Journal of Scientific & Industrial Research Vol. 62, November 2003, pp 1079-1085

Characterization of Yeasts for Ethanolic Fermentation of Molasses with High Sugar Concentrations

B K Bajajt *, Vikas Taank2 and R L Thakur2

I Department of Biotechnology, University of Jammu , Jammu 180006

2Department of Mi crobiology, SBS (PG) Institute of Biomedical Sciences , Bal awala, Dehradun 248 161

Received: 22 April 2003 ; accepted: 19 September 2003

Five yeast isolates obtained from mol assess ( I ,3 A and 3B) or jaggery (G I and G2) and three Saccharolllyces cerevisiae strains, S. cerevisiae MTCC 172, S. cerevisiae MTCC 174, and S. cerevisiae HA U- II were exami ned for their ethanol production ability in molasses with hi gh sugar concentrations and other desirable fermentati on characteri sti cs. Four strains, isolate 3B. S. cerevisiae HA U-I I, S. cerevisiae MTCC 174 and S. cerevisiae MTCC 172, gave hi gh effi ciency of eth anol production , i.e .. 71.0, 67.0, 66.7 , and 6 1.5 per cent, respectively, in concent rated mol asses (40 per cent sugars). Vi abilit y of the yeast strai ns was quite high in diluted molasses hut decreased drasti call y with increase in concentrati on of sugars in the medium and also by prolonged incubation. The four superi or strains (3 B, S. cerevisiae MTCC 172, S. cerevisiae MTCC 174, and S. cerevisiae HA U- I I) showed viability between 57 to 71 per cent in mol asses with sugar concentration of 35- 40 per cent. Although isolates G I and 3A produced maximum biomass but they were poor ethanol producers, while the lo ur strains (3 B, S. cerevisiae MTCC 172, S. cerevisiae MTCC 174, and S. cerevisiae HA U-II ) produced less biomass but yielded hi gher ethanol content. Further, these strains adapted faster in fermentation media with high sugar concentrations as co mpared to other yeast cultures. These 4 strains were found to be highl y ethanol tolerant (16 per cent, v/v), whi le rest of the strains could not grow in the presence of ethanol content of more than 10 per cent. None of the yeast strains studied showed high tl occul ence, it varied between I to 4.

Keywords: Yeast, Ethanol fermentation , Molasses, Ethanol tol erance, Flocculence, Adaptability

Introduction

Bioethanol (ethanol produced by fermentation) has emerged as the strongest candidate to be used as the potential renewable fuel source . Recent Indian government policy of blending ethano l @ 5-10 per cent in petrol has revived the interest in fermentative ethanol production among researchers and e thano l manufacturers . India produces approximately 3000 mL of ethano l annua ll y utilizing less than ha lf of its total installed capaci ty . There are nearly 300 distilleries where eth ano l is produced large ly by conventiona l batch process using dilute molasses or hydrolysed starchy material s as the subst rate and strains of yeast Saccharomyces cerevisiae as the fermenting organi sm. Molasses has been the most preferred substrate for industria l ethano l production, bu t blackstrap molasses which is used for ethanol production contains hi gh level of inhibitory

* Author for co rrespondence E-mai l: bkbajaj l @reditlmail.com

substances for yeast growth and fermentation that is why, it is used e ither in dilute concentrati ons ( 12- 17 per cent fermentable sugars) or afte r various pretreatments 1.2. These treatments make ethano l production from molasses an expens ive process. Further, fermentation of diluted molasses resul ts in the producti on of la rge quantities of e ffluent (approx. 12 LlL of abso lute e thano l), which is diffi cult to di spose off due to its hi gh BOD (60,000-80,000). One poss ible solution to thi s problem could be fermentation of worts of very high gravity [V ery Hi gh Gravity (VHG)-Fermentat ion Techno logy]. This technology has immense potenti a l due to many advantages like, increase in the plant efficiency, increased ethanol yield per unit of fermented broth , decrease in the total energy input , labour, capita l, cost and amount of efflu ent production. But most o f the yeas t strai ns employed at the indust ri a l level can not grow in and ferment molasses of high gravi ty because these conditions subject them to high osmot ic stress which reduce their growth and increase the loss of ce ll viability'.

