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JOURNAL OP FERMENTATION AND BIOENOINEERING Vol. 81, No. 2, 158-162. 1996 Evaluation of Support Matrices for an Immobilized Cell Gas Lift Reactor for Fermentation of Coal Derived Synthesis Gas SUDIPTA CHATTERJEE,‘* ANDREW J. GRETHLEIN,’ R. MARK WORDEN, AND MAHENDRA K. JAIN’ Michigan Biotechnology Institute, 3900 Collins Road, P.O. Box 27609, Lansing, MI-48909l and Department of Chemical Engineering, Michigan State University, East Lansing, MI-48824,2 USA Received 21 February 1995IAccepted 4 December 1995 The fermentation of synthesis gas by Butyribacterium methylotrophicum is limited by the poor solubility of the substrate in water. Gas lift reactors allow for increased gas liquid mixing and may be employed in synthesis gas fermentations to enhance gas mass transfer rates. Studies were conducted to evaluate different matrices for the immobilization of B. methylotrophicum in a gas lift reactor. The performance of celite, molecular sieves, alumina, activated carbon, wood, and ion exchange was analyzed on the basis of cell growth, cell attachment, product formation, and minimum fluidization velocity. Maximum cell growth was evidenced with molecular sieves, whereas celite proved best for cell attachment. The total electron content of the products was higher in the molecular sieves system than in the ion exchange or celite systems. However, the total available electron normalized to protein content varied between 25-33 meq/Z/mg for celite, ion exchange and molecular sieves. The minimum fluidization velocity of molecular sieves>celite>ion-exchange. Based on the results, both celite and molecular sieves were found to be effective for cell immobilization. [Key words: synthesis gas, adsorption, supports, total protein, fluidization] In recent years, concerns about depletion of petroleum resources and increased political restrictions imposed on the import of petroleum have intensified the quest for alternative routes for energy, fuels, and chemicals pro- duction. In the United States, the large domestic coal reserves, the low cost of coal, and the availability of a large variety of coal processing technologies have engen- dered a great deal of interest in the development of coal based alternative energy production routes. Lately, the bioprocessing of coal derived synthesis gas, for the production of chemical feedstocks and fuels, via anaero- bic fermentation has generated considerable interest (1, 2). Catalytic processing of synthesis gas by direct and indirect routes, such as Fisher Tropsch process and methanol/CO/Hz conversions are limited by catalyst selectivity, high operating temperatures and pressures, wide product distributions and the potential risk of catalyst poisoning by the sulfur gases present in coal de- rived synthesis gas. Biocatalytic conversion of synthesis gas using sulfur tolerant microorganisms provides an alternative to conventional metal catalysis. Recently, the use of sulfur tolerant microorganisms capable of con- verting glucose, methanol, CO and HZ-CO2 to acids and alcohols has gained tremendous impetus in the field of anaerobic fermentation technology (1, 3-5). The poten- tial for reduced capital and operating costs has triggered a number of laboratory scale investigations. Conversion of synthesis gas to a mixture of acids and alcohols has already been demonstrated (1, 6-7; Chatterjee, S. et al., Abstr. Fourth International Symposium on Biological Processing of Fossil Fuels, Alghero, Italy, 1993). Choice of bioreactor for fermentation of synthesis gas Single carbon gas bioprocessing presents unique chal- lenges from the standpoint of bioreactor design, as the * Corresponding author. Present affiliation: Department of Applied Technology, Bldg. 830, Brookhaven National Laboratory, Upton, NY-l 1973, USA. substrates (such as CO and COZ) are sparingly soluble in water. In existing commercial-scale aerobic fermen- tations, gas mass transfer is typically rate limiting, and the capital and operating costs associated with mass transfer comprise a substantial fraction of the overall fermentation costs. Traditional approaches to enhancing gas mass transfer may not be well suited for synthesis gas fermentations. Standard flat bladed turbine impellers generate high shear fields and thus high mass transfer coefficients near the impeller, but they also exhibit high power consumption rates and poor liquid mixing charac- teristics in large fermentors. Because extremely large fermentors would be needed to produce commodity fuel additives and octane enhancers, power requirements for mechanical agitation could be excessive. For simple fermentations that produce relatively low-value products and cannot justify unusually expensive fermentors, pow- er input in the form of compressed gas rather than mechanical agitation is preferred (8). Hence, the use of bioreactors without mechanical agitation may be advocat- ed for synthesis gas fermentations. The conversion of coal gas (65% CO, 22% Hz, 1% C02, and 2% CH.,) and acetate by Peptostreptococcus productus and Methanotrix, respectively, has been exam- ined in fixed film plug flow reactors in which the cells are attached to an inert support (9). A gas lift reactor provides an alternative design in which gas is sparged into a draft tube to provide the necessary mass transfer and to induce liquid mixing. Further, gas-lift reactors can be easily scaled up, and may be used commercially. Gas lift fermentors are suited for either immobilized or free cell operations. Immobilization of biocatalysts not only offers advantages of ease of separation of cells from the reaction mixture, reuse, and improved opera- tional and storage stability, but also, in certain cases, en- hanced plasmid stability (lo), and tolerance to water mis- cible organic solvents (11). The ideal cell support parti- cles for a gas lift fermentor should be inexpensive and have a low enough terminal settling velocity to be sus- 158

