition.

The general stoichiometry equation for aerobic microbialgrowth with the product formation considered in this work is:

aC6H12O6 þ bNH4 þ kO2 ! CH1;94O0;76N0;17 þ cCO2 þ dH2O

þ rC2H6O ð1Þ

where a, b, k, c, d and r are the stoichiometry coefficients (C-molcompound per C-mol biomass), which were computed by the C, H,O and N balances and taken as reference to the experimentalCO2 and TRS rates. The elemental dry cell composition (CH1.94

O0.76N0.17) was taken from Silva-Santisteban, Converti, andMaugeri (2006). The corn steep liquor, used as a nitrogen source,is a complex mixture of free amino acids, peptides, proteins, inor-ganic salts, organic acids and many other compounds. A detailedstoichiometry model for the process could be very complicated,and in practice one would lump all the components of the CSL intoone nitrogen-containing substrate as the ammonia (Nielsen &Villadsen, 1994).

The above stoichiometry is valid considering that the oxygen isnot limiting inside the bioreactor (Mitchell, Krieger, Stuart, &Pandey, 2000) and that the yeast metabolism is essentially aerobic(Fonseca, Gombert, Heinzle, & Wittmann, 2007). Based on the stoi-chiometry, the oxygen concentration in the outlet air stream, theglobal metabolic water and ethanol production in the moist solidmedium were calculated considering the CO2 measurement inthe outlet air stream. It is important to emphasise that these com-pounds could be calculated from the TRS data, assuming that theTRS concentration is basically composed of glucose or fructose.However, it can be mentioned that the experimental error associ-ated with the determination of TRS concentration in the medium(±10%) is higher than the CO2 measurement (<1%).

The metabolic heat generated (Qgen) was calculated by a properenergy balance in the bioreactor assuming negligible loss of heatby the wall and that all the metabolic heat generated by the micro-organism leaves the bioreactor in the outlet stream.

The microbial growth was calculated considering the results ofoxygen uptake rate, according to the following equation (Brandet al., 2001):

Xn ¼YX=O � Dt � 1

2dO2dt t¼0 þ

dO2dt t¼n

� �þPi¼n�1

i¼1

dO2dt t¼i

� �þ ð1� a

2Þ � X0 � a �Pi¼n�1

i¼1X

1þ ða=2Þ

where

a ¼ m � YX=O � Dt ð2bÞ

The procedure to estimate the biomass content in a certain time(Xn) requires the knowledge of the biomass yield based on the oxy-gen consumption (YX/O) and maintenance coefficient (m). Themaintenance coefficient was 0.0031 gO2 gcell�1 h�1, as describedby Brand et al. (2001). The main yielding coefficients, such as theyield coefficient of substrate in biomass (YX/S), oxygen in biomass(YX/O), respiratory quotient (RQ) and metabolic heat yielding(YQ/X), were computed as:

YX=S ¼1a

ð3aÞ

YX=O2 ¼1k

ð3bÞ

RQ ¼ ck

ð3cÞ

YQ=X ¼Q gen

Xnð3dÞ

3. Results and discussion

3.1. Stoichiometry

Table 1 presents the stoichiometry and the yielding coefficientscalculated for the seven experimental runs carried out to investi-gate the kinetics of inulinase production by the SSF in packed-bed bioreactor.

The values for the yielding coefficient of the substrate in bio-mass (YX/S) ranged from 0.24 to 0.31 g of cells per gram of TRS con-sumed amongst the runs. The YX/S values increased at highvolumetric air flow rate, except for run 5. The highest YX/S valueswere obtained at runs 3 and 6 at an inlet air temperature of33 �C. The values for YX/S obtained in this study are similar to thatreported in the literature for the growth of the yeast of genusKluyveromyces in submerged fermentation. Silva-Santistebanet al. (2006) reported YX/S in the range of 0.16–0.32 grams of cellsper gram of TRS consumed for the K. marxianus ATCC 16045 grownin synthetic medium. Fonseca et al. (2007) obtained the YX/S of0.51 g/g for batch fermentation and in the range of 0.45–0.49 g/gfor continuous fermentation of the yeast K. marxianus NRRLY-7571 with medium composed of glucose as a carbon source.Treichel et al. (2009) and Makino et al. (2009) obtained YX/S of0.22 and 0.42 g/g, respectively, for K. marxianus NRRL Y-7571growth in a medium composed by agroindustrial residues as pre-treated molasses and corn steep liquor.