1080 J SCI INO RES VOL 62 NOVEMBER 2003

It has been reported that if some osmoprotectants are added,-5 or yeast biomass is immobilized in ge l matrix6 the solu ti ons of high grav ity could be successfully fermented and thereby high concentrat ion of ethar:o l could be obta ined in the fermented broth . Although these strategies make the yeast to fe rment concentrated worts but the ir usage at industri al leve l seems impractical due to high cos t. Therefore, more appropria te is to develop or isolate such strains of yeas t, wh ich are capable of withstanding hi gh osmotic pressure in concentrated worts. Yeast iso lates from nature have been examined for their ability to ferment concentrated worts7

.8

. Also, attempts have been made to develop osmoto lerant strains by e lectrofusion or by protoplast fu sion

. ') 10 techniques' .

In the present study, yeast stra ins were isolated from sugary sources like, molasses or jaggery and exa mined for the ir ability to fe rment concentrated molasses solutions. Bes ides, other characteristi cs like viab ility, floccul ati on, adaptat ion , biomass forming ability, and ethanol tolerance were also studied.

Materials and Methods

Chemicals. Media and Media Components

Che mica ls, medi a or media components used in thi s study we re procured from companies like, Sigma Chemica ls Ltd , St Louis , USA; Difco Chemical Co ., Detro it , USA ; HiMedi a Laboratories Pvt Ltd, Mumbai , India ; Ranbaxy Fine C hemicals Ltd , DeTI hi , India; Qual igens Fine Chemicals, Mumbai , India ; and Merck & Co. , Inc ., USA.

Molasses was procured from local di stillery at Kuanwala, Dehradun. Total reducing sugars 111

I . d" . II II mo asses was es timate tItnmetnca y .

Yeast Strains

F ive yeast isolates, three from molasses (l,3A and 3B) and two from j aggery (G I and G2); one strain each of Saccharomyces cerevisiae MTCC 172 and Saccharomyces cerevisiae MTCC 174 procUl'ed from Microbial Type Culture Collec tion and Gene Bank, Institute of Microbia! Technology, Chandigarh ; and one strain of Saccharomyces cerevisiae HAU- II obtained from Department of Microbiology, SBS (PG) Institute of Biomedical Sciences, Balawala, Dehradun , were used in thi s study .

Isolation of Yeast

For isolating medium l2 was used .

yeast, mineral enrichment 20 g of molasses or jaggery

sample was inoculated in 150 mL of mineral enrichment medium in 250 mL Erlenmeyer f lasks and the fl asks were incubated at 25 °C on orbital shaker @

200 rpm for 48 h. The e nriched sample broth was then spread plated on YEPD agar (yeast ex tract 1.0 per cent, peptone 2.0 per cent, dextrose 2 .0 per cen t, and agar 2.0 per cent , pH 5 .0). After 24 h of incubat ion at 25 °C, co lonies appeared on the plates were picked up and examined microscopically and purified by restreaking. Isolates were maintained on YEPD agar slants at 4 DC with regu lar transfers.

F ermentatioH Studies

Molasses containing approxi mate ly 6 per cent sugars was used as inocu lam medium and fo r fe rmentation medium sugar concen trati on was adjusted to 20 to 40 pe r cent. Molasses medi a was always supplemented with urea (0 .3 per cent) and sodium dihydrogen phosphate (0. 15 per cent). To run the fermentati on experiment inoculam was deve loped by inocul at ing 24 h old ce ll s and incubat ing under shaking conditions (200 rpm). After 18-20 h, log phase biomass was inoculated into the fermentation medium @ 10 per cent (v/v) whic h equ als to ce ll

population of approximately 5 x 107/mL. Fermenta­ti on was conducted in fermentati on bottl es ( 1.25 L capacity) under stat ic conditi ons and e than ol content was determined colorimetrically , as described

. I 11 prevIOus y . .

Determination of Ethanol Tolerance'-I

Growth of yeast st ra ins was determined spectrophotometrically 111 the presence of exogeneously added e thanol uSing followin g equation :

Total growth = Xmax - Xo,

where Xmax = Maximum growth at particular time and Xo = Growth at '0 ' time.