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Page 1: Evaluation of support matrices for an immobilized cell gas lift reactor for fermentation of coal derived synthesis gas

JOURNAL OP FERMENTATION AND BIOENOINEERING Vol. 81, No. 2, 158-162. 1996

Evaluation of Support Matrices for an Immobilized Cell Gas Lift Reactor for Fermentation of Coal Derived Synthesis Gas

SUDIPTA CHATTERJEE,‘* ANDREW J. GRETHLEIN,’ R. MARK WORDEN, AND MAHENDRA K. JAIN’

Michigan Biotechnology Institute, 3900 Collins Road, P.O. Box 27609, Lansing, MI-48909l and Department of Chemical Engineering, Michigan State University, East Lansing, MI-48824,2 USA

Received 21 February 1995IAccepted 4 December 1995

The fermentation of synthesis gas by Butyribacterium methylotrophicum is limited by the poor solubility of the substrate in water. Gas lift reactors allow for increased gas liquid mixing and may be employed in synthesis gas fermentations to enhance gas mass transfer rates. Studies were conducted to evaluate different matrices for the immobilization of B. methylotrophicum in a gas lift reactor. The performance of celite, molecular sieves, alumina, activated carbon, wood, and ion exchange was analyzed on the basis of cell growth, cell attachment, product formation, and minimum fluidization velocity. Maximum cell growth was evidenced with molecular sieves, whereas celite proved best for cell attachment. The total electron content of the products was higher in the molecular sieves system than in the ion exchange or celite systems. However, the total available electron normalized to protein content varied between 25-33 meq/Z/mg for celite, ion exchange and molecular sieves. The minimum fluidization velocity of molecular sieves>celite>ion-exchange. Based on the results, both celite and molecular sieves were found to be effective for cell immobilization.

[Key words: synthesis gas, adsorption, supports, total protein, fluidization]

In recent years, concerns about depletion of petroleum resources and increased political restrictions imposed on the import of petroleum have intensified the quest for alternative routes for energy, fuels, and chemicals pro- duction. In the United States, the large domestic coal reserves, the low cost of coal, and the availability of a large variety of coal processing technologies have engen- dered a great deal of interest in the development of coal based alternative energy production routes. Lately, the bioprocessing of coal derived synthesis gas, for the production of chemical feedstocks and fuels, via anaero- bic fermentation has generated considerable interest (1, 2).

