continuous alcohol production from whey permeate using immobilised cell reactor systems
TRANSCRIPT
Continuous Alcohol Production from Whey Permeate Using lmmobilised Cell Reactor Systems Satwinder S. Marwaha* and JohnF. Kennedy
Research Laboratory for the Chemistry of Bioactive Carbohydrates and Proteins, Depurtnient of Chemistry, Universiry of Birmingham, PO Box 363, Birmingham B152TT, U K
*Present address: Department of Microbiologv, Punlab Agricultural University, Ludihiana- 141004, Punjab, India
(Manuscript received 15 November 1984: accepted3 December 1984)
The present paper reports the performance of a bioreactor packed with alginatc-entrapped Kluyveromyces marxianus NCYC179 for continuous fermentation of whey permeate to ethanol. A maximum ethanol productivity as 28.21 g I . ' h-' was attained at 0=0.42 h-' and 75% lactose consumption (substrate feed rate in the inflowing medium was 200glactose I- ' ) . However, the higher dilution rates ( 0 . 6 1 . 0 h - ' ) resulted in poor productivities and higher substrate washout in the effluent samples. The maximum specific ethanol production (qpi ) and maximum specific lactose uptake (qsi) of the immobilised Kluyveromyces marxianus NCYC179 was found to be 3.88 g ethanol/g immobilised cell/hxlO-' and 8.75 g lactose consumed/g immobilised cell/hxl0-* respectively. A bead size of 2.5nim in diameter and activation period of 24h of alginate beads in lactose solution (10%) prior to their packing in column reactor were found to support the efficient working of the bioreactor. The immobilised cell bioreactor system was operated continuously at a constant dilution rate of 0.15 h- ' and 10% lactose for 562 h without any significant change in the efficiency (varied from 84 to 88% of thcoretical) and viability of the entrapped yeast cells (dropped from 84 to Xl%).
Keywords whey permeate,
ethanol production, immobilised cell reactor, continuous fermentation
1 INTRODUCTION
I n previous papers,'-' ethanol fermentation of whey perme- ate in batch systems by immobilised yeast cells, the effect of immobilisation technique on the morphological characters of the culture, and the influence of fermentation conditions employed during the course of studies on the properties of gel, have all been described. In the batch process,' the experiments designed. included studies of the optimisation of the cell immobilisation technique, fermentation para- meters for maximising the productivity, and the feasibility of reusing the irnmobilised biocatalyst in repeat runs.3 The whey permeate medium containing lactose (lo%, w/v) as substrate was successfully subjected to ethanol fermentation with a conversion efficiency of 82% (theoretical);',' howev- e r , higher lactose levels inhibit the product formation by the immobilised cells. The analysis of the results suggest that whey, which is a low lactose-containing by-product of the dairy industry (contains up to 5% lactose), after concentrat- ing (to increase the lactose content) or supplementing with lactose, appears to be an ideal raw material for ethanol production. The promising results of the batch process, therefore, laid the foundation for the development of a process for continuous ethanol production from whey.
A literature survey reveals the applications of immobilised cell bioreactor systems for ethanol fermentation using molas- ses or whey permeate or other substrate as raw but the data reported in the literature are always insufficient to allow either the comparison of results, or for extrapola- tion of the results from one organism to another or from one substrate to another. The various pratices used to calculate the residence time of the lactose with the cells further complicates the problem. Therefore the prime objective of the present work was to standardise the process conditions for continuous ethanol fermentation while using immobilised Kluyveromyces marxianus NCYC179 cells. The specific details studied were: (i) determination of the optimal operating conditions for continuous ethanol fermentation, (ii) the influence of pre-activation of immobilised yeast cells
on the performance of the bioreactor, (iii) the effect of bead size on the conversion efficiency and stability of the entrap- ped cells, (iv) the effect of various lactose concentrations and dilution rates on the ethanol productivity, and (v) the effect of prolonged fermentation operation with immobilised cells on ethanol productivity.
2 MATERIALS AND METHODS
2.1 Organism
Kluyveromyces marxianus NCYC179 obtained from the National Collection of Yeast Cultures, Norwich, England, was maintained on lactose-agar slants as described in our earlier work.'
2.2 Medium The whey permeate supplemented with lactose (to increase the lactose content of whey to lo%, w/v, unless otherwise mentioned) and calcium chloride (0.025%, w/v), both sterilised at 121°C for 15 min, were used as the substrate for ethanol fermentation. The initial p H of the whey medium was adjusted to 5.0.
