Heat transfer in citric acid production by solid state fermentation

Process"Biochemistry Vol. 31, No. 4, pp. 363-369, 1996

Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 S15.00 + 0.00

ELSEVIER

0032-9592(95)00071-2

Heat Transfer in Citric Acid Production by Solid State Fermentation M. G u t i & r e z - R o j a s , a* S. A m a r A b o u l H o s n , a R. A u r i a , b S. R e v a h b & E. F a v e l a - T o r r e s a aDepartamento de Biotecnologia;bDepartamento de Ingenier/a de Procesos e Hidr~iulica,Universidad Aut6noma Metropofitana-Iztapalapa, Apdo. Postal 55-535, 09340 M6xico, D.E (Received 29 May 1995; accepted 6 August 1995)

Different mechanisms of heat removal (conductive, convective and evaporative) were studied in a SSF process using a packed bed reactor with an inert support (Amberlite IRA-900) previously inoculated. The cylindrical bioreactor was incubated in a temperature controlled chamber (30 °C). Average temperature gradients obtained during the culture were 1"3 and 0"42°C cm - 1 in the radial and axial directions, respectively. During maximal metabolic activity of Aspergillus niger the medium temperature rose from 32 to 48°C, and a maximum radial temperature gradient of 3"l °C cm- 1 was achieved. A t this stage, the unsteady-heat balances indicated that conductive heat transfer was the least efficient mechanism (8.65%) when compared with convective (26"65%) and evaporative (64.7%) mechanisms. Sugar consumption and glycerol and citric acid production were strongly affected by temperature.

NOTATION

hin hout

A0 Heat transfer specific area, orthogonal to the air flow (m 2 m-3 packed) Ap Heat transfer specific area, parallel to the air flow (m 2 m-3 packed) Cp Medium heat capacity (J kg -1 dry matter

k

Inlet air enthalpy, evaluated at Tin (J kg-') Outlet air enthalpy, evaluated at Tout (J kg- 1) Bioreactor wall thermal conductivity (J m-1 S-1 o c - 1 )

Pr

Dimensionless outside Prandfl number at Tc ( Cpg/.tg~g ')

Qw Latent heat of vaporization (J kg-') Re Dimensionless outside Reynolds number at T~ (De pgVl~g ') Tc Temperature of the incubation chamber (°C) T b Temperature of the medium (°C) t Time (s) U Overall heat transfer coefficient (J m - 2 s- 1

°C-1 )

g Air heat capacity (J kg- 1 °C- 1) Cps Support heat capacity (J kg- ' °C- l) Cpw Water heat capacity (J kg- 1 ° C - 1) Do Bioreactor outside (m) G Air mass velocity (kg m - 2 s - 1) G' Specific air flow rate (m 3 air m - 3 packed s - 1) hi Inside film coefficient (J m - 2 s- 1 °C- 1) h0 Outside film coefficient (J m - 2 s- 1 °C -')

oc-, )

v

Air velocity within the incubation chamber

(ms-') Xs

*To whom correspondence should be addressed. 363

Support mass fraction in the medium (kg support kg- 1)

M. Guti~rrez-Rojaset al.

364

Xw Water mass fraction in the medium (kg water

kg-1)

Yin Water mass fraction of inlet air (kg water

kg-') Your Latent heat of vaporization at outlet temperature (J kg- 1)

Greek symbols Bioreactor wall thickness (m) Bioreactor void fraction Mean particle diameter (m) 2 Medium effective thermal conductivity (J m -1 s-1 o c - 1 ) 2g Air thermal conductivity (J m -1 s -1 °C-i) /~g Air viscosity at inlet temperature (kg m -1 S -1 ) Pin Inlet air density (kg m -3) Pout Outlet air density (kg m - 3) Pb Medium bulk density (kg m -3 packed) pg Air density at Tc (kg m -3) 6 e

INTRODUCTION The use of inert supports for solid state fermentation (SSF) studies has received wide attention. 1,2 In this system, microorganisms use water and nutrients contained in the culture medium absorbed onto the inert support, colonize available surfaces, produce biomass 3 and, under appropriate conditions, commercial metabolites such as citric acid. 4,5 Besides potential applications, inert supports can be considered as ideal media for the development of mathematical and heat transfer studies.6,7 Heat transfer problems in SSF induce temperature gradients that may cause: 7-12 (i) belated microbial activity, (ii) dehydration of the medium and (iii) undesirable metabolic deviations. Heat removal difficulties are due to low transfer coefficients and low thermal conductivity of the heterogeneous materials used in this process. Different methods have been explored to remove the excessive heat produced. Among conventional methods, convective mechanisms are more efficient than conductive ones, 11 but require large scale aeration rates which may result in undesirable dehydration of the media. Other less conventional methods make use of evaporative cooling (latent heat of water vaporization) to eliminate metabolic heat accumulation.13 This mechanism is attractive and includes partially saturated inlet air at low temperatures.