Catalytic processing of synthesis gas by direct and indirect routes, such as Fisher Tropsch process and methanol/CO/Hz conversions are limited by catalyst selectivity, high operating temperatures and pressures, wide product distributions and the potential risk of catalyst poisoning by the sulfur gases present in coal de- rived synthesis gas. Biocatalytic conversion of synthesis gas using sulfur tolerant microorganisms provides an alternative to conventional metal catalysis. Recently, the use of sulfur tolerant microorganisms capable of con- verting glucose, methanol, CO and HZ-CO2 to acids and alcohols has gained tremendous impetus in the field of anaerobic fermentation technology (1, 3-5). The poten- tial for reduced capital and operating costs has triggered a number of laboratory scale investigations. Conversion of synthesis gas to a mixture of acids and alcohols has already been demonstrated (1, 6-7; Chatterjee, S. et al., Abstr. Fourth International Symposium on Biological Processing of Fossil Fuels, Alghero, Italy, 1993).

Choice of bioreactor for fermentation of synthesis gas Single carbon gas bioprocessing presents unique chal- lenges from the standpoint of bioreactor design, as the

* Corresponding author. Present affiliation: Department of Applied Technology, Bldg. 830, Brookhaven National Laboratory, Upton, NY-l 1973, USA.

substrates (such as CO and COZ) are sparingly soluble in water. In existing commercial-scale aerobic fermen- tations, gas mass transfer is typically rate limiting, and the capital and operating costs associated with mass transfer comprise a substantial fraction of the overall fermentation costs. Traditional approaches to enhancing gas mass transfer may not be well suited for synthesis gas fermentations. Standard flat bladed turbine impellers generate high shear fields and thus high mass transfer coefficients near the impeller, but they also exhibit high power consumption rates and poor liquid mixing charac- teristics in large fermentors. Because extremely large fermentors would be needed to produce commodity fuel additives and octane enhancers, power requirements for mechanical agitation could be excessive. For simple fermentations that produce relatively low-value products and cannot justify unusually expensive fermentors, pow- er input in the form of compressed gas rather than mechanical agitation is preferred (8). Hence, the use of bioreactors without mechanical agitation may be advocat- ed for synthesis gas fermentations.

The conversion of coal gas (65% CO, 22% Hz, 1% C02, and 2% CH.,) and acetate by Peptostreptococcus productus and Methanotrix, respectively, has been exam- ined in fixed film plug flow reactors in which the cells are attached to an inert support (9). A gas lift reactor provides an alternative design in which gas is sparged into a draft tube to provide the necessary mass transfer and to induce liquid mixing. Further, gas-lift reactors can be easily scaled up, and may be used commercially.

Gas lift fermentors are suited for either immobilized or free cell operations. Immobilization of biocatalysts not only offers advantages of ease of separation of cells from the reaction mixture, reuse, and improved opera- tional and storage stability, but also, in certain cases, en- hanced plasmid stability (lo), and tolerance to water mis- cible organic solvents (11). The ideal cell support parti- cles for a gas lift fermentor should be inexpensive and have a low enough terminal settling velocity to be sus-

158

Page 2: Evaluation of support matrices for an immobilized cell gas lift reactor for fermentation of coal derived synthesis gas

VOL. 81, 1996 CELL IMMOBILIZATION FOR SYNTHESIS GAS FERMENTATION 159

pended by the upflowing gas and liquid streams. Cell im- mobilization may be carried out by entrapment, covalent coupling to supports, or by adsorption to inert supports. Gel entrapment of cells can increase resistances to mass transfer. Hence for synthesis gas fermentation, which is limited by the poor solubility of gases in water, gel en- trapment techniques for cell immobilization may not be useful. Covalent coupling and adsorption both allow for the attachment of cells on the surface of the supports. Whereas covalent coupling is stable, cell desorption may be a problem with the adsorption method. However, the overall simplicity of the adsorption technique makes it very attractive for low-cost industrial applications.