2.3 Immobilisation
The yeast cells used for immobilisation were cultivated in D-glucose-yeast extract medium in a 60-litre capacity Iuniglas Fermenter (Mason and Morton Ltd, England) for 36h at 30°C. The composition of the medium is given in Table 1. The cells were harvested by centrifugation using a Sharples centrifuge and were stored at 4°C. The yeast cells were immobilised in sodium alginate (2%) as described elsewhere.' Alginate beads (with entrapped yeast cells) of uniform size (average diameter of 2.5 mm, unless otherwise specified) and shape were prepared using a proportioning pump (Technicon Instruments), t o extrude the mixture from which the beads were produced.
60 BRITISH POLYMERJOURNAL,VOL. 17, NO. 1 1985
Table 1 Composition of o-glucose yeast ex- tract medium (per litre medium)
D-G h o s e 50 9 ("&SO4 5 9 Na2HP0, l g MgS047H20 0.1 g FeS047H20 0.001 g Yeast extract 5 9
2.4 Continuous fermentation
The fabricated fermenter (detailed schematic diagram is shown in a previous paper from our laboratory3) was a jacketed glass column (17.5cm height and 5cni diam.) fitted with stainless steel sieves at the bottom and in the middle (at a height of 10.6cm) to hold the alginate beads (with entrapped Kluyveromyces marxicanus NCYC179 cells) in a packed form. Steam sterilised whey permeate medium (containing 10% lactose, unless otherwise mentioned) was pumped fom the bottom by a peristaltic pump through silicon tubing (sterilised at 121°C for 30 min) at dilution rates ranging from 0.147 to 1.05h-' (flow rates 5&360rnlh-'). The column was packed with activated beads (activated by incubating in 10% lactose solution for 24 h prior to loading into the column reactor) loaded t o a cell density of 120 g dry weight I-'. Effluent samples were collected at each dilution rate at various time intervals and assayed for lactose (reducing sugar assay) and ethanol (gas chromatography) contents. A steady state of the reactor was assumed when the alcohol and reducing sugar concentrations levelled off as evidenced by three successive samples.
2.5 Optimisation of reactor operator parameters
The time course of ethanol production, lactose consumption and viability of the immobilised cells were studied by inoculating the lactose solution (lo%, w/v) with alginate- entrapped yeast cells, to find out the optimum activation period of the cells entrapped in alginate gel, and for attaining maximum efficiency and stability of their perform- ance in the continuous reactor system. By varying the size of the Tygon@ tubing and syringe needles for extruding yeast- alginate slurry through the proportioning pump, beads of 2.5-5.0mm in size were prepared to examine their stability and the effect of bead size on ethanol yield and ethanol productivity in the continuous process.
In our earlier work,' alginate beads loaded to a cell density of 40gdryweightl-' were found to utilise the lactose effi- ciently for ethanol fermentation u p to a concentration of 10% w/v lactose. Since the cell load in the beads employed for continuous process was increased to 120gdry weight I-', the experiment was designed to investigate the effect of high substrate levels (100-200 g I-' was tested) at various dilution rates (0.15-1.0h-') on the ethanol productivity of the immobilised yeast in a packed bed reactor. A continuous run was carried out for 562h, to investigate the operational stability of the reactor.
2.6 Analysis
The yeast viability at the start and during the course of fermentation was determined as detailed in an earlier paper.3 Alginate beads drawn at various time intervals from the reactor (through a sampling port a t the base of the column, Fig. 1 , ref. 3), were dissolved in sodium pyrophos-
phate solution (1 M) to which an equal amount of methylene blue (0.025, w/v) had been added. The non-viable cells stained blue, whereas the viable cells remained colourless. Cell counts were taken using a haemocytometer.
Reducing sugar contents of the whey medium and the effluent samples were determined by using the 3,5- dinitrosalicylic acid (DNS) assay.14
Ethanol concentrations in the effluent samples were deter- mined by gas chromatography using a Pye model 104 gas chromatograph fitted with a glass column (loft X -%6 in ext. diam.), packed with Phasepak P44/60 mesh. The tempera- tures of the column, injector and detector were kept at 200, 250 and 250°C respectively. Nitrogen was used as a carrier gas at a flow rate of 60 ml min-'. The combustion gases were hydrogen and air.