The yielding of oxygen in cells coefficient (YX/O) ranged from1.08 to 2.06 g of cells per gram of oxygen amongst the runs. The in-crease in inlet air temperature decreased the YX/O values for all vol-umetric air flow rates investigated, except for run 5. Analysing runs2, 5 and 7 at the same inlet air temperature (30 �C) and flow ratesof 2.0, 3.0 and 2.4 m3 h�1, respectively, there was an enhancementof YX/O values until the flow rate of 2.4 m3 h�1decreasing consider-ably at flow rate of 3.0 m3 h�1. The YX/O value is higher than thatreported by Fernández-Fernández and Pérez-Correa (2007)(0.94 g/g) for the growth of Gibberella fujikuroi by SSF in a pilot

i

ð2aÞ

166 M.A. Mazutti et al. / Food Chemistry 120 (2010) 163–173

packed-bed bioreactor with a working capacity of 200 kg of drysolids and lower than reported by Brand et al. (2001) (3.81 g/g)for the growth of Aspergillus niger by SSF in a packed-bed bioreactorfor 40 kg of dry solids.

The respiratory quotient (RQ) ranged from 3.53 to 2.17 g of CO2

per gram of oxygen amongst the runs. The results indicated that anincrease in the inlet air temperature caused a decrease on RQ, inde-pendent of the volumetric air flow rate employed. This behaviouroccurred probably because at low temperatures the oxidativemetabolism of the yeast decreases, consequently decreasing theconsumption of oxygen without alters the CO2 production. TheRQ values obtained in this work are higher than that reported inthe literature for the growth of K. marxianus NRRL Y-7571 in sub-merged fermentation that is 1.0 g/g, considering glucose as a car-bon source (Fonseca et al., 2007). The cause of the high RQ valuesobtained in this study is that the microorganism quickly depletesthe TRS concentration in the medium, requiring other carbonsources to grow and to maintain the endogenous metabolism.However, due to the medium heterogeneity it is difficult to carryout the quantification of all compounds involved during thegrowth.

The metabolic heat yielding (YQ/X) is an important parameter tobe evaluated for the process scale-up, since the greatest challengeduring the operation of packed-bed bioreactors is the metabolicheat removal. In this context, lower values for YQ/X are preferableto make possible the scale-up packed-bed bioreactors for SSF. Inthis work, the YQ/X ranged from 10.41 to 16.65 kJ per gram of bio-mass. The highest values for this parameter were verified at lowvolumetric air flow rate (2 m3 h�1) and temperature (27 �C). TheYQ/X values obtained are higher than that commonly reported inthe literature. For the growth of A. niger using wheat bran as sub-strate, a value of 8.37 kJ per gram of biomass was obtained (Ashley,Mitchell, & Howes, 1999; Fanaei & Vaziri, 2009; Meien & Mitchell,2002; Meien et al. 2004). It is very difficult to compare the YQ/X ob-tained here with the literature, since there are no works reportingthe kinetic evaluation from yeast growth in SSF.

3.2. Kinetic evaluation

Fig. 1 presents the temperature profiles obtained as a functionof bed height for the seven experimental runs. As expected, thetemperature gradients increase along the bioreactor height and,at the maximum gradient, the outlet air temperature reached val-ues 20 �C higher than the inlet air temperature. The variation in thehighest temperature obtained for the outlet air stream were slightdifferent amongst the runs. In the first three runs (volumetric airflow rate of 2 m3 h�1) the highest outlet air temperature wasaround 50 �C, while in runs 4, 5, 6 (volumetric air flow rate of3 m3 h�1) and 7 (volumetric air flow rate of 2.4 m3 h�1) the maxi-mum temperature was around 47 �C. As can be seen, the increasein volumetric air flow rate did not significantly contribute to themetabolic heat removal, due to the low heat capacity of the air,as already described in the literature (Meien & Mitchell, 2002).The main contribution to metabolic heat removal is due toevaporation (Ashley et al. 1999; Fanaei and Vaziri, 2009; Meien& Mitchell, 2002; Meien et al. 2004).

Increasing the inlet air temperature from 27 to 33 �C caused aconsiderably decrease on the time where the highest temperaturegradients were verified. In runs 1–3 these times were 9.6, 8.3 and7.8 h, respectively, while in runs 4–6 the times where 9.3, 8.7 and7.4 h, respectively, and in run 7 the time was 9.3 h. The volumetricair flow rate had little influence on the times of the maximum tem-perature gradients, as can be seen comparing the runs 1 and 4, 2, 5and 7 with 3 and 6. The higher the inlet air temperature the morequickly the moist substrate temperature reached the optimumvalue for the growth of K. marxianus NRRL Y-7571, that is 36 �C

(Treichel et al., 2009). Analysing the temperature profiles insidethe biorreator it is possible to verify that the critical height is20 cm, since above this height the medium temperature reachedvalues higher than 36 �C in the first 3 h or fermentation.

