Immobilization of Clostridium thermocellum cells on bituminous coal particles

ANALYTICAL

BIOCHEMISTRY

129,72-79

(1983)

Immobilization of Clostridium thermocellum on Bituminous Coal Particles PAUL N. HORNE

AND HSIEN-WEN

Cells

Hsu

Department of Chemical, Metallurgical, and Polymer Engineering, University of Tennessee, Knoxville, Tennessee 37996-2200 Received July 2, 1982 The immobilization isotherms of Clostridium thermocellum cells on bituminous coal particles of approximately 0.15 to 0.18-mm diameter were experimentally measured at 60,45, and 30°C with a pH value of 7.0 and with pH values of 6.0 and 5.0 at 6O’C. The immobilization data were correlated into Langmuir forms and their characteristic coefficients were obtained. A method to obtain thermodynamic quantities AG, AH, and AS for the immobilization is also demonstrated.

In an effort to reduce dependence on foreign oil and to avoid competition with food products such as corn or sugar, the develop ment of a technique to produce chemical feedstock to replace petrochemical feedstock from other forms of biomass is essential. One of the most publicized approaches toward biomass utilization is enzyme-catalyzed hydrolysis of cellulose and hemicellulose to low-molecular-weight sugars that can serve as substrates for fermentation to fuels or chemicals (2,4,5,8,10,12,14,15,17,19). Clostridium thermocellum and Clostridium thermosaccharolyticum have recently been applied for the direct conversion of cellulose to alcohol (5). C. thermocellum is an anaerobic, thermophilic bacterium capable of hydrolyzing cellulose and of fermenting the hexose hydrolysates to primary ethanol, acetic acid, hydrogen, and carbon dioxide. Continuous enzymatic reactions by immobilized microbial cells represent a novel approach to fermentation processes. To evaluate the potential for developing such an immobilized cell bioreactor for cellulosic biomass conversion, the immobilization of C. thermocellum cells to bituminous coal particles was investigated. 0003-2697/83/030072-08$03.00/O Copyright 8 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

MATERIALS AND EXPERIMENTAL PROCEDURES (11)

Bacterium. C. thermocellum ATCC 27405 used in the experiment was obtained from the American Type Culture Collection in Rockville, Maryland. Culture medium. The composition of the culture medium was that modified by GarciaMartinez et al. (9) from the CM3 medium of Weimer and Zeikus (20). The modified CM3 medium contained (grams/liter): KH2P04, 1.5; K,HPO,, 2.9; (NH&Sod, 1.3; MgClz . 6H20, 1.O; CaClz * 2Hz0, 0.15; yeast extract (Difco, Detroit, Mich.), 2.0; cysteine hydrochloride hydrate, 1.O; FeSOa * 6H20, 1.25 mg/liter and 1 ml of a 0.2% (w/v) resazurin (Eastman-Kodak, Rochester, N. Y.) aqueous solution. The cellobiose (EastmanKodak) concentration was 1.O% (w/v), which served as the carbon source. The procedures recommended by the VP1 Anaerobe Laboratory for preparation of prereduced anaerobically sterilized (PRAS) media were followed by the method of Dowel1 and Hawkins (7). The pH was adjusted with 8 N NaOH or 5 N HCl to 7.0 before sterilization. For plat72

IMMOBILIZATION

OF

ing, 2.0% (w/v) agar was added to the medium prior to sterilization. Cultivation of bacteria. The bacteria were cultivated in 16 X 125mm Hungate-type anaerobic culture tubes (Bellco, Vineland, N. J.) and in petri dishes placed in anaerobic jars (BBL, Cockeysville, Md.) using a Gas-pak (BBL) for anaerobiosis. The working volume of the Hungate tubes was 10 ml and transfers were made using the syringe method of Macey et al. ( 13). The medium was autoclaved in two portions in Hungate tubes: a 1.O% (w/v) cysteine solution and a 9-ml solution containing the rest of the components suspended in 90% of the final volume. After sterilization, 1 ml of the cysteine solution was added to the 9-ml aliquots and the complete medium was preincubated in a water bath at 60°C. Anaerobic conditions as indicated by the resazurin-dye color change (from pink to colorless) were reached after approximately 5 min. The medium was then innoculated with 0.5- 1.O ml of lag phase bacteria and the culture temperature maintained at 60°C by a dry incubator. To cultivate the bacteria in petri dishes, 12 ml of the 90% final-volume medium with agar in Hungate tubes was sterilized and 1.5 ml of the cysteine solution added. The plates were inoculated with 1 ml of a bacterial culture dilution and the reduced medium was poured and mixed. The plates were placed in anaerobic jars, medium side down, and allowed to incubate for 4-6 days at 60°C. Stock cultures were prepared in Hungate tubes by growing a cell population on 10 ml of the reduced medium. They were maintained by transferring approximately 0.5 ml of a less than 24-h-old culture to 10 ml of fresh medium. Viable cultures were also preserved for several months by storage in a refrigerator at 4°C. Growth and cell number estimation. Growth was estimated by optical density at 525 nm utilizing a Gilford Model 240 spectrophotometer with an uninoculated medium sample serving as the blank. The slit width of the spectrophotometer, which refers to the width