This paper compares and evaluates the immobilization of B. methylotrophicum, by adsorption, on different sup- ports for the anaerobic fermentation of synthesis gas in a gas lift reactor. The effects of celite, molecular sieves, ion-exchange, wood, activated carbon and alumina on cell growth, cell attachment, and fermentation product profile are investigated. In addition, minimum fluidiza- tion velocities and densities of molecular sieves, ion-ex- change and celite are determined. The results can pro- vide the basis for selecting an immobilization support for synthesis gas fermentations.

EXPERIMENTAL PROCEDURES

Materials and methods Microorganism A CO-utilizing strain of B. meth-

ylotrophicum, previously developed in our laboratory (12), was adapted to grow on 100% synthesis gas at 40°C and 150rpm in a gyratory shaker (New Brunswick GlO, New Brunswick, Edison, NJ, USA). This adapted strain was used as the biocatalyst for the anaerobic fermentation of synthesis gas.

Culture medium A phosphate buffered basal medi- um (PBBM) containing primarily inorganic salts, nutri- ents, vitamins and yeast extract and reduced by Na#, was used to grow B. methylotrophicum. This medium has been described in detail elsewhere (13). All the com- ponents of the culture medium were sterilized under strictly anaerobic conditions at 20 psig and 121 “C.

Synthesis gas A synthesis gas mixture (65% CO, 30% Hz, 3% COZ, 1.2% H2S, and 0.8% COS) simulat- ing synthesis gas derived from coal (Illinois #6), was purchased from AGA Gas Inc. (Cleveland, Ohio, USA).

Chemicals All chemicals used were of analytical grade and were purchased from Sigma Chemical Com- pany (St. Louis, MI, USA) and Aldrich (Milwaukee, WI, USA). A bicinchoninic (BCA) protein assay kit, pur- chased from Pierce Chemical Company (Rockford, IL, USA) was used for total protein determinations.

Supports for cell immobilization Ion exchange mix- ed bed resins, 20-80 mesh (Bio pad Labs., Richmond, CA, USA); molecular sieves, 3 A, l/16” pellets (Fluka, Switzerland); alumina, neutral (Fisher Sci., Fairlawn, NJ, USA); celite (Manville Corp., Denver, CO, USA); wood powder (poplar); and activated carbon, Filtrasorb 400 (Calgon Corp., Pittsburgh, PA, USA) were used to immobilize B. methylotrophicum cells.

Preparation of support (i) Sizing Celite (10/14), wood powder (125/150),

and alumina (125/150) were sized by dry screening, us- ing standard sieves (USA Standard Testing Sieves). (The numbers in parentheses designate two mesh sizes, e.g. in the case of celite, the fraction of the particles passing

through sieve size 10 and retained on sieve size 14 were used for the experiments). Activated carbon, molecular sieves and ion exchange resins were used as obtained.

(ii) Sterilization Approximately log of each sup- port was weighed out separately into 158 ml serum vials and covered with 1OOml of PBBM, pH 7.0. The sup- ports were then degassed for approximately 2-3 h to eliminate all air from the system. Next vitamins, inorgan- ic salts, nutrients, yeast extract and Na$ were added to the vials (13). The supports were then placed under N2 and left to equilibrate at 4°C for 8-10 h, prior to sterili- zation at 20 psig and 121°C.

Cell immobilization Cells were grown in the pres- ence of the different supports to allow for self-adhe- sion. Approximately 1 g of support (wet weight), was weighed out into 58 ml serum vials (Wheaton, Millville, NJ, USA) and covered with 10ml of PBBM, to which all nutrients and a 2% (v/v) actively growing inoculum had been added previously. The headspace of the vials was then filled with the substrate, synthesis gas, to a pres- sure of 15 psig. Cells were incubated at 40°C and shaken at 150rpm. Five replicate vials were set up for each kind of support. One vial from each set was analyzed for total protein at each of the following times: 4, 11, 15 and 20 d. The remaining vials were repressurized with synthesis gas to 15 psig after 4, 11 and 15 d in order to ensure the continuing availability of substrate to the cells. Control experiments were run without supports.

