Appl Microbiol Biotechnol (1984) 20:100-104

Ethanol production in a hollow fiber bioreactor using Saccharomyces cerevisiae

Mohamed A. Mehaia and Munir Cheryan

Department of Food Science, 382D Agricultural Engineering Sciences Building, University of Illinois, 1304 W. Pennsylvania Avenue, Urbana, IL 61801, USA

Summary. A new approach for continuous pro- duction of ethanol was developed using a Hollow fiber fe rmentor (HFF). Saccharomyces cerevisiae cells were packed into the shell-side of a hollow fiber module. Using 100 g/1 glucose in the feed gave an op t imum ethanol productivity, based on total H F F volume, of 40 g ethanol/1/h at a dilution rate of 3.0 h -1. Under these conditions, glucose utilization was 30%. However , at 85% glucose utilization the productivity was 10 g ethanol/1/h. This compares to batch fe rmentor productivity of 2.1 g ethanol/1/h at 100% glucose utilization.

Introduction

As world pe t ro leum reserves are depleted, new sources of carbon and hydrogen must be found to supply our chemical and energy needs. Large quan- tities of biomass are available in most parts of the world and could be used as an energy mechanism or as raw material for chemical manufacturing.

The ancient art of alcohol fermentat ion has been documented for many years, and the well-known fermentat ion processes for converting sugar to alco- hol by using yeast and bacteria can be found readily in the past and current literature. Several new techni- ques have been developed for economic continuous ethanol production. These include immobilized mi- crobial cells (McGhee et al. 1982; Ghose and Bandyophadhyay 1980; Wada et al. 1980; Margaritis et al. 1981), cell recycle (Cysewski and Wilke 1977; Ghose and Tyagi 1979; Rogers et al. 1980) and vacuum fermentat ion (Ramal ingam and Finn 1977; Cysewski and Wilke 1978). An alternate approach involves the use of synthetic semipermeable mem- branes in the appropriate configuration to either

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continuously separate and recycle the biocatalyst (Mehaia et al. 1984; Cheryan and Mehaia 1983; Deeslie and Cheryan 1981) or to entrap it within the membrane separation module itself (Inloes et al. 1983; Mehaia and Cheryan 1984; Kohlwey and Cheryan 1981; Roy et al. 1982). This paper discusses the development of a plug-flow type of membrane bioreactor, based on hollow fiber membrane tech- nology, for the continuous fermentat ion of glucose to ethanol.

Materials and methods

Microorganism and feed medium. Saccharomyces cerevisiae NRRL-Y-132 was obtained from the U,S. Department of Agri- culture, Peoria, IL, USA. It was maintained on malt extract-yeast extract-glucose-peptone (MYGP) slants (3 g malt extract, 3 g yeast extract, 50 g glucose, 5 g peptone and 20 g agar in 11 of water). All ingredients were obtained from Difco Laboratories, Detroit, Michigan, USA, Table i gives the composition of the medium used in our studies.

Batch fermentation. The batch experiments were performed in a New Brunswick Microferm Fermentor using 2 1 volume. The

Table 1. The composition of the medium a

Compound Amount g/1 of tap water

Glucose (anhydrous) 100 Yeast extract (Difco) 8 NH4C1 8 Na2HPO4 • 7 H20 3.9 KH2PO 4 0.5 MgSO4 0.5 CaC12 0.28 Citric acid b 4.3 Sodium citrate b 1.25 Antifoam (Sigma, no. 5758) 0.5 ml

a All salts and glucose reagent grade b Citric-citrate buffer at pH 4.0

M. A. Mehaia and M. Cheryan: Hollow fiber fermentor 101

fermentor and its contents were sterilized in an autoclave at 120 ° C for 15 min. The medium was sterilized through a 0.2 ~tm microfilter (Gelman Acroflux Capsule) and added to the fermentor aseptically before inoculation. Nitrogen was passed through the fermentor vessel to maintain anaerobic conditions during these experiments. All experiments were conducted at 28 ° C.

Hollow fiber fermentor (HFF). The overall scheme of the hollow fiber fermentor system is shown in Fig. 1. There were basically two stages in the system: a sterilization stage, where the medium is "cold sterilized" by passing it through a cross-flow 0.2 ~tm microfilter (Gelman Acroflux Capsule) and a fermentation stage, where the sterilized medium was converted to ethanol in the hollow fiber fermentor. The hollow fiber fermentor was a hollow fiber module (currently designated the "short-short" hollow fiber cartridge by the manufacturer, Romicon, Inc., Woburn, MA, USA). It contained about 660 fibers each with a 50,000 molecular weight cut-off. Membrane surface area was 0.7 m 2. The shell-side volume was 430 ml, while the fiber volume was 195 ml, giving a total volume of 625 ml. The HFF was loaded with yeast cells by "backflushing" the unit through inoculation port P1 with a yeast suspension that had been grown on MYGP broth as described earlier (Mehaia et al. 1984).

