Characteristics of Zymomonas mobilis immobilized by photo-crosslinkable resin in ethanol fermentation

JOURNAL OF FERMENTATIONAND BIOENGINEERING Vol. 75, No. 1, 28-31. 1993

Characteristics of Zymomonas mobilis Immobilized by Photo-Crosslinkable Resin in Ethanol Fermentation TAKAMITSU IIDA,* MASAHIRO SAKAMOTO, HITOSHI IZUMIDA, AND YOSHIMI AKAGI The Fuel Alcohol Research Association (FARA ), Technical Research Laboratory, Kansai Paint Co., Ltd., Higashi- Yawata, Hiratsuka 254, Japan Received 30 September 1991/Accepted 11 September 1992

Zymomonas mobiUs, an ethanol-producing bacterium, was immobilized in hydrophilic photo-crossfinked resin gels to form a blocatalyst. The molecular structure of the photo-crosslinkable resin could be modulated so as to minimize a disadvantage of this bacterium--poor-tolerance to salts in molasses. Characteristies of Z. mobllls immobilized by photo-crossllnkable resin gel, such as fermentability, cell growth in gel, the potential of gel materials, diffusion of materials, and salt distribution are discussed. ENTG-3800 photo-crosslinkable resin was selected as the most suitable entrapping material for Z. mobtlt~, especially in using molasses.

Ethanol production from biomass resources is likely to attract further public attention since ethanol is now available as a fuel blended with petroleum. Ethanol fermentation is usually carried out batchwise with yeast like Saccharomyces species. In order to develop a novel and energy-saving process, attempts have been made to develop a fermentation system and ethanol-producing microbes. Zymomonas mobilis, which exhibits a higher fermentation rate compared with that of yeast (1), has disadvantages such poor-tolerance to salts in the medium (2) and selectivity for fermentable sugar (3). We have attempted to develop continuous ethanol fermentation by immobilized Z. mobilis with high productivity. The immobilization technique using photo-crosslinkable resin prepolymer has two characteristics--one of which is the controllability of the entrapping gel structure and mild gel formation by ultra-violet ray irradiation. Thus, gel formed by photo-crosslinkable resin can provide a specific reaction field for microbes without damage due to activity. Immobilization of microbes with photo-crosslinkable resin is considered appropriate for improving tolerance to salts, adaptability to molasses, and cell growth. In this paper, the selection of a photo-crosslinkable resin suitable for these purposes and the characteristics of the immobilized gels are described.

leneglycol (PEG, molecular weight 4000) and polypropyleneglycol (PPG, molecular weight 4000) with photocrosslinkable ethylenic double bonds at both ends, and ENTV-500 in which the backbone structure is polyvinylalcohol (PVA, molecular weight about 20,000) with ethylenic double bonds at the side chains, as shown in Fig. 1. The backbone of ENTV-500C is composed of PVA containing 5 mol% and over of acrylamide derivatives and showed cationic with ammonium salts. Immobilization by photo-crossilnkable resins 10 parts by weight of photo-crosslinkable resin solution were mixed with 0.08 parts by weight of photo-initiator, 2 parts by weight of 3% sodium alginate, 2 parts by weight of distilled water and 2 parts by weight of centrifuged Zymomonas cell suspension. This solution was dropped in 1.5% calcium chloride solution to form beads (Hasegawa, E. et al., US Patent 4605622, 1986). The initial cell concentration in this mixture was about 4 x 107 ceUs/ml. The beads thus formed were irradiated by ultra-violet rays with a wavelength of 300-400 nm (Hasegawa, E. et al., US Patent 4605622, 1986). After irradiating for five minutes, the immobilized Z. mobilis beads obtained were washed with sterile water. Preparation