Quantitation of cell growth and cell immobilization Cell growth in the presence of the different supports was assessed by determining the protein contents of free and immobilized cells. One vial (from each set) was sacrificed after 4, 11, 15 and 20 d, and the contents were filtered to separate the supports from the media. A 50 micron Dutch twill (100 x 80) filter cloth (Envirotech Corpora- tion) was used for this purpose. The filtrate and residue were separately tested for protein content. A known volume of the filtrate and a known weight of the residue (support) were analyzed separately for total protein by the BCA protein analysis method (14). The sum of the protein contents of the filtrate and residue was taken as a measure of the total protein synthesized in a given system. The protein content of the support was taken as a measure of cell immobilization. Control experiments were analyzed at time zero to assess the protein contents

Water in-

t

2.54 cm

-I I

-Water out

Packed bed

FIG. 1. Apparatus for determination of fluidization velocities (figure not drawn to scale).

Page 3: Evaluation of support matrices for an immobilized cell gas lift reactor for fermentation of coal derived synthesis gas

160 CHATTERJEE ET AL. J. FERMENT. BIOENG.,

of the sterile supports. Determination of minimum JIuidization velocity

The minimum velocity necessary to suspend the supports in an upflowing liquid stream was determined in a 100 cm long, 2.54cm i.d. glass column (Fig. 1). The column was initially packed with 75 g of wet support which had been previously degassed. The liquid flow rate was controlled by varying the pressure of the inlet water stream. Linear regression plots of bed height versus superficial velocity were extrapolated to the fixed bed height, and the corresponding superficial velocity was taken as a measure of the minimum fluidization velocity.

Determination of densities of wet support Densi- ties of the degassed supports were determined by measur- ing the volume of water displaced by 1 g of wet sup- port. The wet supports were carefully wiped with filter paper to remove any entrapped water prior to the densi- ty measurements.

Analysis of product The production of acids and alcohols by B. methylotrophicum was monitored periodi- cally. Liquid samples (filtrate) were analyzed using a Hewlett Packard (Avondale, PA) 5890 series gas chro- matograph equipped with a 1.2 m Chromosorb 101 @O/100 mesh) packed column and an FID detector. All samples were acidified with a drop of 50% HzS04 and centrifuged to precipitate the cells prior to analysis. The concentrations of products were determined by compari- son to standard samples which were run concurrently on the gas chromatograph.

RESULTS AND DISCUSSION

Eflect of supports on cell growth The effects of different supports on the growth of B. methylotrophi- cum were analyzed by measuring the total protein synthe- sized in a given system. Table 1 shows the amount of free and immobilized protein synthesized in the different systems over a period of 20d. In all the systems, except molecular sieves, protein synthesis leveled off to an almost constant value after 11 d. The cells apparently grew during the first 11 d and then entered the stationary phase. However, in the case of molecular sieves, the cells apparently continued to grow even after 20d. The poor cell growth with activated carbon could have been caused by the adsorption of media components by the support, giving rise to a nutrient-poor system. The differ- ent growth patterns observed in the different systems may be the result of a combination of factors such as:

10 15 20 Time (d)

FIG. 2. Effect of supports on protein immobilization. Symbols: ffl, celite; 0, molecular sieves; 0, wood; A, alumina; A, ion exchange.

TABLE 1. Effect of supports on protein synthesis

Weight protein immobilized (mg)

Support 4d lld 15d 20d

la Fb la Fb la Fb Ia Fb

Celite 0.98 6.88 2.93 22.86 2.65 19.44 3.43 16.70 Wood 0.00 20.46 0.00 29.10 0.00 33.42 2.27 24.20 Activated carbon 0.05 12.15 0.06 nd 0.08 nd 0.00 3.89 Ion exchange 0.05 13.12 1.10 20.20 1.13 23.33 0.41 19.10 Molecular sieves 1.20 10.83 1.43 30.20 1.50 33.66 2.10 41.56 Alumina 0.22 14.88 1.10 29.58 1.75 ND 2.60 23.60 Control NA 7.12 NA 25.86 NA 21.5 NA 26.70

a I: Immobilized protein, refers to the protein content of 1 g sup- port.

b F: Free or unimmobilized protein, refers to the protein content of 10 ml filtrate.

