European J Appl Microbiol Biotechnol (1982) 14:216-219 Appl,ed Microbiology and Biotechnology �9 Spr inger -Ver lag �9 1982

Studies on Continuous Ethanol Fermentation of Sugar Cane Molasses

I. A System for Cont inuous Fermentation

Ake Haraldson 1 and Carl-Gustaf Ros6n 2

1 Department of Biochemistry and Biotechnology, Royal Institute of Technology, S-100 44 Stockholm, Sweden* 2 Alfa-Laval AB, Box 500, S-147 00 Tumba, Sweden

Summary. A new laboratory system for continuous fer- mentation is described. It is well suited for fermenting concentrated substrates such as moderately dilute mo- lasses. A rotating microporous filter, which is annexed to the fermentor vessel, allows the free escape of metabolic products while retaining yeast in the fermentor.

The slop is recirculated after removal of ethanol by distillation leading to a build-up of non-fermentables. The concentration of these and of yeast cells is checked by a controlled bleed. The described system is a useful tool for small-scale experiments on continuous ethanol fer- mentation.

Introduction

The fermentation of sugar to ethanol is carried out in a number of different ways. The most common route for producing ethanol by fermentation on an industrial scale is a batch process. Traditional methods generally suffer from drawbacks such as product inhibition and great con- sumption of process water and a concurrent effluent problem. New fermentation techniques have been design- ed to meet these problems, such as in the Vacuferm pro- cess (Boecheler 1948; Ramalingam and Finn 1977; Cysews- ki and Wilke 1977), in which the product is continuously removed under reduced pressure from a continuous stir- red tank reactor (CSTR).

Since the ethanol concentration is kept low, product inhibition is kept at a minimum, and concentrated sub- strates may be fermented, leaving a highly concentrated slop. Equivalent results may be reached by fermentation

* Present address: SWECO, P.O. Box 5038, S-10241 Stockholm, Sweden

Offprint requests to: A. Haraldson

at ambient temperature, if the yeast is retained in a closed loop, while the broth is recirculated through a heat ex- changer system, in which ethanol is continuously boiled off. An industrial scale process of this type has been de- scribed by Cook (1980).

Whereas separation of yeast, necessary for recircula- tion, is easily achieved on an industrial scale using gravi- tational or centrifugal sedimentation, continuous recovery of cells in a laboratory size fermentation unit is often a problem. In this paper we describe a bench-scale fermen- tation system, which incorporates a microporous filter for yeast retention and a distillation unit, which conti- nuously removes ethanol from the yeast free phase, which is recirculated to the fermentor. In a separate study the equipment has been used to investigate the influence of various parameters on fermentation and growth character- istic of yeast in continuous runs (Haraldson and Ros6n 1981)~

Fermentation Unit

The system, of which a simplified flow diagram is given in Fig. 1, has been built around a Chemoferm, High Yiel fermentor equipped for control of pH, temperature, aera- tion, oxygen tension, liquid level, and impeller speed. The separation device indicated in Fig. 1 is a cylindrical vessel into which a rotating, cylindrical, microporous stainless steel filter has been fitted. The filter is an MCS 1001 pHH type manufactured by Pall Trinity Micro Corp. and has a nominal pore size of 5/~m. The metabolic products in the broth are continuously removed through the rotating membrane, while the yeast cells can be retained inside the fermenting system (Margaritis and Wilke 1978).

The yeast free phase is transferred to the product stripping unit, where ethanol is removed as a 25-35% w/v vapour. Although fermentation and ethanol stripping are physically separated in this process, a return flow

0171-1741/82/0014/0216/$01.00

A. Haxaldson and C.-G. Ros6n: System for Continuous Ethanol Fermentation 217

Jtt

Fig. 1. Schematic diagram of the fermentation system

(I/h) t. i w I t

FIo_ E~ STRIPPERB I s

Fig. 2. Fluxes of the fermentation system

from the product stripper back to the fermentor per- mits control of the ethanol concentration in the fermen- tor, independent of the concentration of substrate in the feed. Inert material flow through the system is balanced by bleed-off from the stripper.