of

photo-crosslinked

resin

membrane

A mixture of 10 g photo-crosslinkable resin, 0.08 g photoinitiator and 6 g distilled water was laid between polypropylene films and controlled at about 0.5 mm thickness. After irradiating the mixture with ultraviolet rays for 3 rain and tearing off both films, the naked photo-crosslinked resin membranes obtained were used for the measurement of membrane potential and permeability. However, a membrane of calcium alginate did not have sufficient mechanical strength to enable the apparatus to be set up. Measurement of membrane potential The selective ion-permeability dependent on the electric potential of the membrane, was measured as one of the functions of the photo-crosslinked resin with the apparatus illustrated in Fig. 2. With the vessels A and B separated from each other by a photo-crosslinked resin membrane, when a salt solution of concentration C~ was put into the former (vessel A) and a salt solution of concentration Cz into the latter (vessel B), the difference in potential between the two faces of the membrane was represented by the following equation:

MATERIALS AND METHODS Cultivation of microbes Zymomonas mobilis NRRL B-14023 was cultivated using GY-5 medium, composed of 50 g/l glucose and 5 g/l yeast extract. After 24 h static cultivation at 30°C, cells were harvested by centrifugation, and used for immobilization. In the case of immobilized Z. mobilis, 3 g of immobilized Z. mobilis beads and 20 ml of substrate, for example, GY-15 medium composed of 150g/l glucose and 5g/l yeast extract, 15% molasses medium ( CM-15 ) with a sugar concentration of 15% and supplemented with 1 g/! ammonium sulfate, were cultivated without stirring in a 100 ml conical flask at 30°C. Photo-crossiinkable resins The photo-crosslinkable resins used in these experiments were ENTG-3800 which has a backbone composed of a combination of polyethy* Corresponding author. 28

VOL. 75, 1993

CHARACTERISTICS OF IMMOBILIZEDZYMOMONAS .

o

CH2 CHCO(CH2)2OCNHC.2~."

NH~O(CH2CH20)gl~NH0t Oi

29

o

H2NH~0(CH2)20~CH'CH2 CH3-"'CH3

CH 3 CH 3

~H3 ---(¢HCH20)52---

ENTG-3800 Prepolymer

CH3CH2 ( - ( CH2'CI'I) I nCH2CH I )m-CH2CH2OH

OH

!H 2

CH3CH2 ( - ( CH2(~- ) P- ( CI"I2~H ) nCH2~ tt)mCH2CH2OH ~:=O OH!

NH

I

C=Ol CH2

ENTV-500 Prepolymer

NH " ~+

/

N~

R2

R3 X-

H2 NH

R: alkyl group

c=ol

X-: anionic group

I

~H CH2

R4

ENTV-500C Prepolymer

FIG. 1. Structure of photo-crosslinkable resin prepolymers. The main chain of ENTG-3810 is a mixture of polyethyleneglycoland polypropyleneglycol(= 80/20). The main chains of both ENTV-500and ENTV-500Care polyvinylalcohol. E = - ( R T / Z F ) (t + - t - ) In (C2/C1)

t + + t - = 1 (1)

where E is the potential of the membrane; R, the gas constant=8.31 (J/K.moD; t, the absolute temperature; Z, the valency; F, the Faraday constant =9.65 x 104 (C/tool); t +, the cation transport number; and t - , the anion transport number. Measurement of permeation coefficient The equipment for measuring the permeation coefficient is shown in Fig. 3. A solute of initial concentration Co was put into vessel A of capacity V(cm a) which was combined with vessel B of the same capacity V(cma), separated by a membrane of thickness ! (cm) and area S (cm2), and distilled water was put into vessel B. The concentration of the liquid in vessel B after the diffusion of the solute through the membrane in t h is represented by Ct, and the diffusion coefficient D (cm2/s) was calculated by the following equation according to Fick's law: D = (Vx I x

Ct)/[S x

t x ( C o - CO]

(2)