Clostridium thermocellum

73

of the emitted spectrum, was adjusted to the value necessary for a zero absorbance reading of the blank. The number of cells in solution was determined by developing a calibration curve of absorbance vs viable colony count expressed as cells/milliliter. The count was estimated by serial dilution and plating of culture samples for which absorbance had been determined (3). The diluents consisted of 9 ml of a 0.1% peptone solution with resazurin and 1 ml of a 1% cysteine solution in Hungate tubes. Transfers of 1 ml were made by syringe method. Samples of the desired dilution were plated in petri dishes and incubated in anaerobic jars as described. Only plates with 30300 colonies were counted. The assumption that one viable cell results in one colony was made. Immobilization. Of the several methods of whole-cell immobilization, bacterial adsorption is perhaps the simplest to implement. Coal was chosen over other adsorbents because of its availability and reported use in an immobilized cell bioreactor (16,18). The adsorbent used was bituminous coal obtained from the reserves at the University of Tennessee power plant facilities. The coal was pulverized and seived to a particle size of 0.15- to 0.18mm diameter (80/ 100 Tyler mesh), washed several times with deionized water, and dried at 100°C for 24 h. To conduct an experiment, known and equal weights of coal were placed in two Hungate tubes. The weight of coal used from one experiment to another ranged between 0.12 and 0.15 g. Cells used for the experiments were obtained by centrifugation of approximately 12-h-old cultures at 5,OOOgfor 10 min. The supernatant was decanted and the cells were resuspended to the desired cell concentration in fresh CM3 medium. Five milliliters of the cell suspension was added to the first coal sample and 5 ml of uninoculated medium was added to the second sample, which served as the control. The remaining cell suspension and uninoculated media were used for an absorbance measurement of the original cell concentration. The pH of the two coal suspensions were adjusted to the desired

74

HORNE AND HSU

value and agitated in a Dubnoff metabolic shaking incubator at the desired temperature for 20 min. Temperature was maintained by circulation of water from a Haake constanttemperature bath. The suspensions were allowed to settle for several minutes and a sample of each supernatant was filtered through a Sweeney filter with a metal screen, which retained coal particles but allowed free passage of cells. These samples were used for an absorbance measurement of the equilibrium cell concentration. RESULTS

AND

ANALYSIS

Batchwise cultivation. C. thermocellum cultured in Hungate tubes containing 15 ml of modified CM3 medium were taken by syringe and monitored for absorbance at 525 nm and for pH at several time intervals until growth ceased. The sample taken was approximately 1.5 ml of the culture for each measurement. The growth curve and pH of the culture during the cellobiose cultivation are presented in Fig. 1. The maximum specific growth rate of 0.19 h-‘, which indicated a maximum doubling time of 3.7 h, was maintained over the first 12 h of growth. Culture growth ceased

01 0

I 8

I

I

I

10

24

30

Time FIG.

1. Growth

[hr

1

and pH variation

tridium therrnocellum on celloboise

after about 30 h of cultivation and was accompanied by a decrease in pH from 7.0 to 5.15 in the medium. A calibration curve of absorbance to the viable colony count is given in Fig. 2. Immobilization. An initial adsorption experiment was conducted at 60°C with a pH value 7.0 to determine the time required to reach an equilibrium with two cellular concentrations in the liquid phase. The number of cells adsorbed per gram of coal, C, , for coalparticle incubation times of 10, 20, 40, and 60 min are presented in Fig. 3, which indicated that equilibrium can be attained in about 20 min for both liquid concentrations. Since the doubling time of the bacteria was much longer than the time required for an adsorption of bacteria cell on the coal particle, it was assumed that reproduction did not significantly affect the results. Adsorption isotherms. The mass of bacteria immobilized on a unit mass of coal particles was characterized with adsorption isotherms. Adsorption isotherms between C. thermocellum cells and bituminous coal particles at 60, 45, and 30°C with a pH value of 7.0 and with pH values of 6.0 and 5.0 at 60°C are presented in Figs. 4A and B, respectively. The

