Characterization and continuous production of ethanol

using immobilized yeast cells

Kethineni Chandrika, S. F. Choragudi, Ravipati Srimukhi and Rusumdar Salma

Department of Biotechnology, Koneru Lakshmaiah Education Foundation, Guntur, India

ABSTRACT

In this study, characterization of the immobilized yeast cells for ethanol production by varying the initial sugar and

yeast cell concentration was performed. The results have indicated that the effective fermentation can be achieved

using 1% of the immobilized yeast cells with 10-12% of initial total reducing sugar concentration. The role of plant

and fungal based chemicals as activators were studied and the studies indicated that Chitin and Rhizopus Oryza bio-

mass has shown signi cant effects of increase in fermentation rate (47% and 23.94%) in free and immobilized cell

–activator systems respectively. Continuous ethanol production studies with immobilized yeast cells suggested that

the productivity can be improved up to 100% by reducing retention time. Also, these studies con rmed the reusability

of beads for up to 16 days without losing activity.

KEY WORDS: ETHANOL FERMENTATION, MOLASSES, IMMOBILIZATION, ACTIVATORS, REDUCING SUGAR

571

Biotechnological

Communication

Biosci. Biotech. Res. Comm. 11(4): 571-576 (2018)

INTRODUCTION

The gradual depletion and the environmental deteriora-

tion resulting from the over consumption of petroleum

derived transportation fuels have gained great attention

across the world. Hence, it is necessarily important to

develop alternatives that are both renewable and envi-

ronmentally friendly. Ethanol is one of the most exotic

synthetic oxygen-containing organic chemicals. It has

unique properties as a fuel, a solvent, a beverage, a ger-

micide, an antifreeze that reduces greenhouse gas (GHG)

emission, lessens the dependence on fossil fuels, and

improves vehicle performance. It is generally manufac-

tured by three types of agricultural raw materials viz.,

saccharine materials, starchy materials and lignocel-

lulosic biomass using bacteria and yeast. Fermentation

using yeast and sugarcane molasses is one of the widely

used and ancient fermentation systems.

Despite the great advantages and importance, fuel

ethanol is not competing to cheaper oil derivatives

ARTICLE INFORMATION:

Corresponding Authors: kkchandrika@kluniversity.in

Received 1

Nov, 2018

Accepted after revision 21

Dec, 2018

BBRC Print ISSN: 0974-6455

Online ISSN: 2321-4007 CODEN: USA BBRCBA

Thomson Reuters ISI ESC / Clarivate Analytics USA

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NAAS Journal Score 2018: 4.31 SJIF 2017: 4.196

Online Contents Available at:

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DOI: 10.21786/bbrc/11.4/6

Kethineni Chandrika et al.

572 CHARACTERIZATION AND CONTINUOUS PRODUCTION OF ETHANOL BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

because of its high production costs due to low pro-

ductivity. Hence, attempts cells, immobilization of whole

cells, vacuum fermentation and optimization of operat-

ing parameters to reduce production costs. Several stud-

ies show that ethanol productivity can be increased by

the addition of ergosterol (Patil, 1985, Kanellaki 1989,

Patil 1990, Jozef 2003, Giuliano, 2004, Kara. 2005) chi-

tin and polysaccharides (Neetu 2006), mineral kissiris

skim powder and fungal mycelium

Different working parameter condition shows the

optimal condition of initial sugar concentration, pH,

temperature, dilution rate and bead size on maximum

production of ethanol (Bardi,1994). Using continuous

immobilized yeast fermentation, microalgal hydrolysate

was converted into ethanol at a yield of 89% (Kyoung

2014) conversion of biomass into ethanol varies consid-

erably depending on the nature of feedstock primarily

due to the variation in biochemical composition, and so,

only a few feedstocks have been exploited commercially

(Zabed 2017).

In the present study, the effect of initial sugar concen-

tration, yeast cell concentration on an immobilized cell

system and the effect of different activators (saw-dust,

chitin and Rhizopus-Oryzae) on free and immobilized

cell system is studied. Continuous production of Ethanol

was done to study the effect of  ow rate and the reus-

ability study. The reduction of bead size increased mass

transfer of substrates from the liquid to the immobilized

cells accelerating sugar consumption and ethanol pro-

duction (2018). Fermentation of sugar for production of

ethanol was carried out using Saccharomyces cerevisiae

cells immobilized in calcium alginate  lms, (Leal 2018).

MATERIALS AND METHODS

MICROORGANISM

Commercially available fresh baker’s yeast was used for

experimentation. The culture was obtained as a cake.

The same was stored under 0-4

C temperature till it was

further used.