Recent studies ~2,~4 with Rhizopus oligosporus growing on corn grit have shown the effectiveness of such evaporative methods. However, swift changes in the temperature of the medium provoked strong effects on biomass morphology, ~2 which may result in associated metabolic deviations of the organisms, therefore, a compromise between drastic, but efficient, alternatives versus less efficient solutions which keep the biological system unmodified should be made. Temperature control in SSF systems is still a problem without a clear and promising solution. Metabolic heat has been scarcely quantified and almost stoichiometrical constants values such as 16 ×106 J kg -1 dry matter 9 or rates of heat produced by Aspergillus niger in cassava flour of 3.3 × 105 J h -1 kg -1 dry matter 8 were reported. Since the global values change during fermentation processes, heat accumulation must be quantified to develop appropriate cooling strategies for SSF processes. The objective of this work was to evaluate unsteady-state heat accumulation as well as conductive, convective and evaporative contributions to metabolic heat removal and its effects on sugar consumption, citric acid and metabolite production in SSF on an inert support.

MATERIALS AND METHODS Microorganism

Aspergillus

niger-lO was propagated in potato-dextrose-agar slants at 30°C and transferred monthly. Inocula for SSF was prepared as previously reported? Support and culture media Amberlite IRA-900 (Technical grade Rohm and Haas, Mrxico) was used as support. The pretreatment of the support material was as reported earlier. 3 A culture medium for fermentation with high initial sucrose concentration was chosen to amplify the effects of heat and citric acid production. The composition was (in kg m -3 packed): Amberlite, 240; sucrose, 144; (NH4)2SO4, 12; urea, 5.4; KzHPO 4, 4.4; MgSO 4. 7H20, 2"2; KC1, 2-2; FeSO4.7H20, 0"04 and water, 360. The pH of the medium was adjusted to 2.7 and then sterilized at 115°C for 15 min. Spore suspension was added up to 1011 spores kg -~ dry support. The final pH, after sterilization, was 5"5.

Heat transfer in citric acid production Solid state fermentation

The experimental set-up for SSF is shown in Fig. 1. A cylindrical bioreactor (0-095 m i.d. and 0.25 m height) fabricated in acrylic material 6 mm thickness) was used. The bioreactor (1.53kg imbibed support) was provided with a baffled air feeding chamber in which a perforated plate supported the packed bed and a set of 10 k-type thermocouples distributed in differential radial and axial positions. Compressed air was saturated with water by passing through a series of air humidifiers before entry to the reactor. The aeration rate was fixed at 0.03 m 3 air m -3 packed s -1. The bioreactor was incubated (under non-sterile conditions) for 78 h within a closed air chamber built-up in polycarbonate and glass (0.7 m, height; 0.9 m, width; 0.9 m, depth). The temperature inside the chamber was controlled at 30°C by means of an on/off heating resistance (800 W). To achieve good air distribution within the chamber, a 0"11 m diameter ventilator (0"03 HP) and a vertical baffle (0.6 m, height; 0.7 m, width) were used (see Fig. 1). At the end of the fermentation, the bioreactor was removed and the contents carefully sliced at different sections throughout radial and axial directions for analysis.

365

extracted (four-fold extraction) from wet samples (30 min with 300 g litre -1 ammonium sulphate solution); determinations were made with a refraction index detector (Perkin EImer LC-30 RI) using 6 mM sulphuric acid at a flow rate of 0.6 ml min -1 as mobile phase through a Rezex Organic Acid (Phenomenex) column.

THEORETICAL CONSIDERATIONS H e a t balances

Heat accumulation (qac) within an inoculated packed bed with an inert support, can be written by the sum of individual conductive (qcond), convective (qconv), evaporative (qev) and metabolic (-AHr) heat contributions, as:

qac=qcone,+qconv+qev-AHr .

(1) Considering only maximal gradients in the system, the unsteady-state heat accumulation can be written as:

+ G'( Pinhin - Pouthout) (2)

-t.- a A o O w ( Y i n - Y o u t ) - m I ~ F . Analytical m e t h o d s

At the end of the fermentation, sucrose, glucose, fructose, citric acid and glycerol were determined by HPLC (Perkin Elmer LC-250). 4 Citric acid was

P a r a m e t e r evaluation

The heat capacity of the medium (Cp) was estimated using the expression:

Cp =xwCpw+ xsCps.