3 RESULTS AND DISCUSSION
Figure 1 depicts ethanol productivity, and ethanol and carbohydrate concentrations as a function of dilution rate. A maximum ethanol concentration of 44.4 g ethanol I-' was recorded at D=0.147h-', which declined slowly to 32.8 g ethanol l-' at D=0.42 h-' and finally dropped to ll.OgethanolI-' at D=1.05 h-'. The maximum ethanol productivity of 13.77 g I- ' h-' was achieved at D=0.42 h-' and 74% carbohydrate utilisation; the productivity decreased correspondingly with the further increase in the dilution rate. However, the minimum value for the productivity was obtained at D=0.147 h-' and complete, loo%, utilisation of carbohydrate. The residual carbohydrate content varied from
I W I I I I ' 0
0 075 0.30 0.45 0.60 0.75 0.90 1.05 Dilution rote, D (h-')
Fig 1 Ethanol concentration (0-O), residual carbohydrate (*---*) and ethanol productivity (x-X) as a function of dilution rate for immobilised cells of Khyveromyces marxianus NCYC179.
0.00-26.65 gl-' for D=0.147-O.42 h-' and increased sharply to 75gl-' at D=1.05 h-'. The ethanol productivity values recorded during the present studies are in line with the results of earlier worker^,^ who also have made similar observations and have obtained an ethanol productivity of 13.4gl-'h-', while using molasses syrups containing 10.5% reducing sugars as substrate.
The specific ethanol production rate (qp i ) and specific carbohydrate consumption rate (qsi) of immobilised Kluyveromyces marxianus NCYC179 cells as a function of dilution rate are presented in Fig. 2. Gradual increases in
BRITISH POLYMER JOURNAL,VOL. 17, NO. 1 1985 61
0 0.15 0.30 0.45 0.60 0.75 0.90 1.05 Dilution rate, D (h- ' l
-
Fig 2 Specific ethanol production rate (.---a) and specific lactose uptake rate (0-0) of immobilised K . marxianus NCYC17Y cells as a function of dilution rate.
100 U .- c e P g
- 7 5
A
-50 .E " .- - c -25 .S
r w 0
-0 0
qpi and qsi were recorded with the increase in dilution rate up to D=0.42 h- ' ; thereafter, values for both the functions declined. A maximum qpi value of 3.88 g ethanol/g immobil- ised cells/hx10-2 and a maximum qsi value of 8.75g ethanol/g immobilised cells/h X lo-* were recorded at 0=0.42h-'. However, in earlier work,13 with K. marxianus, higher values of qpi than our present results were recorded. The differences in qpi recorded can be reasoned on the basis that the substrate used for ethanol fermentation in our studies differs from the substrate used by other w ~ r k e r s ' ~ and also these substrates may be having different diffusion rates (transfer of substrate into the alginate beads carrying entrapped yeast cells and transport of product into the outer medium), because of difference in their molecular weights, thereby resulting in different specific ethanol production rates.
To find out the optimum period of activating the alginate- entrapped yeast cells, before loading them into the reactor for continuous ethanol fermentation, the alginate beads were incubated in lactose solution (10%) till the ethanol
W " I -
Incubation period ( h )
Fig 3 Time course of activation process of alginate entrapped yeast in a medium containing 10% lactose solution. Ethanol (0-0); residual carbohydrate (*---o), viable cell number (x-x).
production, lactose consumption and viable cell count level- led off. The course of the cell activation is shown in Fig. 3. The complete substrate utilisation and maximum ethanol production was observed at the completion of 18h of incubation. However, no significant change in the viability of the culture was recorded. On the basis of the above results a time period of 24 h was selected for activation of beads prior to their loading into the reactor. The activated beads were further found to reduce the non-steady state period in the continuous process. Activation periods of 20 and 36 h have been reported", '* optimum to regain the activity (of alginate-entrapped yeast cells) lost during 10 and 20 days of continuous ethanol fermentation, respectively, in packed bed reactor systems.
'To improve further the operation of the bioreactor, beads of different sizes (2.5-5.0 mm) were utilised to find out the optimum bead size. The data on ethanol concentration, carbohydrates consumption and conversion efficiency of the immoblised cells as a function of bead size are presented in Fig. 4. The beads of 2.5-3.5mm in size were found to have no effect on the ethanol production, carbohydrate consump- tion, ethanol yield (YP/S) and conversion efficiency (theore- tical) of the trapped culture. However, further increase (4.G5.Omm) in the bead size had an adverse effect on all the functions, which could be assigned to the mass transfer limitation (diffusion of substrate into the interior of big size beads and product out into the media). Moreover, disrup- tion of beads or slit formation in beads due to C 0 2 evolution were also recorded in beads ranging from 4.0 to 5.0mm in size. The results of this experiment suggest that the beads of small size are more stable and suitable for operating an immobilised cell reactor for prolonged periods. From the above results, beads of 2.5 mm in size were selected to carry out fermentation in future experiments.