Determination of Viability

Viability of yeast strains in different med ia was determined, using methylene blue method l5.

Measurement of Yeast Biomass

Biomass producing ability of yeast was determined by inoculating 24 h old ce ll s to YEPD medium (dextrose 2 per cent) and al lowing the m to grow for 48 h under shaking conditions at 30 DC. Cell suspension was then centrifuged and wet weight of the biomass was determjned.

BAJAJ el al. : CHARACTERIZATION OF YEASTS 108 1

Yeast Adaptation in Different Media

Log phase yeast biomass was inoculated into 100 mL mol asses fermentati on media contained in 125 mL fermentation bottl es and incubated statically at 30 °C. Fermentation bottles were observed at different time intervals for s igns of activity, I.e ., effervescence or boiling appearance.

Determination of Flocculence of Different Yeast Strains

Flocculence of the different cultures was measured by the method of Johnston and Reader

l 6.

The culture was grown in YEPD medium ( 10 mL) at 30°C fo r 48 h and the cells were pelleted out by centrifugation . The pe lle t was suspended in sodium acetate buffer (0 .05 M , pH 4.5) containing 5 mM calcium chloride and the suspension was mixed vigorously on vortex mixer and allowed to stand at room temperature for 30 s. Flocculation was observed and expressed on a subjective scale, ranging from zero (non-fl occulent with non visible floc s, totally turbid suspension) to five (highly flocculent with clearl y visible flocs) clearing of top 3.5 cm tube after 30 s.

Results and Discussion

Ethanol Production and Viability

Ethanol producing ability of all yeast strains was tested in molasses media with different sugar concentrations and results are shown in Table 1. High ethanol production effi c iency was found in diluted molasses and with increase in concentration of sugars

in the medium, e ffi c iency decreased . Four yeast cultures, isolate 3B, S. cerevisiae MTCC 172, S. cerevisiae MTCC 174, and S. cerevisiae HAU-II showed maximum ethanol production effi c iency, i.e., 86 .5 , 89.6, 93 .6 and 92 .5 per cent, respecti vely, in medium with 20 per cent concentrati on of sugars (Table I). However, in concentrated molasses with 40 per cent sugars, hi ghest ethanol production efficiency was shown by isolate 38 (7 1 per cent) followed by S. cerevisiae HAU- II (67 per cent), S. cerevisiae MTCC 174 (66.7 per cent), and S. cerevisiae MTCC 172 (6 1.5 per cent) . Max imum yie ld of e thanol was given by yeast isolate 38 ( 15 .27 per cent, v/v) fo ll owed by S. cerevisiae HAU- II ( 14.4 per cent), S. cerevisioe MTCC 174 ( 14.3), and S. cerevisiae MTCC 172 ( 13.2 per cent). Yeast isolate I could not grow in molasses with 40 per cent sugars. Rest of the iso lates showed poor ethanol producti on effi c iency in concentrated molasses. Reduction of ethanol yield in concentrated molasses may be attributed to multiple reasons, viz. , high content of saccharides in concentrated molasses worts results in increased osmotic pressure, which is detrimental for yeast. Hi gh gravity molasses has reduced water activity, which also inhibits yeast growth . Bes ides thi s, molasses has inhi bitory substances like, coloring matter and heavy meta l ions, concentrations of which are high in concentrated molasses and de leteriously effect yeast growth and fermentation 2

.

Fermentation of concentrated worts to get enhanced e thanol yie ld per unit of ferme nted broth has been attempted by many researchers. Ce rtain

Table I - Ethanol production by yeast strains in molasses media with different sugar concentrat ions"

Yeast strains Sugar concentration (per cent , w/v)