The equipment is designed for continuous fermenta- tion under automatic control with a minimum of atten- dance. The rotating filter was placed in a separate vessel outside the fermentor in order to make inspection, clean- ing or change of the filter simple and without interrupt- ing the fermentation. Particularly with molasses as sub- strate but also because yeast cells and fragments tend to clog the pores, the filter has to be changed about every 24 h.

The separation efficiency - normally around 90 per cent - is dependent on rotation speed as well as flow rate through the filter. Since the density difference between cells and medium is also of importance, recirculation of cells is possible only with broths containing less than about 20 per cent dry substance. Partial clogging of the triter also decreases the efficiency due to the fact that the flow rate through the active parts of the filter incre- ases. A detailed analysis of separation by means of a ro- tating filter ~ be presented elsewhere.

Characterization of the System

For the quantitative study of ethanol yield, yeast growth, sugar consumption, accumulation of non-fermentables,

etc. it is useful to characterise the system in terms of a few equations, which describe mass balance involved.

The fermentor proper and the filter unit may for all practical purposes be regarded as a single, well-mixed fer- mentor. The volume of this fermentor, VF, approximates the volume of the entire system. Substrate with concen- tration So g/1 enters the system at a rate of F 1/h (Fig. 2). Fluid leaves the system as distillate and slop (bleed), i.e. under steady state conditions

F = L + B (1)

where L and B are distillate and slop flow rates, respec- tively.

Although the system is in principle closed as regards ceils, a certain bleed of cells is necessary to balance growth and to allow the escape of dead cells. A certain proportion of cells is (unavoidably, c.f. Haraldson 1981) allowed to escape through the filter into the distillation unit, where they are destroyed.

Since no cells enter the system, the overall cell mass balance is described by

dX P X F V F - EX~ =G'c-VF (2)

where p is the specific growth rate h - 1, E is the flow rate (l/h) from the fermentor to the distillation unit, XE is the yeast concentration (v/v) in that flow, and XF the yeast concentration in the fermentor.

To maintain steady state conditions, growth has to compensate for cells lost not only by escape through the filter but also for ceils which die in the fermentor, a pro- cess which is accelerated under the stressed conditions, which prevail for instance when the osmotic pressure is high. The necessary yeast growth may be calculated from Equation (3):

P X F V F = E X E + a X F V F (3)

where a is the specific death rate (killed cells/-total num- ber of cells, h).

218 A. Haraldson and C.-G. Rosgn: System for Continuous Ethanol Fermentation

t g 30- g ul

~, 2 0 -

10-

o 55 ~o I~O 26o 3o0 Hours

Fig. 3. Theoretical accumulation of non-fermentables for different distillate: bleed ratios by continuous fermentat ion o f 40 ~ Bx molasses. Dilution rate 1.71 1 0 - 2 . x, distillate : bleed, 1 : 2; o, dis- tillate : bleed, 1 : 1; +, distillate : bleed, 2: 1

In a typical run XF may be 5% (v]v), VF = 141, E = 21 h -1 , X E = 0 .5% (v/v) , and a = 0.01 h -1 . By means of Eq. (3), a necessary growth rate of 2.4% h- 1 is calculated from these values.

The product, i.e. ethanol, leaves the system predomi- nantly as distillate but also, to a minor degree, in the bleed. The mass balance on product is thus expressed as

dP VVF -- LPL - BPB =--77, VF

a t (4)

where v is the volumetric productivity (g 1-1 h - 1), and PL and PB are the product concentrations in the distillate and the bleed, respectively.

Similarly, the mass balance pertaining to substrate is expressed as

dS FS F - BS B - / ] V F = -;zrV F

( I t (5)

As stated in the Introduction, one of the advantages of the type of fermentation system here under study is that it leaves a concentrated slop. The crucial problem of yeast survival and metabolism under conditions of the re- sulting low water activity has been presented as a separate study (Haraldson and Ros6n 1981). From the point of view of process design it is necessary to describe the accu- mulation of non-fermentables taking place in the system described in this paper.

When steady state conditions have been reached

FIo = BIB (8)

assuming that the only escape of non-fermentables is in the bleed and that no non-fermentable end products are formed in the fermentation. I o and I B are here the Con- centrations of non-fermentables in the feed and bleed, respectively. In the initial phases of a continuous run, however, non-fermentables are accumulated as describ- ed by

dlF VF = F I o - B I B (9) dt

If the initial concentration of non-fermentables is Is, integration between 0 and t gives an expression from which the concentration of non-fermentables may be calculated as a function of fermentation time.