ND: Not determined, nd: none detected; NA: not applicable.

adsorption of nutrient, cells and products by the sup- ports; pH fluctuations; and random variations between replicates. Inconsistent growth patterns of B. methylo- trophicum, even under identical conditions, have been reported previously (Grethlein, A. J., Ph.D. thesis, Michigan State University, 1991). The apparent decrease in total protein content with time, in certain cases, could also be an artifact because different vials were used for different time point analyses.

As shown in Fig. 2 and Table 1, there was a steady in- crease in percentage protein immobilized with time for celite and alumina. The decrease in percentage immobili- zation with molecular sieves, despite an increase in im- mobilized protein synthesized, is due to a far greater increase in free protein than immobilized protein after 4 d. This probably indicates a saturation of available sites for immobilization after the fourth day. However, in the case of ion exchange, the decrease in percent immobiliza- tion is due to a decrease in immobilized protein. This may have been caused by cell desorption, poor growth in that particular vial, or an experimental error in pro- tein estimation. Cell desorption would have led to an increase in free protein; however, as both free and total protein also decrease with time, it appears to be a case of poor growth.

The maximum percentage immobilization for celite, wood, ion exchange, molecular sieves and alumina was 17, 9, 2, 5, and lo%, respectively. Although wood showed 9% immobilization at the end of 20d, cell

g OZ Time (d)

FIG. 3. Weight protein immobilized per unit external surface area versus time. Symbols: q , celite; 0, molecular sieves; 0, wood; A, alumina; A, ion exchange.

Page 4: Evaluation of support matrices for an immobilized cell gas lift reactor for fermentation of coal derived synthesis gas

VOL. 81, 1996 CELL IMMOBILIZATION FOR SYNTHESIS GAS FERMENTATION 161

-50 0 so 100 150 200 250

Superficial velocity (cmlmin)

FIG. 4. Bed height versus superficial velocity for fluidization of celite.

attachment was not apparent during the first 15 d. The characteristics of a support, such as the size of the pores (as compared to the size of the cells), pore size distribu- tion, internal and external surface area, and the number of adsorption sites, determine its cell adsorption proper- ties. Figure 3 represents the weight of protein immobi- lized per unit external area of support. External surface areas and volumes were calculated assuming the particles to be of regular shapes. The molecular sieves were cylin- drical in shape (6 mm length x 1 mm diameter). The rest of the particles were assumed to be spherical, with an average diameter corresponding to their mesh size. The weight of protein immobilized/unit area was calculated from the wet density of support, surface area and volume of the supports using the following equation:

Mass protein immobilized Surface area support

= Mass protein immobilized Unit weight of support

x Density of support

Volume of support ’ Surface area of support

On the basis of total protein synthesis (cell growth) and percentage immobilization, celite, alumina, ion ex- change and molecular sieves seem to be best suited for immobilization of B. methylotrophicum.

Minimum fluidization velocity The fluidization pro-

60 k

0 Lb- -100 0 100 200 300 400 500 600

Supetilcial velocity (cm/min)

FIG. 5. Bed height versus superficial velocity for fluidization of molecular sieves.

60

50

P 2 40

E: .p 30

i 20

10

p-’

c

Superficial velocity (cm/min)

FIG. 6. Bed height versus superficial velocity for fluidization of ion-exchange.

perties of celite, molecular sieves, and ion exchange resins, are shown in Figs. 4, 5, and 6, respectively. At- tempts to Auidize alumina resulted in its entrainment from the top of the column as the alumina particles were much smaller (125/150 mesh) than the other supports. Table 2 depicts the minimum fluidization velocities, den- sities and diameters for molecular sieves, ion-exchange, and celite.

The minimum fluidization velocity of molecular sieves > celite >ion exchange. As expected the minimum fluidization velocity decreased with increasing density from ion exchange to molecular sieves.