Cleaning procedure. After operation the hollow fiber module was submitted to the following cleaning cycle: (1) rinse with about 201 of distilled water; (2) backflushed with about 10 1 of distilled water; (3) cleaned with Tergazyme (Alconox Inc., New York, USA) solution overnight; (4)rinsed with distilled water for 15rain; (5) stored in 1% formaldehyde. The hollow fiber module was rinsed with 20 1 of sterile water before re-use. A hollow fiber cartridge can be used several times. This particular cartridge has been used 50 times.

Analyticalprocedures. Ethanol concentration was measured by the alcohol dehydrogenase method as described in Sigma Technical

Feed

FEED TANK

Product BALANCE TANK Bleed

Sterilized ~ _ ~ ! !PI Medium X

Hollow Fiber

~c: Sterilization )1~: Fermentation

Fig. 1. Schematic of the hollow fiber fermentor

Bulletin No. 332-UV (Sigma Chemical Co., St. Louis, MO) and by gas chromatography as described earlier (Mehaia et al. 1984). Glucose concentration was determined by the dinitrosalicylic acid (DNS) method (Summer and Somero 1949). Cell concentration is expressed as yeast cell dry weight per unit volume (g/l). The cell concentration was measured optically at 525 nm and converted to g/1 units using a calibration curve.

Results and discussion

Batch fermentation

Table 2 shows batch fermentation results using 100 g/1 glucose. With an initial cell concentration of 2.6 g/1, fermentation was completed in about 24 h and the final ethanol concentration was 49.5 g/1 (about 6.1% v/v). Productivity varied from an optimum of about 4.3 g ethanol/1/h at 11 h of fermentation to about 2.1 g/1/h at the end of the fermentation. Maximum specific growth rate was 0.22 h -1 and the yield of conversion of glucose to ethanol was 95% of theoretical yield.

Continuous fermentation using HFF

All data reported here for HFF where obtained when the system reached steady-state conditions, after five reactor volumes of medium passed through the system. This is adequate since this bioreactor prob- ably approximates plug-flow behavior (Kohlwey and Cheryan 1981).

For comparison purposes, ethanol productivities for the HFF were evaluated using two different reactor volumes, as suggested by Inloes et al. (1983). In one approach, productivity was expressed in terms of the fiber volume (Fig. 2B). The fiber volume is calculated from the fiber outer diameter, the number of fibers and the fiber length between the epoxy end-seals. On this basis, the optimum productivity was 126 g ethanol/1/h. In the other approach, the total reactor volume was used, which includes the fiber

Table 2. Batch fermentation by Saccharomyces cerevisiae using 100 g/1 glucose

Time Glucose Ethanol Biomass (h) concentration concentration (g/l)

(g/l) (g/l)

Yp/s a Productivity (g/l/h)

0 100 0.0 2.6 - 0.0 2 91 4.4 2.9 0.49 2.2 4 76 11.2 3.7 0.47 2.8 7 42 28.4 7.1 0.49 4.1

11 0 47.9 8.6 0.48 4.3 19 0 49.0 9.5 0.49 2.5 24 0 49.5 9.4 0.49 2.1

a Theoretical ethanol yield (YP/s) = 0.51

102

volume and the shell-side volume accessible to yeast cells between the epoxy end-seals. On a total volume basis (Fig. 2A), optimum productivity was 40 g/1/h at a dilution rate of 3 h -1. However, glucose utilization at this point was only 30%. Reducing dilution rate to 0.25 h -1 resulted in an increase in glucose utilization to 85% but reduced the ethanol productivity to 10 g/1/h.

4O

2c

N

8 0

_= 6 0

, i

80 A

£

, ~ , ~ , ~ ,

B

20 • Ethanol •

i I ) I I i , i ' 0 1 ' 2 O0 2 4 6 8 1

Dilution Rate (D), h - 1

40

30

20

J=

"r. i 60

2 0 m

8 0

4 0

Fig. 2A, B. Fermentation kinetics of hollow fiber fermentor in continuous (single-pass) mode. Feed was synthetic glucose medium (100 g/l). Initial cell concentration = 100 g/1. 0 , Glucose; A, ethanol; vq, productivity. A Calculation of productivity based on the total volume of the fermentor. B Calculation of productivity based on fiber volume

M. A. Mehaia and M. Cheryan: Hollow fiber fermentor

Figure 3 shows data from an experiment evaluat- ing the long-term stability of the HFF. Feed con- centration was 100 g/1 glucose and the dilution rate (D) was 0.5 h -1 (expressed in term of total HFF volume). The productivity during the long-run was about 17 g/t/h and glucose utilization was 66%. The shell-side was initially loaded with 100 g/1 yeast. After 93h of operation, the yeast concentration had increased to about 260 g/l, which probably represents an extremely close-packed arrangement in the shell-side (Inloes et al. 1983). This led to poor cell-substrate contact, inadequate mixing and rela- tively low productivity. However, productivity was still better than conventional fermentation systems (batch, continuous culture), see Table 3.