As the permeate in vessel A passed through the film of photo-cross[inked resin gel to vessel B, the concentration of permeate in vessel B was measured succ~sively. The liquids in the two vessels were stirred homogeneously by a magnet at a ~ temperature of 30°C. The concentratiom of ethanol and glucose as permeates were measured by gas chromatograph and Gluco-Test C Wako (enzy-

matic method), respectively. Assay of magnesium ions as permeate was carried out by Magnesium Test Wako (chelate-forming method). The initial concentration of substrates (Co) was 1.05 tool/! of MgCI2, 0.55 mol/l of glucose, and 2.17 mol/l of ethanol. Measurement of distribution of salts between gel and liq. uid After the photo-crossiinked gel beads were immersed in a constant concentration of magnesium chloride solution for two weeks at 30°C sufficient to produce equilibrium of the salts, the magnesium ion contents in both the bead gel and solution were determined. Magnesium ions in the bead gel were extracted five times by distilled water and concentrated to a degree capable of measurement by heating. Magnesium chloride, which was easy to assay was used as the representative of cations. Assay of magnesium ions also carried out by Magnesium Test Wako. Observation of immobilized Z. mobilis After two weeks continuous fermentation in molasses medium (CM15), using a bioreactor packed 20% by volume with immobilized Z. mobilis beads, the immobilized bead gel was taken out of the bioreactor, fixed with glutaldehyde, and washed with the phosphate buffer. The bead gel was dehydrated with ethanol and acetone. When it was sufficiently dried the bead gel was fractured by a metal knife Sampling

mV meter

I I L--~ul

Resin sheet

Constant temperature bath ( 30 °C ) 3N-KCI Vessel A Vessel B 3N-KCl FIG. 2. Apparatus for meusurmnent of membrane potential. Concentration of KCI solution in vessels A and B, separated by photo-crosslinked resin membrane, is I mol/l and 0.01 mol//, respectively. The potential of the membrane is measured by the voltmeter to

calculate the cation transport number by Eg. 1.

FIG. 3. Apparatus for measurement of diffusion coefficient. A

solute of conc. Co in vessel A (Vcm3) diffused into the sheet (thickness: lcm, area: Scm2) and moved into vessel B (Vcm3) to a conc. of Ct over a period of time t. The liquids in the vicinityof the sheet surface in both A and B vessels were stirred magneticallyby a motor.

30

IIDA ET AL.

J. FERMENT. BIOENG.,

and spatter coated with gold metal. The specimen was observed with a JOEL JSM-T220-type scanning electron microscope for better observation of microbes in the gel from all angles.

~ so

Effect o f salts on ethanol fermentation In molasses medium of 15% sugar concentration (CM-15), the ethanol fermentation rates of Z. mobilis NRRL B-14023 were 1.2 adsorbed on Celite 545, 1.0 on Duolite A-7 resin, 2.3 entrapped in calcium aiginate, and 3.2 in ENTV-500, with the rate of free cells represented by 1.0. These results show that the fermentation rate in molasses was high in the case of entrapping, especially, in photo-crosslinkable resin. From the results of the fermentation for 23 h at 30°C of immobilized Z. mobilis NRRL B-14023 in GY-10 medium, GY-10 medium supplemented with 1.2% salts including K +, Na +, Ca 2+ and M g 2+ equivalent to the concentration of salts in 20% molasses, GY-10 medium supplemented with 1.5% salts, and a CM-20 medium of which the sugar concentration was 20% in molasses, using ENTG-3800, ENTV-500 and ENTV-500C, the fermentation in GY-10 medium was less than 50 g/l ethanol in every case, and the alcohol concentration was reduced by the addition of salts. The reduction was the smallest in both GY-10 and CM-20 medium in the case of ENTV-500C, which proved the best carrier in tolerance of salts. Alcohol fermentation was repeated batchwise up to five times, and the yields of alcohol after 6-h fermentation were compared. Figure 4 shows the relative yields, with the first fermentation yield represented by 100. The yields of alcohol after 24-h fermentation were the same as at 6 h. In the fermentation in a molasses medium, the fermentation rate tended to decline with the number of repeats. While no change in fermentation rate was observed when ENTG-3800 was used, the yield dropped with ENTV-500 or ENTV-500C. Although the reason for this trend has not been clarified, conceivable causes of the drop include