Fig. 2 presents the moisture profiles obtained in four zones in-side the bioreactor for the seven performed experimental runs.The results showed an increase of 10% in the moisture content ofthe medium after 24 h of fermentation (independently of the oper-ational conditions employed). This aspect is interesting, since theoptimal range presented in the literature for inulinase productionby SSF varied from 60% to 75% (Mazutti et al., 2006; Selvakumar& Pandey, 1999). Analysing the moisture profiles we can verify thatthe water content of the solid substrate decrease with the bioreac-tor height, probably due to the increase in the air temperature(consequently increase the carrier capacity of the air), which iscaused by the microbial growth. The substrate drying effect wasverified mainly in zones 3 and 4, which are located above the crit-ical height. Nevertheless, it is important to emphasize that themoisture content did not reach values lower than 65% throughthe fermentation. The content of water in the inlet air used in thisstudy was about 90–100% of the saturation, what contributed forthe increase in the moisture content of the solid.

Fig. 3 presents the inulinase profiles obtained in four zones in-side of the bioreactor for the seven experimental runs. The inulin-ase production decreased with the bioreactor height, with the mostpronounced reduction verified at zones 3 and 4. This reduction inthe production is a consequence of the deactivation caused bythe high temperatures achieved in these zones, which are locatedabove the critical height, as will be discussed later. Moreover, themicrobial cells are affected by temperatures higher than 36 �C,resulting in lower rates of enzyme production. This statement isconfirmed when the activities obtained in zones 1 and 2 are ana-lysed, where we can observe that the medium temperature didnot exceed the optimal growth temperature for the yeast, whichwere similar between them and considerably higher than that ob-tained in zones 3 and 4. The highest inulinase production was ob-tained in run 5, where the mean activity (mean amongst the fourzones) was 463 U gds�1. In this condition, the activity of the en-zyme on inulin was determined. The ratio between the activityon sucrose and the activity on inulin (S/I) was lower than 50, char-acterising this enzyme as an inulinase rather than an invertase(Ettalibi & Baratti, 2001).

Similar results were previously reported by Xiong et al. (2007),that optimised the nutrient levels for the production of inulinaseby a new strain of Kluyveromyces S120 in SSF. The average inulin-ase activity under optimal conditions was 410 U gds�1 (using100 g of dry solids). Ayyachamy et al. (2007) reported the inulinaseproduction in SSF by Xanthomonas campestris using garlic peels andonion peels (10 g of dry solids). The maximum inulinase activityobtained was 117 U gds�1. Mazutti et al. (2006, 2007) investigatedthe inulinase production in conical flasks containing 5 g of sub-strate and obtained a maximum inulinase activity of 392 U gds�1

(72 h of fermentation) and 250 U gds�1 (24 h of fermentation),respectively.

Fig. 4 presents the profiles of TRS consumption, metabolicheat and biomass for the experimental runs carried out. Regard-ing the metabolic heat production, two distinct behaviours wereidentified in the seven experimental runs. In runs 1, 2 and 3(flow rate of 2.0 m3 h�1) we have verified the highest valuesfor the metabolic heat (in mean 676 kJ), while in runs 4, 5 and6 (flow rate of 3.0 m3 h�1) smaller values were observed (inmean 535 kJ). In practice, at elevated flow rates the heat dissipa-tion in the bioreactor is considerably improved and the retentiontime is increased. In addition, the microorganism tends to pro-duce less heat if the dissipation is sufficient and it can accumu-late energy to perform other metabolic activities, justifying the

Fig. 1. Profiles of temperature obtained in the seven experiments carried out. Of down to up: temperature measured at inlet and outlet air stream (first and fifth lines) andinside of the bioreactor at 10 cm (second line), 20 cm (third line) and 30 cm (fourth line). Experimental conditions for each run presented in Table 1.

M.A. Mazutti et al. / Food Chemistry 120 (2010) 163–173 167

lower metabolic heat produced in the runs at the highest flowrates. For example, in runs 4, 5 and 6 the inulinase activity(mean values) was higher than that obtained in runs 1, 2 and3, corroborating the above mentioned statement. Besides, thetemperature profiles (Fig. 1) showed that the medium tempera-ture in the runs 4, 5 and 6 (at 10, 20 and 30 cm inside of biore-actor) are lower than obtained in the runs 1, 2 and 3.