Determination of the effect of support on product profile During fermentation of synthesis gas by B. methyiotrophicum, acetic and butyric acids and trace amounts of ethanol and butanol are produced (Chat- terjee, S. et al., Abstr. Fourth Inter. Symp. Biologic. Proces. Fossil Fuels, Alghero, Italy, 1993). Figure 7A and B compare the total electron contents of the products (acetate+butyrate) formed in the presence of molecular sieves, celite and ion-exchange resins over 20 d of fermentation. The total number of available electrons (EJ contained in the products was calculated by the fol- lowing equation:

where, Et : total available electrons, meq/l C : concentration of product, mM N : the number of carbons in a molecule of product y : reductance degree A, B : subscripts referring to acetate and butyrate,

respectively. The reductance degrees for acetate and butyrate are 4 and 5, respectively (15). The reductance degree is defined as the number of electron equivalents available for transfer to molecular oxygen during combustion, ex- pressed on a C-mole basis. The number of carbon atoms

TABLE 2. Minimum fluidization velocities and densities of supports

support Minimum fluidization velocity Density Diameter (cm/min) k/ml) (cm)

Molecular sieves 161.00 1.70 0.100 CeIite 60.70 1.43 0.140 Ion exchange 10.20 1.08 0.025

Page 5: Evaluation of support matrices for an immobilized cell gas lift reactor for fermentation of coal derived synthesis gas

162 CHATTERJEE ET AL. J. FERMENT. BIOENG.,

L 8 1 1000

4 % 5 506

B Z

0 0 5 10 15 20 25

Time (d)

0 5 10 15 20 25 Time (d)

FIG. 7. Effects of celite, molecular sieves, and ion-exchange on metabolic output of the cells. Symbols: M, celite; 0, molecular sieves; A, ion exchange.

in acetate and butyrate are 2 and 4, respectively. Figure 7A shows the variation of total available elec-

trons vs time. The total available electrons are higher in the molecular sieves system than in the ion-exchange or celite systems. These results are in agreement with the cell growth patterns evidenced in the respective systems. However, the total available electrons normalized to pro- tein content (Fig. 7B) varied between 25-33 meq/l/mg for the three supports (at the end of 20d).

Choice of support for immobilization of B. methylo- trophicum Table 3 compares molecular sieves and celite based on a number of factors. In respect of cell growth (given by total protein synthesis), molecular sieves was found to be superior to celite. However celite gave a higher percentage immobilization of protein (17%) as opposed to molecular sieves (5%) in 20d. Since the fermentation of synthesis gas with B. methylotrophicum is very slow (typical batch fermenta- tions have a logarithmic growth phase of two weeks [Chatterjee, S. et al., Abstr. Fourth Inter. Symp. Biolog- ic. Proces. Fossil Fuels, Alghero, Italy, 1993]), the steady increase of percentage immobilization with celite is a definite advantage. In addition, celite gave a maxi- mum immobilization (mg protein/g support) which was -1.6 times that of molecular sieves.

Alumina and ion-exchange resins were not considered because of entrainment from top of the fluidization column and total product formation, respectively. Never- theless it may be speculated that the optimum choice of particle size and operating conditions for alumina and ion-exchange, respectively, may have yielded better results with the two supports. However such detailed

TABLE 3. A comparison of celite and molecular sieves based on overall results

Celite Molecular sieves

Total protein synthesis (mg/g support) Maximum protein immobilization

(mg/g support)

20.00 44.00 3.43 2.10

Maximum protein immobilized (%) 17.00 5.00 Density (g/ml) 1.70 1.43 Minimum fluidization velocity (cm/min) 60.00 161.00

investigations were outside the scope of this research.

ACKNOWLEDGMENTS

Funding for this research was provided by DOE-PETC via the award of contract #DE-AC22-92PC92117. The authors wish to thank Hong Ye and Jeff Carroll for their technical assistance.

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