Table 3 is a summary of ethanol productivity data obtained using different fermentation systems. It is

.~ 100

80

-6

.= 60

40

8 20

PD a---O"

• • Ethanol - - i - - - - m

Glucose

' 1 'o ' ~o ' ~o' ~o '5~ ' go ' / o , I , 0 |

80 90

Time (h)

2 0 .=,

lo -'¢

2

Fig. 3. Long-term stability of hollow fiber fermentor. Feed was synthetic glucose medium (100 g/l). Initial cell concentration was 100 g/1 and dilution rate was 0.5 h -1. O, Glucose; A, ethanol; [3, productivity

Table 3. Comparison of ethanol productivity from different fermentation systems (based on total bioreactor volume)

System Feed Sugar Microorganism Ethanol Sugar Produc- concen- concert- utili- tivity tration tration zation (g/l/h) (g/l) (g/l) (%)

Reference

Batch Glucose 100 S. cerevisiae 49 100 2,5 Glucose 250 Z. mobilis 102 80 3,4 Lactose 150 K. fragilis 75 100 3,5

Continuous Glucose 160 S. cerevisiae 31 38 4.1 culture Glucose 100 Z. mobilis 40 80 8.0

Immobilized Glucose 197 S. cerevisiae 71 74 25 Glucose 100 Z. mobilis 44 97 29 Glucose 150 Z. rnobilis 74 98 57

Membrane Glucose 100 Z. mobilis 44.5 90 120 recycle Glucose 100 S. cerevisiae 49 100 100 fermentor Lactose 150 K. fragilis 71 97 75

Hollow Lactose 50 K. fragilis 24 100 24 fiber Glucose 89 S. cerevisiae 12 27 26 fermentor Glucose 100 S. cerevisiae 40 85 10

This work Rogers et al. (1980) Mehaia and Cheryan (1984)

Ghose and Tyagi (1979) Rogers et al. (1980)

Ghose and Bandyopadhyay (1980) Margaritis et al. (1981) Klein and Kressdorf (1983)

Rogers et al. (1980) Mehaia and Cheryan (1984) Cheryan and Mehaia (1983)

Mehaia and Cheryan (1984) Inloes et al. (1983) This work

M. A. Mehaia and M. Cheryan: Hollow fiber fermentor 103

apparent that significant improvements in productiv- ity can be made using continuous systems, especially immobilized cell and membrane bioreactors. Among membrane bioreactors, the membrane recycle fer- mentor appears to perform better than the hollow fiber fermentor. This could be due to several practical problems that arise during the operation of hollow fiber fermentors. Because the substrate and the biocatalyst are separated by a barrier, the rate-lim- iting step becomes the diffusion of substrate into the shell-side and diffusion of products back into the lumen (tube) side. We attempted to minimize diffusion limitations by applying a back-pressure of 70-100 kPa (10-15 psig) in the feed stream and leaving Port P2 slightly open to simultaneously remove the gas and a small fraction (about 1 - 5 % ) of the shell-side contents. Visual inspection, however, indicated that the yeast cells were mostly attached to the fibers or were localized to an annular volume just around the fibers, similar to what was observed by Roy et al. (1982). Thus our calculated productivity would have been much higher if we had used the "active" fermentor volume or the "fiber" volume instead of the total reactor volume to calculate dilution rate.

The continually growing mass of cells gives rise to several problems. Since the cells far from the fibers were probably starved of nutrients and dying or undergoing lysis, cell debris must be continuously removed, such as through our bleed stream through Port P2. If not, the high cell density will eventually give rise to pumping problems and/or rupture the relatively sensitive fibers (Roy et al. 1982; Inloes et al. 1983). Attempting to slow down cell growth by limiting essential nutrients (Roy et al. 1982; Inoles et al. 1983) is counterproductive in growth-associated Gaden Type-I fermentations since it reduces the activity of the microorganisms, and thus lowers bioreactor productivity.

When evaluating the data in Table 3, it should be borne in mind that Zymomonas mobilis has been reported to ferment sugars more rapidly and result in higher ethanol concentrations than yeasts (Rogers et al. 1982). Also, the higher productivity obtained with HFF in our earlier study with Kluyveromyces fragilis was due not only to higher sugar concentration (150 g/1 lactose) but also because we used a hollow fiber cartridge with a much higher fiber density (2,150 fibers vs 660 fibers in this study). This resulted in a higher surface area-to-volume ratio (17.8 cm2/cm 3) than the cartridge used in the present trials (11.2 cm2/cm3), which probably improved substrate-cell contact and led to improved productivity.

The performance of a hollow fiber fermentor for ethanol production can be improved by decreasing the flow rate of the glucose medium, applying some

back-pressure in the feed stream (to minimize diffusion limitations), continuously removing the gas and some of the shell-side content (to improve mixing) and by increasing the fiber density (a higher surface area-to-volume ratio) in the cartridge. Because of problems discussed above, the hollow fiber fermentor is not as efficient as membrane recycle fermentor. Unless these problems can be worked out, the hollow fiber fermentor will not be practical for ethanol fermentation with real feeds (of "natural" origin) since these problems may be magnified.

Acknowledgement. This research was supported in part by the Illinois Agricultural Experiment Station, University of Illinois, Urbana, USA.

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Received November 18, 1983