In order to increase the productivity of ethanol, higher lactose concentrations (100-200 g I-') were employed. Fi- gure 5 shows data on ethanol productivity plotted against dilution rates at various lactose concentrations. An increase in productivity was observed at all the lactose concentrations tested, up to D=0.42 h-', but thereafter ethanol productiv- ity values declined very sharply. A maximum ethanol productivity of 28.21 g I-' h-' was attained at D=0.42 h-' and 75% lactose utilisation in a medium containing 20% w/v lactose. The low ethanol production recorded at higher dilution rates may be due to higher levels of substrate washout in the effluent (50-60% and 7.545% of the lactose offered was unconsumed at D=0.6 h-l and D=l.O - ' respec-
m Bead size (rnrnl I
Fig 4 Ethanol concentration (0-o), carbohydrate utilisation (*---*), ethanol yield YP/S (O---o) and conversion efficiency (X-X) of immobilised K . marxianus NCYC179 as a function of alginate bead size.
62 BRITISH POLYMER JOURNAL, VOL. 17, NO. I 1985
A
75
60
25
i
- 10 .5
- - 0.4 x-x-x+-x-x -X-X-X-X-X
- 0.3
- - 0.2
- 0. I
I I I 0
1 I I I J 0 0.3 0.6 0.9 1.05
Di lu t ion rate , D(hi)
Fig 5 Ethanol productivlty plotted against dilution rate at various lactose concentrations. Lactose concentration (g1-l); (*), 1OOg; ( 0 ) .
125g: (x), 150g; (0 ) . 175g; (A) , 200g.
tively). In our previous paper' it was reported that a higher substrate level (>lo% lactose) has an inhibitory effect on the conversion efficiency of both immobilised and free yeast cells in a batch system, but the above results (Fig. 5) reveal that, in continuous ethanol fermentation process, higher substate levels (100-2OOg I-') have no adverse effect on the efficiency of the organism. The ability of the culture to metabolise high lactose concentrations could be attributed to the increased cell density in alginate beads (in the batch system a cell load of 40gdrywt1-' was used, whereas in the present studies the cell density was 120gdry wt I- ' ) .
The stability of the immobilised cell reactor for prolonged
)I
6 loor
Time (h)
Fig 6 Continuous operation of immobilised cell reactor system at D=0.15 h-' and IOOglactose 1. I (0). conversion efficiency; ( O ) , viabil- ity; ( x ) , ethanol yield.
operation was investigated by monitoring the ethanol pro- duction and conversion efficiency as a function of time at a dilution rate of 0.15 h- ' and 100glactosel-'. The ethanol concentration in the effluent varied from 41.8 to 44.0 g I-' during the 562 h monitoring period. The values of conver- sion efficiency (84.03-88.23%, of theoretical) and the viabil- ity (84.29-81.03%) also showed very little change during the course of the continuous run (Fig. 6). However, slight leakage of cells and disruption of alginate bead was observed.
The ethanol productivities recorded while using whey lactose as substrate for ethanol fermentation during the course of the present investigation are comparable to the productivi- ties obtained with molasses (a conventional substrate) for alcohol p rod~c t ion .~ These observations suggest that the lactose from whey permeate can be used successfully as an alternate substrate for fuel grade alcohol production. The data on operational stability of the reactor further shows that the immoblised Kluyveromycs marxianus NCYC179 cells can withstand the operational conditions for prolonged periods and thus can be employed successfully for ethanol fermentation from whey. No doubt the results of the experiments carried out in our laboratory are encouraging, but for the scale-up of the process extensive investigation of reactor design is required to obviate the gas and biomass hold-up problems of the reactor.
4 ACKNOWLEDGEMENTS
S. S. Marwaha is thankful to the Commonwealth Scho- larship Commission in the UK and Ministry of Education and Culture, Government of India, India, for the award of a postdoctoral fellowship to carry out research in the Research Laboratory for the Chemistry of Bioactive Carbohydrates and Proteins, Department of Chemistry, at the University of Birmingham, Birmingham, England.
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