20 25 30 35 40

EY FE EY FE EY FE EY FE EY FE

7.88 73 .3 9.26 68.9 8.76 54.3 5.87 3 1.2

3A 8.1 5 75.8 9.24 68.7 9.3 1 57.7 8.24 43.8 6.77 31.5

3B 9.30 86.5 11 .08 82.4 12.83 79.5 14.04 74.6 15.27 71.0

GI 7.49 67.7 8.07 60.0 8.63 53.5 7.41 39.4 5.76 26.8

G2 7.8 1 72.6 9.37 • 69 .7 8.36 5 1.8 9.7 1 5 1.6 6.43 29.9

S. cerevisiae MTCC 172 9.64 89.6 11.70 87.4 12.20 75 .6 13.08 69.5 13.23 6 1.5

S. cerevisiae MTCC 174 10.07 93 .6 12.2 91.2 12.71 78 .8 11 .95 66.8 14.37 66.7

S. cerevisiae HAU- II 9.95 92.5 12.5 86.6 12.60 78.1 11 .58 67.6 14.4 1 67 .0

"EY= Ethanol yield (per cent v/v) FE= Fermentation efficiency (per cent )

1082 J SCI IND RES VOL 62 NOVEMBER 2003

osmoprotectants like, glyc ine, beta ine, pro line, soy fl our, soy bean, mea l castor o il seed meal or ground nut meal have been reported to enhance not only the abi lity of yeas t to successfu lly fe rment concent rated worts but a lso increase the fe rmentation effic iency of yeast H I2. 17. These additi ves a lthough gave des irab le

result s but cost in vo lvements a re so high that these a re not prac ti cable at industri a l leve l. Therefore, development of osmoto lerant yeast with ability to ferment concentrated worts w ith high effi c iency is sti II the need of the hour. Carid i ef ai. 7 have iso lated certa in Saccharomyces cerevisiae iso lates, which were capable of fe rmenting concentrated worts of 30-400 B. Simi la rl y, Kiran Sree e! al. B have obta ined a few isolates from natu re, w hich were capable of fermenti ng g lucose concentrati on of up to 35 per cent. Besides , atte mpts have been made to construct os moto le rant strai ns in the laboratory. Sa lek and Arno ld') have deve loped highly osmotoletant Saccharomyces cerevlSlae stra ins for e thanol production by e lectro fu s ion. Lucca ef ai. ID

have constructed osmoto le rant hybrids of Saccharomyces cerevisiae by protoplast fu sion with Toruiaspora de fbreuckii, an osmoto le rant stra in . Immobilization of yeast in so lid matrti x like, ca lc ium alginate ge l has been reported to protect the yeas t aga inst osmot ic stress and enhance the ir ability to fe rment concentrated worts of up to 30 per cent (w/v) .

V iabil ity of yeast cu ltures was hi gh in diluted molasses and reduced drastica lly w ith inc rease in concentrati on of sugars in the med ium (Table 2) . In

molasses medium with 40 per cent sugars viab ility was mini mum. Yeast stra in I cou ld not grow in mo lasses with 40 per cent sugars while Saccharomyces cereVlSlae MTCC 172 showed max imum viabili ty (62 per cent) fo ll owed by yeast iso late 3B (6 1 per cent), Saccharomyces cerevisiae HAU- I I (59 per cent) and Saccharomyces cerevisiae MTCC 174 (57 per cent). Rest of the s tra ins d ispl ayed still lower viability. Vi abi lity was found to decrease with prolonged incubati on, i.e., afte r 48 h viability declined further.

Loss of viability in concentra ted molasses is again due to high os motic pressure of medium with reduced water acti vity and inhi bitory substances of mo lasses.

Add iti ves li ke, g lyc ine, pro line, betai ne, soy flour, groundnut meal or castor oi I seed mea l are reported to he lp ma inta in hig h viab ility even in

d 14 1? 17 A I . b' i" t· concentrate worts ' " -' . so, Im1l10 I Izalion 0 yeast b iomass in calcium a lg inate ge l enhances the viab ility in concentrated worts by partl y re liev ing the osmotic st ress6

.