PREPARATION OF IMMOBILIZED YEAST BEADS

About 4g of sodium alginate was weighed and dissolved

in 100mL of boiling distilled water and made slurry. In

another beaker 8g of fresh baker’s yeast was dissolved in

distilled water. The two solutions were mixed and made

slurry. The  nal volume was adjusted to 200mL with dis-

tilled water. The slurry was extruded drop wise into 0.2M

CaCl

solution with the help of a micro-tip connected to

silicone tubing, which passes through peristaltic pump.

The beads were left in calcium chloride solution over

night for curing. The beads were then taken out, washed

and stored till they were further used.

PREPARATION OF MOLASSES SOLUTIONS

Pre-activated yeast beads of 1% concentration were

taken in four conical  asks (250mL), each containing

different concentrations of molasses solution (i.e., 6%,

10%, 12%, 16% and 20%). The activity of immobilized

yeast was observed for different time interval like 24,

48, 72, 96 and120 hours. The amount of total reducing

sugars was measured at each time interval.

PREPARATION OF YEAST CELL

CONCENTRATION

A series of batch experiments was conducted to test

the effect of yeast bead concentration on alcohol yield.

Yeast beads of various concentrations were taken (i.e.,

0.5%, 1%, 2%, 3%, 4%, and 5%) and activated for over-

night in the medium separately. Each of the beads was

then transferred to 12 percent molasses solution and the

reduction of total reducing sugars was estimated.

FERMENTATIONS WITH THE USE OF

ACTIVATORS

Experiment on external activator compounds were con-

ducted by using 1% of activators (i.e., chitin, sawdust

and Rhizopus-Oryzae biomass) with 1% free and immo-

bilized yeast cells in conical  asks (250mL). The fermen-

tation was allowed for 10 hours. The amounts of total

reducing sugars were measured after every two hours.

EXPERIMENTAL SETUP FOR REACTOR

Initially, the fermentation was carried out batch wise

using immobilized yeast cells until the substrate was

depleted and was then switched to a continuous mode

with the feeding of molasses. Fresh molasses was contin-

uously supplied to the fermenter with a peristaltic pump

and at the same time cell free liquid was removed from

the bio-reactor through the  lter module. As a whole,

the volume of broth in the fermenter was controlled at 5

liters by making the total  ltrate  ow (outlet) rate coun-

ter balance the total feed  ow rate. The fermentation

was carried out at a room temperature.

ANALYTICAL METHODS FOR SUGAR AND

ETHANOL ESTIMATION

Amount of total reducing sugar was estimated by the

DNS method (Milller,1985). The ethanol concentration

was measured by gas chromatography (Thermo sher,

8610) using a chromosorb 101 column (80 to 100 mesh

of packing material, 1/8 inch outer diameter and 6 ft

long stainless steel tube) with  ame ionization detector.

Temperatures of the detector, injector and column oven

Kethineni Chandrika et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS CHARACTERIZATION AND CONTINUOUS PRODUCTION OF ETHANOL 573

were 300

C, 200

C, and 150

C respectively. Nitrogen was

used as a carrier gas. The data reported for sugar con-

centration and percent alcohol were the mean value of

at least two independent samples analyzed in duplicate.

RESULTS AND DISCUSSION

EFFECT OF SUGAR CONCENTRATION OF

ETHANOL FERMENTATION RATE

It was evident from the Fig.1 that total reducing sugar

level in the fermentation broth has in uence on the fer-

mentation rate. The maximum reduction in the amount

of total reducing sugar was observed after 24 hours of

the fermentation in the broth with 12 % TRS. As this

total reducing sugar level increased beyond 12 percent,

the ef ciency of the yeast fermentation has reduced,

indicating the high sugar concentration decreased the

fermentation rate.

observed to be increasing with decrease in amount of

immobilized yeast crystal concentration. In case of fer-

mentation with the 5 % immobilized yeast, the ferment-

able sugars were completely utilized within 4 h of fer-

mentation time.

The data also indicated that the effective fermenta-

tion can be achieved using 1% of immobilized yeast

crystals with 12% TRS level in the fermentation broth.

However, the economic feasibility in ethanol fermenta-

tion can be achieved with 5 percent yeast crystals. The

data acknowledge that 12% concentration broth is more

productive for ethanol production.

EFFECT OF ACTIVATORS ON THE

FERMENTATION RATE

As evidenced from the ( gure 3), in the free cell sys-

tem it was observed that control has shown more effect

than activators for the initial 2 hours of fermentation

time however , the activator effect was observed start-

ing from 4 hours which was pronounced only at the

end of 6 hours of fermentation time where chitin sup-

plemented broth showed 47% increase in fermentation

rate followed by Sawdust (20.86%) and Rhizopus Ory-

zae (12.5%) over control where maximum sugars were

degraded.