200

"~7o 0

RESULTS AND DISCUSSION

o

~80

O

rO 4J e~

~40

~a 20 lo o 15

20 Sugar

25

content

30

in m o l a s s e s ( % )

FIG. 5. Dependence of fermentation by Z. mobilis NRRL B14023 on sugar content in molasses at 30°C. Fermented by immobilized cells in ENTG-3800 after 24 h (©), and by free cells after 24 h ( o ) , and 167 h (A), respectively.

no salt-proof function, and the adsorption and accumulation of fermentation inhibitors, contained in molasses, on the carrier. These findings indicated that, for repeated or continuous fermentation in a molasses medium, ENTG-3800 was the most suitable carder in terms of the retention of fermentative activity and durability. Ethanol fermentation by free and ENTG-3800 immobilized Z. mobilis NRRL B14023 was carried out with varied concentration of molasses as illustrated in Fig. 5. The activity of free Z. mobilis decreased when the sugar concentration was higher than 20%, while that of immobilized Z. mobilis was retained at higher concentrations. The immobilized Z. mobilis generated 60 g/l of ethanol in 24 h after fermentation was started in molasses with a sugar concentration of 28%, while about 70°A based of fermentable sugar was available for ethanol in the medium with a sugar concentration of 15-23%. Observation of immobilized Z. mobilis Along with improving the functions of the carrier for immobilizing

o 150 O U e.

~ 100 r,

.~

i!ii

50

"-A

0

I

0

I

I

3 Number

of

|

5

cultivations

FIG. 4. Repeated ethanol fermentation by immobilized Z. mobillis NRRL B-14023 at 30°C. Z. mobil& was immobilized by ENTG-3800, ENTV-500, and ENTV-500C, indicated by circles, triangles, and squares, respectively; fermentation media were GY-15 (open symbols) and CM-15 (closed symbols). Ethanol concentrations after 6 h cultivation are shown, with the first fermentation yield represented by 100.

FIG. 6. Section of a Z. mobilis bead immobilized by photocrosslinkable resin ( × 50). After the growth of Z. mobilis in molasses, a section of a bead immobilized by photo-crosslinkable resin was observed by scanning microscope.

CHARACTERISTICS OF IMMOBILIZED ZYMOMONAS

VOL. 75, 1993

TABLE 1. Membrane potentials and transport numbers of photo-crossiinked resin sheets Photo-crosslinked resin ENTG-3800ENTV-500 ENTV-500C Membrane potential (mV) -5 -6 -10 Cation(K+) transport number t + 0.48 0.47 0.45 Anion (C1-) transport number t- 0.52 0.53 0.55 TABLE 2. Diffusion coefficients (D) of solute into photocrosslinked resin sheets D (cm2/s)x 10~