Regarding the production of biomass, the direct quantificationof the growth in SSF is a hard task and most studies presented inthe literature have used an indirect approach to quantify the cellgrowth. In a previous work we have used the glucosamine contentto correlate the microorganism growth (Mazutti et al., 2006). How-ever, this method has shown high associate experimental errors. Inthis work we have adopted the procedure described by Brand et al.

Fig. 2. Profiles of moisture for the four zones inside of bioreactor obtained in the seven experimental runs carried out. Experimental conditions for each run presented inTable 1.

168 M.A. Mazutti et al. / Food Chemistry 120 (2010) 163–173

(2001), where the microbial growth is calculated based on the oxy-gen consumption data. The increasing in the mass of cells was low-er compared to the previous works with the same strain insubmerged fermentation (Treichel et al., 2009), but a directcomparison is difficult to perform, due to the different characteris-tics of each evaluated processes. However, the approach used toestimate the mass of cells showed some interesting connectionwith the overall process. The final mass of cells obtained in this

study ranged from 45.2 g (run 1) to 56.0 g (run 5). The greatest bio-mass productions were verified at runs 2, 5 and 7, 50.4, 56.0 and49.2 g, respectively. In these runs the temperature of the inlet airwas 30 �C and the volumetric air flow rate ranged from 2 to3 m3 h�1. Analysing the biomass profiles presented in Fig. 4 it ispossible to identify the microbial growth phases, where the lagphase occurs at 0–6 h, exponential phase at 6–11 h and stationaryphase from 11–24 h. Amongst the experimental runs, the most

Fig. 3. Profiles of inulinase for the four zones inside of bioreactor obtained in the seven experimental runs carried out. Experimental conditions for each run presented inTable 1.

M.A. Mazutti et al. / Food Chemistry 120 (2010) 163–173 169

attractive results in terms of process scaling-up were obtained inrun 5, since in this experiment the highest inulinase and biomassproduction were obtained and, mainly, the lowest value for themetabolic heat yielding coefficient (YQ/X) was achieved. This resultshowed that it is possible to manipulate the operational variablesin a manner that the metabolic heat production is reduced; a rele-vant effect on the final mass of cells is not observed, as presented inrun 5.

The direct comparison of the results referring to the biomassprofiles obtained in this work with the literature is difficult, sincethere are no works reporting the growth of the yeast K. marxianusNRRL Y-7571 in SSF. However, it is possible to compare the resultswith growth profiles obtained in submerged fermentation for thesame strain, taking into account the distinct characteristics of eachprocess. Treichel et al. (2009) used the same strain in submergedfermentation to produce inulinase from pre-treated molasses, corn

Fig. 4. Profiles of TRS, production of metabolic heat and biomass obtained in the seven experimental runs carried out. Experimental conditions for each run presentedin Table 1.

170 M.A. Mazutti et al. / Food Chemistry 120 (2010) 163–173

steep liquor and yeast extract as substrates. In the optimised con-dition, the initial mass of cells and TRS were 14 and 200 g, respec-tively, what yielded 48 g of final mass of cells after 24 h offermentation. Comparing the results of these works it is possibleto verify that the growth in SSF presented a better performancethan in submerged fermentation, since it was possible to obtaintwo times more cells in the same conditions. The best performanceof the yeast in SSF is due to the fact that the oxygen is not limiting

in the process and, conform suggested by several authors, themicroorganisms showed better adaptation capacity in SSF than insubmerged fermentation (Couto & Sanromán, 2006; Pandey, 2003).

Related to the TRS profiles presented in Fig. 4 it is important toemphasize that the initial mass of TRS contained in the moist solidparticles presented a little variation amongst the experimentalruns. This can be related to the experimental errors associated withthe measurement and, mainly, the greatest variability in the

Fig. 5. Profiles of ethanol, CO2, O2 and metabolic water obtained in the seven experimental runs carried out. Experimental conditions for each run presented in Table 1.

M.A. Mazutti et al. / Food Chemistry 120 (2010) 163–173 171

substrate composition, since several agroindustrial residues wereused as substrate. Initially the kinetics of TRS consumption in thefour zones inside the bioreactor (similar to that presented for theinulinase) was monitored, but the variations in the results werewithin the experimental errors. Then, we decided to express the re-sults in means terms. The kinetics obtained in SSF is similar to thatreported for the same strain in submerged fermentation, where themass of sugar is quickly consumed after 8 h of fermentation(Makino et al., 2009; Treichel et al., 2009).