(3)

The overall heat transfer coefficient (U) was calculated as: 1

U-a

2

- - -,-II

4

1 6" --+--+hi h0 k

(4)

The inside film coefficient (hi) for packed bed columns with laminar air flow (the internal Reynolds number was 0.5 ), was calculated with: 15

hi = 3.6 J,g ( ~ G ] °'365 - -

\ flge ]

Fig. 1. Experimental set-up: (1) temperature controlled air chamber, (2) packed bioreactor, (3) air flow-meter, (4) air humidifiers, (5) perforated plate support, (6) thermocouples, (7) temperature indicator and control, (8) standard rheostat, (9) heat resistance, (10) forced air ventilator, (11) vertical baffle, and (12)exhausted air exit.

"

(5)

The outside (h0) film coefficient, for an immersed cylinder into a laminar air environment is given by: 15 h0 = 0"9 1 1(Re)°385(pr) 1/3.

(6)

Partial terms: qac, qcond, qconv and qev can be experimentally measured and the metabolic heat

M. Gutidrrez-Rojaset al.

366

term (AHr) readily obtained by solving eqn (2). To solve eqns (3)-(6) and, finally, eqn (2) the value of constant parameters were:

50 45 4O

Cps = 1210 J kg -~ °C -1 CPw= 4181 J (kg-1 oc-1 ) k = 0 . 0 5 8 6 j m-1 s-a oC-1

30

25 20

cI, = 0"00055 m

40

60 Time, h

~.g= 0.026 J m - ~ s- 1 °C- 1 /~g = 1.8El0 -5 kg m -~ s -~

80

50

e=0.4

45

v = 0 . 0 2 m s -1

40 35

RESULTS AND DISCUSSION

30

25

At the end of the fermentation (78 h) sucrose was completely hydrolysed, but glucose and fructose accumulated in the culture medium (stoichiometrical amounts of both sugars indicated that 70% of the initial substrate was consumed). During the fermentation, the temperature increased notably. Substrate consumption and product formation should be affected as a result of changes in metabolic activity; therefore we studied the temperature gradients formed and the removal of excess heat by different mechanisms.

Temperature patterns Experimental temperature patterns are depicted in Fig, 2. Radial temperature profiles (see Fig. 2(a)), showed a maximum between 32 and 44 h when the temperature rose to 48°C at the bioreactor radial centre, while in the vicinity of the inside wall the average temperature was 33°C throughout the fermentation. Axial profiles (see Fig. 2(b)), exhibited a similar pattern to radial profiles and a maximum temperature of 48°C was reached after 30-40 h at the upper zone and centre of the bioreactor, respectively. The maximum radial temperature gradient observed (3"1°C cm -~) was very close to those reported in similar SSF systems ( 3 ° C c m - 1 , 9 2.5°C cm-1, 8 4-5°C c m - l , l l ) . Thus axial and radial temperature gradients were not constant and rapid heat accumulation followed by a slow heat removal was also observed. These results suggest that a considerable quantity of metabolic heat is produced during fermentation affecting substrate consumption and metabolite production.

J

i

|

.

.

20

.

.

.

.

.

.

.

.

.

.

40

.

60

.

.

.

.

80

Time, h

Fig. 2. Temperature kinetics. (a) Top of the bioreactor at different radial positions: (_n--), near to the wall (0.04 m); ( + ) , middle point (0.025 m); and (-- A--), center (0 m). (b) Center of the bioreactor at different axial positions: ( + ) , bottom (0"03 m); ( + ) , middle point (0.16 m); and ( - A--), near to the top (0.21 m).

Effects of temperature gradients on sucrose consumption and metabolite production At the end of fermentation, the final distribution of glucose and fructose, through the packed bed (see Fig. 3(a) and (b)), exhibited almost the same radial and axial profiles. Lower sugar consumption was observed in the centre than at the walls and lower sugar consumption in the bottom than the top. It is worth noting that the excessive gradients found, especially in a radial direction, were 20 kg m -3 for glucose and 13 kg m -3 for fructose. Since high sugar accumulation sites and hot zones correlated well, temperature gradients could explain the sugar gradients observed. Although heat accumulation reduced the complete consumption of sugars, metabolite production was enhanced in the sites where sugar accumulation was observed. High metabolite production might be due to osmoregulatory responses rather than highest temperatures. For example, citric acid production is usually carried out at high initial sugar concentrations which involves osmotic shock effects in microorganisms. 5 In order to counter this problem,

367

Heat transfer in citric acid production ,,:,,

(a)

35

80.00 60.00

? N

5 0

,

40.00 20.00

0.00 -20.00

I

5

10

15

20

-40..00

25

-60.00

Heigth, cm

35 E30 ~= 25

0

10

20

30

40

'9

o

!