Biomass Fo rmillg Ability

Su ffic ient bio mass for mati on is an essentia l prerequI s Ite fo r e ffic ient etha no l producti on. M ax imum bio mass was produced by iso late G I, fo ll owed by 3A, G2, and Saccharomyces cerevisiae HAU-I I (Figure I). It has been reported that growth is linked to the produc t fo rmati on and incase of ethanol fe rmentation a parti a l re lati onship between

Tab le 2 - Viabili ty of yeast strai ns in molasses med ia wi th different sugar concent rations

Yeast strai ns Sugar concen tration (per cent. w/v)

20 25 30 35 40

Time (h), Viabi lity (per cent)

24 48 24 48 24 48 24 48 24 48

95 83 88 71 75 63 4 1 34

3A 93 81 81 75 76 67 47 38 40 35

3B 95 79 89 70 71 61 63 42 61 38

GI 93 75 91 73 67 66 49 35 39 34

G2 96 83 80 75 61 69 50 29 47 29

S. cerevisiae MTCC 172 95 85 89 78 73 72 66 47 62 42

S. cerevisiae MTCC 174 93 81 90 74 77 67 69 51 57 40

S. cerevi.l'iae HAU- I I 96 89 91 82 80 71 71 53 59 4 1

BAJAJ el al.: C HARACTERIZATION OF YEASTS 1083

growth and product formation ex ists. The yeast culture in the initial phase must build up sufficient biomass for efficient product format ion . Sharma and Tauro l8 and Bajaj et al. 19 have reported that slow growing yeasts are poor ethanol producers. However, in the present study we have reported that yeast strains G I and 3A produced maximum biomass but yie lded less ethano l. Thi s shows that bes ides growth rate, ethanol produc ing ability is governed by many other factors like, activity of key enzymes involved in ethanol production , (pyruvate decarboxylase, alcohol dehydrogenase, acetaldehyde dehdrogen ase), me mbrane composition and e thano l to lerance of the yeast strains and severa l other genetic factors which are still obscure, for the alcohol product ion is complex process controlled by large number of genes.

Adaptability of Yeast Stra ins

Adaptation of the organi sm in ' producti on medium has got profound influence on product

sc·s. cerev;s;ae

;t; ~- n ,., ~ ;;-=< ;:: R ,., ::

Y (list Strains - ~

Figure I - Biomass producing abi li ty of yeast strains

formation. Faster an organi sm adapts itse lf in the fe rmentation medium, more is the rate and yie ld of product. Yeast stra in 3B was first to adapt fo llowed by Saccharomyces cerevisiae HAU-II , Sacclw­romyces cerevisiae MTCC 172 and Saccharomyces cerevisiae MTCC 174 (Table 3). Rest of the strai ns took longer time for adaptation (up to 30 h in med ium with 20 per cent sugars). With increase in sugar concentration in the medium adaptation time increased due to obvious reasons that concentrated molasses is detrimenta l for yeast growt h and fermentation. Four stra ins, which produced higher ethanol contents, were able to adapt themse lves in 24-28 h in molasses with 35-40 per cent sugars while other strains took more than 38 to 40 h to adapt themselves. Yeasts abi I ity to adapt in concentrated worts depends primaril y upon the ir osmotolerance. Besides , molasses being crude substrate puts other type of stresses on the organism. Therefore, longer time is requ ired by the o rgani sm to acquaint itse lf in such an environment. It has been reported that if some additives are used like, soybean mea l or groundnut meal adaptability of yeast in concentrated worts could be enhanced by more than 73 per cent 15 . However, it is des irable that commercial yeast strain s should be hardy enough to tol e rate inhibito ry substances of molasses and shou ld adapt quickly without the a id of any supplements.

Flocculence of Yeast Cultures

Flocculence is a one of the most desirable features, which an indu stri a l strain must possess because it omits the need of centrifugation for the cell recovery after completion of fermentation , wh ich is

Tabl e 3 - Adaptation of yeast strains in molasses medi a with differen t sugar concentrations

Yeast st rains Sugar concentrati on (per cent), Adaptation time (h )

20 25 30 35 40

30 30 32 36

3A 30 30 36 38 38

3B 18 18 20 20 24

GI 28 30 34 36 38

G2 30 30 36 40 40

S. cerevisiae MTCC 172 24 24 26 26 26

S. cerevisiae MTCC 174 24 24 24 24 26

S. cerevisiae HAU- II 20 22 22 24 24

1084 J SCI IND RES VOL 62 NOVEMB ER 2003

otherwise a high-energy requiring process. Bes ides, biomass recyc ling can be practi ced with great ease when the stra in happens to be fl occulent one. F locculent stra ins for the industri a l purpose have been

. ?0 ? 1 N fl constructed by diffe rent workers- '- . one 0 t le yeast stra ins analyzed in the present study was found to be highl y fl occulent. F locculence varied be tween 1-4 (Figure 2).