In Fig. 3 this accumulation has been plotted against duration of a continuous fermentation for a number of distillate: bleed ratios, other parameters being selected to represent a typical continuous fermentation. It is seen that the concentration of non-fermentables increases for the first 10-15 days but levels off att about 20-30 per cent, a concentration range, where sugar is still effi- ciently converted into alcohol (Haraldson and Bj6rling 1981).

where SF and SB are the substrate concentrations in the fermentor and the bleed, respectively, and/3 is the sub- strate consumption (g 1-1 h - 1),

Two dimensionless values may be defined, which ex- press the fraction of substrates consumed for yeast growth and product formation, respectively. These are the cell yield factor, Yx/s, and the product yield factor, Yp/s:

dXF dt VF + EX~

Yx/s - (6) FS o - BSB

d p__P VF + LPL + BPB dt

Yp/s = FS o - BSB (7)

Conclusions

The fermentation system described in this paper is suit- able for continuous fermentation of concentrated sub- strate with simultaneous product removal. Non-fermen- tables are accumulated until a steady state level of 20-30% is reached. This represents a considerable re- duction in process water and a corresponding reduction of sewage disposal problems, which are considerable in distilleries (Jackman 1977).

The rotating filter proved to be a suitable device for continuous yeast separation on a laboratory scale. Al- though the efficiency of the filter is not as high as might be desired, it represents a good compromise between efficiency, non-clogging properties, and care of handling. The periodic, simple removal of the filter unit for clean-

A. Haraldson and C.-G. Ros6n: System for Continuous Ethanol Fermentation 219

ing keeps it f r o m clogging, which is r epo r t ed ly the main

p r o b l e m wi th similar system. The 90 per cent r e t en t ion

o f cells results in an average residence t ime o f 40 hours .

Acknowledgements. The authors are grateful to L. MSrtsell for valuable advice and assistance with the instrumentation. This work was supported by a grant from the National Swedish Board for Technical Development.

Nomenclature

B = bleed rate, l/h, E = flow rate from filter to distillation unit, l/h; F = feed rate, l/h; I B = concentration of non-fermentables in the bleed; I F = concentration of non-fermentables in the fermen- tor; 10 = concentration of non-fermentables in the feed; I S = concentration of non-fermentables at start-up; L = distillate flow rate, l/h; PB = product concentration in the bleed, PL = product concentration in the distillate; S B = substrate concentration in the bleed; S F = substrate concentration in the feed; V F = fermen- tot volume, 1; X E = cell concentration in the effluent; XF = cell concentration in the fermentor.

References

Boecheler BC (1948) US Patent No 2,440,925 Cook RM (1980) BIOSTIL. A process for the production of

ethanol from concentrated substrates. Bio-Energy 80, Atlan- ta, Georgia, USA

Cysewski GR, Wilke CR (1977) Ral~id ethanol fermentations using vacuum and cell recycle. Biotechnol Bioeng 19: 1125-1143

Haraldson A, Bj6rling T (1981) Yeast strains for concentrated substrates. Eur J Appl Microbiol Biotechnol 13:34- 38

Haraldson A, Ros6n CG (1982) Continuous alcohol fermenta- tion and product removal in a laboratory scale plant. Eur J Appl Microbiol Biotechnol 14:220-224

Jackman EA (1977) Distillery effluent treatment in the Brazilian national alcohol Programme. The Chemical Engineer, pp 239-242

Margaritis A, Wilke CR (1978) The Rotorfermentor. I. Descrip- tion of the apparatus power requirements and mass transfer characteristics. Biotechnol Bioeng 20:709-726

Ramalingam E, Finn RK (1977) The Vacuferm Process. A new approach to fermentation alcohol. Biotechnol Bioeng 19:583-589

Greek letters

c~ = specific death rate h - 1 ; 13 = specific substrates consumption h - 1 ; v = specific productivity h - 1 ; # = specific growth rate h - I. Received December 23, 1981