5

10

60

70

80

60

70

80

Time,h

(b)

0

50

15

20

25

Heigth, cm

5.00 0.00 -5.00 -10.00 -15.00 -20.00 -25.00 -30.00

-35.00 -40.00 0

10

20

30

40

50

(c) Time, h

~= 25

Fig. 4. Heat evolution kinetics: (a) (--zx--) total accumulated; (-43-) total produced; and ( ~ ) total removed. (b) (--x--) conductive;(---o--),convective;and (--~-) evaporative. 5

10

15

20

25

Heigth, cm

!

0 =

0

5

10

15

20

25

Heigth, c m

Fig. 3. Final substrate and metabolite concentrations as a function of reactor height.(a) Glucose, (b) fructose, (c) citric acid, and (d) glycerol at different radial positions: (--t3---), near to the wall (0"04m); (-#---), middle point (0-025 m); and (--/x--), center (0 m). microorganisms produce compatible solutes such as glycerol, erythritol, arabinol and mannitol acting as osmoregulators:, 16 Figure 3(c) and (d) shows the citric acid and glycerol concentrations found through the packed bed. Concentration gradients for citric acid and glycerol were not as marked as in the case of sugars but enough to be considered. Gradients in the radial direction were 8 kg m-3 for citric acid and 6 kg m-3 for glycerol. The difference in concentration between the extreme points in the bio-

reactor, in terms of citric acid productivity (kg citric m -3 h - ~), results in a higher productivity (up to three-fold) in the centre and the bottom of the packed bed than in the wall and the top. Gradients in final citric acid formation were probably due to heat accumulation since high metabolic activity took place. Therefore, as the SSF progressed, heat should be removed at different magnitudes by different mechanisms. H e a t transfer m e c h a n i s m s

Total heat accumulation was calculated using medium temperature data in the left-hand-side in eqn (2). The derivative values were obtained using finite differences and numerically integrated within the fermentation time domain. Conductive, convective and evaporative heat removal was calculated with the first, second and third righthand-side members in eqn (2) and also integrated. Metabolic heat was calculated by solving eqn (2). The kinetics of total heat produced as well as the total heat accumulated and removed are shown in Fig. 4(a). The metabolic heat quantified during the entire fermentation was 2.0 x 105 kJ m -3, i.e. only 26% of the theoretical expected value (7.6x105 kJ m -3 considering 70% of

M. Guti(rrez-Rojas et al.

368

sucrose consumption to biomass). This may indicate that some of the available energy is retained in the synthesis of metabolites. Figure 4(b) shows conductive, convective and evaporative heat removal. Positive values denote heating and negative values indicate cooling. Two maxima of heat produced can be observed, the first at 42 h can be linked to rapid cell growth, i.e. when the culture exhibits a maximum of metabolic activity and a second self-heating (52-68h) associated with the stationary phase of growth, when spore production was observed. In both cases, the total heat removed was restricted and heat accumulation occurred. During the initial 28 h, the three mechanisms of heat removal were sufficient to avoid heat accumulation. Later, during rapid cell growth and spore production, evaporative cooling values approached the total heat produced, suggesting that this is the main cooling mechanism. A low value of U (1.25 J m -2 s -1 °C-1) was measured as compared to those obtained in small glass packed columns s (75 J m -2 s -1 °C-1). T h i s was due to the low thermal conductivity of the bioreactor walls and the low air velocity (v) of 0-02 m s -1 in the incubation chamber. A simulated increased value of v up to 2.0 m s -1 increases the U value only to 4.2 J m-z s-1 oC-I suggesting that conduction is always limited by the thermal conductivity of the fermented matter and the acrylic walls of the bioreactor. The data in Table 1 give a comparison of the mechanisms of heat removal during the phase of high metabolic activity (38 and 42 h). Evaporative cooling of the fermented matter is the most efficient mechanism as reported earlier, lz CONCLUSIONS The magnitude of the radial gradients showed that heat transfer was limited to a great extent by the Table 1. Comparison of heat removal efficiency

Heat transfer mechanism Conductive Convective Evaporative Total

Heat removal a

(kJ m- ~)

(%)

- 4 800 - 14 780 - 35 900 - 55 480

8.65 26.65 64.70 100-00

aCalculated as an average of 38 and 42 h of culture.

deficient thermal properties of the system. Although a considerable amount of heat was removed, it was not enough to eliminate all the heat generated during the exponential phase of growth. Conduction was less efficient when compared with convective and evaporative cooling. In order to remove all the heat generated, especially during the phase of high metabolic activity, injection of cool-dry air accompanied by moisture replenishment at different points in the packed bed are suggested. The temperature gradients produced extreme gradients in final sugar concentration and, consequently, in metabolite production.

ACKNOWLEDGEMENTS

This work was supported by the Mexican Council for Science and Technology (CONACyT). Thanks are given to Dr P. Gunasekaran for his comments and criticism. The assistance of Rohm and Hass, in providing samples of Amberlite IRA900, is also gratefully acknowledged.

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