Ethanol Tolerance

Maxi mum ethano l producing abili ty of yeas t reflects its ability to to le rate ethanol. For a yeast strain to produce high ethano l yie ld it must be able to to le rate hi gh concentrati ons of ethanol since ethano l is inhibitory for growth . Saccharomyces spp. is be lieved to be the maximum ethano l to lerant organi sm, Four stra ins whi ch produced higher ethano l yie ld were capable of to le rating up to 16 per cent ethano l while rest of the stra ins did not grow in medium with

.;. 3.5

'" 3 :( :::: 2.S c -; 1 -; ~ I.S o ~ I

0.5

~ ~ ,. .,

sc- S. ureJli.\'ia~

I !? D ". rt5 n

;:: ::: ... >-R ~

Yeast Strains -.> ~

Figure 2 - Flocculation abi li ty of di fferent yeast strains

ethano l content o f more than 10 pe r cent (T able 4). These observations are in line w ith earl ie r reports. Sake yeast which produce wines o f 20 per cent ethano l were found to be much mo re ethano l to le rant than brewers yeast whic h produce beers of 4-5 per cent ethano l22

. E thano l inhibition is linked with inhi bition and denaturati on o f glyco lyti c/fe rmentat ive enzymes and modi fication of cell me mbrane.

Membrane lipid compos iti on is mos t dras tica ll y e ffected by exposure of S. cerevisiae to e thano l and ethano l to lerance is very closely assoc iated with membrane lipid compOS ttl on, in parti cul ar the proportion of longer fa tty acy l res idues and ste ro ls with unsaturated a lkyl chains2:1 . Over express ion of OLE I gene enhanced the y ie ld and ethanol productivity of S. cerevisiae24 and thi s was supposed to be partly occurring due to increased unsaturated fatty ac id contents in the OLEI recomb in ant stra ins than wild stra ins which lead to the enhanced e thanol to lerance o f the organi sm and consequent improved growth and e thano l produc ing ability.

It is conc luded from thi s study th at yeas t stra ins, iso late 3B, Saccharomyces cerevisiae MTCC 172, Saccharomyces cerevisiae MTCC 174, and Saccharo­myces cerevisiae HAU-II are capab le o f ferme nting concentrated molasses successfull y, bes ides, these have got most of the other des irabl e cha racte ri st ics mandatory fo r an industria l stra in and could be of great commerc ia l importance. Furth er studi es on genetic bas is of osmotolerance are in prog ress .

Acknowledgements

Dr B K Baj aj is grateful to Director, SBS (PG) Institute o f Bio medical Sciences, Balawala,

Table 4 - Ethanol to lerance of di ffe rent yeast strains

Yeast stra i ns Ethanol concentration (per cent , v/v), GroWth 5411 nm

0 2 4 6 8 10 12 14 16

1.44 1.44 1.37 0.98 0.53 0.03 0.0 1 0.Q2 0.03

3A 1.46 1.43 1.39 1.2 1 0.46 0.23 0.04 0.06 0.04

3B IA9 1.48 IA I 1.24 1.0 I 0.74 0.50 0.2 1 0.19

G I 1.49 l AS 1.4 1 1.06 0.37 0.06 0.06 0.05 0.06

G2 IAI 1.37 1.38 1.1 9 0.69 0.38 008 0.04 0.04

S. cerevisiae MTCC 172 l AO 1.43 1. 28 1.1 0 1.02 0.75 0.46 0. 18 0. 16

S. cerevisiae MTCC 174 IA8 IA I 1.36 1.1 2 1.02 0.74 0.52 0.23 0. 10

S. cerevisiae HA U-II 1.43 1.42 1.1 8 1.1 5 1.02 0.76 0.53 0.26 0. 12

BAJAJ et ai.: CHARACTERIZATION OF YEASTS 1085

Dehradun , and Head, Department of Biotechnology, University of Jammu , Jammu , for all the necessary laboratory fac ilities.

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