X , Absorbance(525nm) of culture with time.

of Clos-

FIG. 2. Calibration curve between and culture absorbance at 525 nm.

viable

colony

count

IMMOBILIZATION

OF Clostridium

75

thermocellum

librium coefficient between the solid-phase concentration of cells, es,, and the liquid-phase concentration of cells, CL, at equilibrium at a given temperature. The free-energy change of adsorption, AG, the adsorption coefficient, K, and their respective concentrations in each phase are given by -AG = RTln K = RTIn(CJci)

[l]

or

Time

lmin

1

FIG. 3. Variations of immobilized cell concentration on coal particles with time at 60°C and pH 7.0 for two liquid-phase concentration of cells.

results show monolayer adsorptions. Based on the monolayer adsorption characteristic, these results were correlated in double reciprocal forms and are presented in Figs. 5A and B, respectively. As the data indicate, there is a breakpoint in each isotherm, thus indicating a departure from the assumptions of the classical Langmuir isotherm (1). For example, if the immobilized cells carry a surface charge, the first adsorbed particle will exert a repulsive force on the approach of the second cell particle to the same neighborhood. Thus, if cells are immobilized on a surface to a certain density, the repulsive force certainly will increase. Then, considering the situation from a higher density immobilized end, which gives the maximum immobilization capacity per unit mass of adsorbent, the deviation from the Langmuir isotherm at the lower concentration region may be interpreted as a reduction in a repulsive potential due to the reduction in immobilized cellular population on a surface. The adsorption coefficient, K,’ is the equi’ Abbreviations used: CL, cell concentration in the liquid phase (number of cells/ml solution); cs, cell concentration in the solid phase (number of cells/g solid); AG, Gibbs freeenergy change @al/g-mol); AH, enthalpy change

[la] K = exp(-AGIRT) = es/CL Note that the units used in the equations for PI and CL are different from the units used in Figs. 3 and 4. In a low-concentration region, the adsorption free energy is increased due to the reduction of a repulsive force, since cell population is lower. Accordingly, the free-energy change in Eq. [l] has to be modified. If one defines the quantities with an asterisk(*) as those quantities for a low-concentration region, then the free-energy change and the adsorption coefficient in a low-concentration region becomes G*=AG-AG1=-RTlnK*

PI

in which G, is the free-energy change due to the change in a repulsive potential. Therefore, one has RTlnK=RTlnK+

+RTlnKI

131

or K = K*K,.

A Langmuir

[W

adsorption

in a double reciprocal form is given as

@al/g-mol); &, proportionality factor (ml solution/number of cells); ki , the Langmuir adsorption isotherm (ml solution/number of cells); K, equilibrium coefficient between cell concentrations in solid phase and liquid phase; R, universal gas constant (1.987 cal/g-mol-OK); AS, entropy change (cal/g-mol-DK); 7’, absolute temperature (OK); superscript * quantity in a low-concentration-region; subscript I contribution due to the repulsive potential.

76

HORNE AND HSU

-

I/

pH 7.0

0

. 1

4 CL x 10

-0

8

5.0 6.0

l

* 30 0 45 . 80 12

4

-’ [cells /ml1

CL x lo-’

60 “C

7:o

I

I

8 [cells/ml]

12

FIG. 4. Immobilization isotherms of Clostridium thermoceiium cells on coal particles (A) at 60, 45, and 30°C with pH 7.0 and (B) at 60°C with pH 7.0, 6.0, and 5.0.

1

1

1

Ei = m&naxm

[4a]

+ aTlax

and for modified form in a low-concentration region, which becomes -=1

[Cl

exp[-GI/RT]

1

hP%x&‘tl

+ K’slmax’

[51

Using only a linear term of the series expansion of the exponential of the repulsive force, Eq. [5] reduces to 1

z

I--AGI/RT

= k~[~slm&i1 + E

1

*

Pal

In Eqs. [4], [5], and [5a], the quantity kl is the Langmuir coefficient, which has a dimension of (milliliters of solution per number of cells, and is different from the adsorption coefficient K defined in Eq. [ 11. The characteristics of the Langmuir isotherm and its modified form at three selected temperatures were obtained from Figs. 4(A) and (B) together with Eqs. [5 J and [5a]. They are presented in Table 1. Thermodynamic properties of adsorption.

In order to interpret the obtained adsorption data cS and CL in terms of adsorption energy, the Langmuir coefficient k, has to be con-

vetted to the dimensionless adsorption coefficient K. If one defines this conversion factor as b, it follows that K = k,/b.