Photo-crossiinked resin sheet

Ethanol

Magnesium chloride

Glucose

ENTG-3800 ENTV-500 ENTV-500C

4.2 3.8 3.8

3.5 3.0 2.1

1.7 1.4 1.7

microbial cells, it is important to increase the multiplication of Z. mobilis in the carrier gel. With a view to examining how Z. rnobilis NRRL B-14023 cells were multiplied in ENTG-3800 gel, a cross-section of an immobilized microbial bead after the completion of continuous fermentation was observed with a scanning electron microscope. A cross-section could not observe by optical microscope, because the preparation of a sample was made difficult by the elasticity of photo-crosslinked resin gel. As shown in the 50-times magnified photograph in Fig. 6, the central part of the bead, a white circle, contained a concentration of Z. mobilis cells. This was probably because the inner part of the bead was less affected than the outer part by the ash in the molasses medium and dissolved oxygen in medium. Therefore, it was presumed that the immobilizing process might have the benefits of making the anaerobic bacteria more resistant to a high ash content and to contamination. Measurement of membrane potential The cation and anion transport number of photo-crossslinked resin membranes were calculated by Eq. 1 to determine the ion permeability in resin gel. The salt solution used for the measurement of the potential was KC1 solution. The potentials of the different photo-crosslinked resin membranes were measured, and the cation and anion transport numbers in each carrier gel were determined on that basis. The results are listed in Table 1. Each photo-crosslinked resin membrane manifested a negative potential with reference to the salts concentration C~ in the vessel A side. A K + ion transport number of less than 0.50, which was the transport number in water, revealed the selective anionpermeability of the membrane or its barrier function against cation permeability. The cation transport numbers of carrier membranes were lower in the order of ENTG3800, ENTV-500 and ENTV-500C. These results suggested the presence of a salt-proof function attributable to urethane, oxiamide and the aminoethylamide bond. Measurement o f diffusion coefficient In order to obtain the diffusiveness of the substrate and salts in the photo-crossllnked resin gel, the photo-crosslinkable resin was prepared in a membrane form, and the diffusion coefficients were measured with the apparatus shown in Fig. 3. For the solute, MgCI2, glucose and ethanol were selected as representative of salts in molasses, substrate and product, respectively. The diffusion coefficients of the photo-crosslinked resin membranes for these solutes are listed in Table 2. With every resin membrane, ethanol showed the highest diffusion coei~cient, followed by MgCI2

31

TABLE 3. Magnesium ion distribution in resin gels and liquids (~) Conc. of magnesium 13 chloride soin. (mg/dl)

144 (mg/di)

1300 (mg/dl)

ENTG-3800

Gel Liquid

16 84

17 83

23 77

ENTV-500C

Gel Liquid

53 47

56 44

52 48

Calcium alginate

Gel Liquid

79 21

67 33

55 45

Resin gel

and glucose in that order, which seems to be the same as the order of molecular weight. Consequently, fermentation in these resin gels must be not affected by product inhibition, because ethanol produced in gel rapidly moves out to the surrounding liquid. There were no appreciable differences in the diffusiveness of glucose or ethanol among these membranes, presumably because non-electrolytes such as ethanol and glucose, unlike electrolytes of MgCI2, were hardly susceptible to the ion-proof effect of the membrane. Measurement of salt distribution between gel and liquid Molasses used in this research contained 6.7% ash and 55% sugar, so molasses of 15-20% sugar concentration had ash of 2.3% and magnesium ions of about 0.1%. The cations such as calcium, potassium and magnesium ions in molasses were always distributed in both gel and liquid. To confirm the concentration of salts in the photocrosslinked resin gel, the distribution of magnesium ion in ENTG-3800 gel was measured and found to be one-fifth smaller than that in liquid, as shown in Table 3. On the contrary, the distribution of magnesium in ENTV-500C gel was almost equal to that in liquid and that of calcium alginate gel was larger than in liquid. From these results, it is seen that the photo-crosslinked resin gels, especially ENTG-3800, had a barrier against the diffusion of salts owing to the charge of the resin gel, and the structure suitable for cell growth. In conclusion, Z. mobilis immobilized by ENTG-3800 photo-crosslinkable resin showed a high fermentation rate, even in molasses, almost equal to that of yeast, suggesting that the resin carrier protected Z. mobilis from the effect of salts. The immobilization technique of this photo-crosslinkable resin method should be applicable to anaerobic bacteria to form a reaction field easily in resin gel. AKNOWLEDGMENT

This research was conducted by The Fuel Alcohol ResearchAssociation jointly with NEDO. REFERENCF~

1. Rogers, P. L. and Tribe, D. E.: Kineticsof alcohol production by Zymomonas mobilis at high sugar concentration. Biotechnol. Lett., 1, 165-170 (1979). 2. Rbee, S.K., Pagan, R.J., Lefebvre, M.F., Wong, L., u d Rogers, P. L.: Ethanol production from desalted molasses using Saccharomyces uvarum and Zymomonas mobilis. J. Ferment. Technol., 62, 297-300 (1984). 3. Lee, K.J., Skotaidd, M.L., Tribe, D.E., and Rogers, P.L.: The kinetics of ethanol production by Zymomonas mobilis on fructose and sugar media. Biotechnol. Lett., 1, 207-212 (1981).