Analysing the TRS profiles obtained in runs 1, 3, 4 and 6, wherethe initial air temperature were 27 and 33 �C (independent of theflow rate), we can easily visualise that the initial rate of sugarconsumption (first 4 h of fermentation) is lower than that obtainedin the runs 2, 5 and 7, where the initial air temperature was 30 �C.The fast initial sugar consumption in runs 2, 5 and 7 resulted inhighest CO2 production, which was 56.5, 65.8 and 54.5 g, respec-tively. The greatest consumptions of sugar were verified at runs1, 2, 5 and 7, where the consumed mass were 107.4, 107.6, 117.8

172 M.A. Mazutti et al. / Food Chemistry 120 (2010) 163–173

and 108.8 g, respectively. For another experimental runs the con-sumed mass was lower than 100 g. An aspect that deserves atten-tion is the high mass of sugar consumed in the run 1 does not agreewith the production of biomass, since in this run the lowest massof cells was obtained. However, the explication come from theanalysis of yielding coefficients presented in Table 1. Run 1 led tothe highest values for YQ/X and RQ. The first coefficient indicatesthat the microorganism increased its capacity to produce meta-bolic heat, what can be proven by analysis of the metabolic heatproduction, as shown in Fig. 4. The second coefficient indicates thatat higher values, the carbon source corruption is higher, justifyingthe high mass of TRS consumed in the process. The lower mass ofcells obtained in this run can be related to the fact that the micro-organism spend energy to generate metabolic heat, decreasing theenergy to the growth. This behaviour was not verified at run 4probably due to the better heat dissipation in the medium, occa-sioned with the high volumetric air flow rate.

Fig. 5 presents the profiles of ethanol, metabolic water, CO2

and O2 for the seven experimental runs. The profiles of CO2 pro-duction indicated that the mass produced is directly proportionalto the mass of cells and TRS, as expected. The maximum produc-tion was verified between 5 and 12 h of fermentation, with thehighest production obtained in runs 2, 5 and 7, where the valueswere 56.5, 65.8 and 54.5 g, respectively. The data obtained forthe O2 consumption indicated that it is not limiting in the pro-cess, since only 39 g in mean was consumed during it. Consider-ing a molar fraction of 21% in the inlet air and the high flow rateemployed, only a little fraction of the inlet oxygen was con-sumed. The analysis of the metabolic water profiles showed thatthe water production due to the respiration ranged from 14.6 g(run 1) to 30.1 g (run 5), which was directly related to the massof cells produced. Regarding the ethanol profiles we can statethat its production was not associated with the oxygen limita-tion in the medium, due to the high volumetric air flow rateand the low amounts of oxygen consumed. The main cause forthe ethanol production can be related to the high initial massof TRS in the medium, which probably changed the normal met-abolic pathway of glycolysis to ethanol production. Concerningthis, several works cited in the literature report the ethanol pro-duction by the yeast K. marxianus, including aerated systems(Fonseca et al., 2007; Silva-Santisteban et al., 2006).

4. Conclusions

The best condition for enzyme production was inlet air temper-ature and volumetric flow rate of 30 �C and 3 m3 h�1, respectively,reaching an activity of around 463 U gds�1. The results presentedhere can be useful in the scale-up and optimisation of thepacked-bed bioreactors by three reasons. Firstly, the volumetricair flow rate had little influence on the dynamic behaviour of theprocess. This is particularly interesting because at higher scalesthe volumetric flow rate should be altered to satisfy the new con-figuration without altering the process characteristics. Secondly,the inlet air temperature can be optimised to improve the overallprocess performance. As an example, the time domain could bepartitioned in N-intervals and the inlet air temperature optimisedat each interval using the dynamic optimisation tool to find betteroperational conditions. Finally, the moisture content of solid sub-strate was higher than 65% (w/w) through the bioreactor. This re-sult is interesting because the influence of evaporative cooling inthe metabolic heat removal can be investigated in the future, sincedry air can be used in determined periods of the fermentation(mainly in the period of the greatest metabolic heat production)to remove the heat generated by the microbial growth withoutdrying the moist solid substrate.

Acknowledgements

The authors are grateful to CAPES (Project PROCAD/CAPES0337/05-6), FAPERGS, CNPq and URI-Campus Erechim for financialsupport of this work and scholarships.

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