PI

The conversion factor kc, has the same units as k,. Furthermore, it was used as a proportionality constant to convert CL to a molar quantity cr in the estimation of molar absorption free-energy changes. By rearranging Eq. [I] together with Eq. [6], one obtains In k, = -AGfRT

+ In ko.

171

If the free-energy change of adsorption remains constant over a small temperature range, the conversion factor k,, and the freeenergy change for the adsorption AG can be obtained from the Langmuir adsorption coefficient, k, vs 1/T relationship. Using the Langmuir isotherm coefficients obtained at three temperatures with pH value 7.0, the conversion factors at two concentration regions were found to be b = 3.6 1 X 10m9ml of solution/ number of cells and ko* = 9.13 X lo-’ ml of solution/number ofcells. The adsorption coefficients for each concentration region were obtained using Eq. [6]. Then, the free-energy changes due to repulsive force reduction were

IMMOBILIZATION

OF Clostridium

pH 7.0

1

I

I

I

OO

2

[ml

x lo8

2

6

i lo8

I

hell1

6

4 (l/C,)

030 I

4 (l/CL)

-0

I

I

77

thermocellum

[ml

lee111

FIG. 5. Double reciprocal plots of immobilization isotherms of Clostridium thermocellum cells on particles (A) at 60, 45, and 30°C with pH 7.0 and (B) at 60°C with pH 7.0, 6.0, and 5.0.

obtained from Eqs. [2] and [3]. Those quantities are presented in Table 2. Values of the enthalpy and entropy changes of adsorption in the two concentration regions were obtained from the adsorption coef-

ficient K and temperature Hoff s equation [6]. InKcwm

coal

relationship,

RT+R*

van’t

AS

PI

TABLE I CHARACTERISTIC

CONSTANTS OFIMMOBILIZATION ON BITUMINOUSCOALPARTICLES

Low-concentration Temperature

k*

region

Pxnu

ISOTHERM OF FOR EQS.

Transition

wualu

Clostridium thermocellum CELLS [5] AND [5a] High-concentration region

concentration

tckmm

k,

Paux

(“C)

pH

60 45 30

7.0 7.0 7.0

12.4 14.4 16.2

5.05 6.00 7.97

2.83 2.77 2.2 I

3.94 4.80 6.23

3.04 3.31 3.76

a.49 10.0 13.7

60 60 60

7.0 6.0 5.0

12.4 7.38 11.7

5.05 8.80 9.00

2.83 5.05 4.31

3.94 6.93 7.51

3.04 0.909 0.497

a.49 22.0 44.0

((ml/cell)

X IO’)

((cell/g)

X lo-*)

((cell/ml)

X IO-‘)

((cell/g)

X IO-*)

((ml/cell)

X 108)

((cell/g)

X lo-*)

78

HORNE AND HSU TABLE 2 THERMODYNAMIC

PROPERTIES OF IMMOBILIZATION OF ONBITUMINOUSCOALPARTKLES

Low-concentration region

Clustridium thermocellum CELLS

High-concentration region

Adsorbate interaction

Temperature m

PH

K*

AC* (kcd/mol)

K

AG (kcal/mol)

KI

AG (kcal/mol)

60 45 30

7.0 7.0 7.0

13.60 15.80 17.70

-1.73 -1.74 -1.73

8.42 9.17 10.40

-1.41 -1.40 -1.41

0.619 0.580 0.588

+0.32 +0.34 +0.32

60 60 60

7.0 6.0 5.0

13.60 8.12 12.90

-1.73 -1.39 -1.69

8.42 2.52 1.38

-1.41 -0.61 -0.2 1

0.619 0.310 0.107

+0.32 +0.78 -1.48

From a linear regression analysis, the van? Hoff s equation for two concentration regions were obtained as follows:

centration above the breakpoint, (ciJtrans, follows one mechanism and a concentration below (cLXrans follows another. Inasmuch as the double reciprocal plots of each isotherm in In K = 22 - 0.033 u = 0.107 191 both regions are straight lines, the mechanism T in both regions may be assumed to be characteristically the same, just a change in a maglnK* =y-0.076 a=0.134 [9a] nitude of binding energies between cells and surface of coal particles. The quantity u is the standard deviation for The difference in the two regions is that an the correlation. Then, the quantities m, AH*, immobilization (adsorption) energy at a highAH1, AS, AS*, and A& were obtained from concentration region is reduced by increasing Eqs. [9] and [9a], which are presented in Tarepulsive forces induced by the immobilized ble 3. cell population on the coal surface after it reaches a certain value (CL),,,, or (cS,Xra,,. DISCUSSION It is interesting to note that the values of As seen from the double reciprocal plots in QL in Table 1 are only to demonstrate that Figs. 5A and B, there is a breakpoint in each the Langmuir-type isotherms at a low cellular isotherm which indicates that a change in the concentration region are also applicable. immobilization mechanism occurred. A con- However, one should not confuse with the limit of the true maximum immobilization TABLE 3 due (Cs)maxobtained from a higher cellular concentration region. Maximum adsorption IMMOBILIZATION ENTHALPIE~ AND ENTROPIES OF increases with a decrease in temperature, Ciostridium thermoceiium ON BITUMINOUS which is the characteristic of exothermic adCOALPARTICLES sorption. This is also the verification of Le AH AS Chaterlier’s principle (6). The magnitudes of Region (kd/g-mol) (cd/g-mol-OK) the heats of adsorption AH and AH* were relatively small; thus, one may conclude that High concentration -1.43 -0.067 Low concentration -1.78 -0.152 a physical adsorption is predominant in the Repulsive = (high - low) +0.35 +0.085 immobilization process.

IMMOBILIZATION

OF Clostridium

The pH effect on the immobilization shows that the maximum immobilization capacity in the solid phase (C?,>,,,,, increases with decreasing pH value. However, the Langmuir coefficient k, decreases as the pH value decreases from pH 7.0 to 5.0. Furthermore, the point of transition in (&)trans due to the reduction in repulsive potential shifted to higher values of (Crjtra,, with the decreasing pH values. A quantitative analysis for these effects is warrant for further study. REFERENCES 1. Adamson, A. W. (1976) in Physical Chemistry of Surfaces, 3rd ed., Wiley-Interscience, New York. 2. Brooks, R., Su, T. M., Brennan, M., and Ftick, J. (1979) in Proceedings of the 3rd Annual Biomass Energy Systems Conference, p. 275, Solar Energy Research Institute, Department of Energy, Golden, Colo. 3. Collins, C. H., and Lyne, P. M. (I 976) in Microbiological Methods, 4th ed., p. 197, Butterworths, London. 4. Cooney, C. L., and Wise, D. L. (1975) Biotechnol. Bioeng. 17, 1119-I 135. 5. Cooney, C. L., Daniel, I., Wang, C., Wang, S. D., Gordon, J., and Jiminez, M. (1978) Biotechnol. Bioeng. 8, 103-l 14. 6. Denbigh, K. (1964) in The Principles of Chemical Equilibrium, 2nd ed., pp. 139, 143, Cambridge Univ. Press, London/New York.

79

thermocellum

7. Dowell, V. R., and Hawkins, T. M. (1974) in Laboratory Methods in Anaerobic Bacteriology, U. S. Govt. Printing Office, Washington, D. C. 8. Flickinger, M. C. (1980) Biotechnol. Bioeng. 22, Suppl. 1, 27-48. 9. Garcia-Martinez, D. V., Shinmyo, A., Madia, A., and Demain, A. L. (1980) Eur. J. Appi. Mcrobiol. Biotechnol. 9, 189-197. 10. Herrero, A. A., and Gomez, R. F. (1980) Appl. Environ.

Microbial.

40, 571-577.

1 I. Home, P. N. (1982) Immobilization of Clostridium thermocellum Bacterium on Coal Particles, MS. Thesis, University of Tennessee, Knoxville. 12. Lamed, R., and Zeikus, J. G. (1980) J. Bucteriol. 144, 569-578.

13. Macey, J. M., Snellen, J. E., and Hungate, R. E. (1972) Amer. J. Clin. Nutr. 25, 1318-1323. 14. Ng, T. K., Weimer, P. J., and Zeikus, J. G. (1977) Arch. Microbial. 114, l-7. 15. Remirez, R. (1981) Chem. Eng. 88(2), 51-55. 16. Scott, C. D., and Hancher, C. W. (1976) Biotechnol. Bioeng. 18, 1393-1403. 17. Shinmyo, A. D., Garcia-Martinez, D. V., and Demain, A. L. (1979) J. Appl. Biochem. 1, 202-209. 18. Sitton, 0. C., and Gaddy, J. L. (1980) Biotechnol. Bioeng. 22, 1735-1748. 19. Wang, D. I. C., Biotic, I., Fang, H. Y., and Wang, S. D. (1979) in Proceedings of the 3rd Annual Biomass Energy Systems Conference, p. 6 1, Solar Energy Research Institute, Department of Energy, Golden, Colo. 20. Weimer, P. J., and Zeikus, J. G. (1977) Appl. Environ. Microbial.

33, 289-297.