FIGURE 1. Effect of initial sugar concentration on

ethanol fermentation rate.

FIGURE 2. Effect of immobilized yeast concentration on

ethanol fermentation rate.

The reduced sugar conversion with the higher con-

centration of molasses is probably due to osmotic effects

(Roukas 1994), decreased water availability and plas-

molysis of cells that leads to the growth inhibition of

cells. Though the initial fermentation rate was less in

16 and 20% broth than in 12 percent broth, it increased

with increase in fermentation time. The amount of TRS

left after 120 hours of fermentation was higher at 16%

and 20% broth than in 12% broth. This suggests that

the fermentation rate decreased with increase in alcohol

concentration.

EFFECT OF IMMOBILIZED YEAST

CONCENTRATION ON ETHANOL

FERMENTATION RATE

As evident from the Fig.2, the rate of fermentation

increased with increase in immobilized yeast concen-

tration. However, the left-over sugar concentration was

FIGURE 3. Effect of activators on the free cell

fermentation rate

Kethineni Chandrika et al.

574 CHARACTERIZATION AND CONTINUOUS PRODUCTION OF ETHANOL BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS

The effect of activators (Fig.4) on immbobilized cell

system shows a similar trend of free cell system was

repeated where control showed more effect than activa-

tors for initial 2 hours while the effect of activators was

observed starting after 4hours , however, unlike the free

cell system where chitin showed much effect here in this

case Rhizopus oryzae has shown signi cant effect, i.e., a

23.94 % increase in fermentation rate followed by saw

dust (16.37%) and Chitin (12.32%) with respect to control

after 6h where maximum sugars have been degraded

Chitin, a cell wall component of yeast increased the

ethanol production, which is probably not contributing

as a nutritional factor needed by yeast cells, but may

have released some nutritional factors that lead to the

partial hydrolysis of complex substances present in the

molasses or may be due to the removal of inhibitory

compounds. The increased in ethanol productivity due

to Sawdust is due to cells immobilized on diligni ed cel-

lulosic material that lead to an active bio-catalyst reduc-

ing the activation energy (Bardi, 1994). Besides, Saw-

dust may have enhanced the catalytic action of some

enzymes involved in the fermentation pathways or any

undesirable substances in molasses may be excluded by

their adsorption on the diligni ed materials.

Patil and Patil found the acceleration of ethanol pro-

duction activity of yeast in cane molasses fermentation

by the addition of fungal mycelium (Patil 1990). Besides,

brewery yield of ethanol was increased by the addition

of Proteo-lipid from Aspergillus and Oryzae promote

yeast growth and yeast durability against ethanol and

salt concentration.In the present work we hypothesize

that the activators in common may have released some

nutritional factors from other complex polysaccharides

(as molasses contain different polysaccharide beside

sugars) which degraded the sugar. This activator effect

is seen more pronounced only at lower, reducing sugar

concentrations (after 50% of initial sugar degradation)

which is clearly re ected in our work, where the increase

in fermentation activity has started at 4 hours of fer-

mentation time both in free and immobilized activator

system. This may probably due to less mass transfer

effect of activators in high sugar concentration.

CONTINUOUS PRODUCTION OF ETHANOL

USING IMMOBILIZED YEAST

To evaluate the sodium alginate- yeast beads for con-

tinuous production of ethanol, the experiments were

planned using 5-L reactor with an inlet and outlet. The

reactor was equipped with an overhead stirrer so as to

provide uniform mixing.

Initially the reactor was run in batch mode for 24h

there after the inlet feeding was initiated at a  ow rate

of 190mL h-1 having residence time of 26.3 hours. The

productivity was observed to be 2.49g L-1 h-1. Once

the stabilization was achieved at this  ow rate it was

increased so as to achieve the residence time of 21.73 h

( ow rate-230mL h-1). It was noticed that at this resi-

dence time the productivity was increased in the out-

let at the tune of 50%. The same was re ected by the

decrease in sugar concentration in the outlet. To evalu-

ate further, experiments was conducted at  ow rates of

270mL h

-1

and 280mL h

-1

with residence time of 18.51 h

and 17.85 h respectively.

As evidenced from the  gure 5, the productiv-

ity increased with increasing  ow rate up to a certain

level (270mL h

-1

) beyond which the increased  ow-rate

resulted in the decrease of productivity.

FIGURE 4. Effect of activators on immobilized cell fermentation rate

Kethineni Chandrika et al.

BIOSCIENCE BIOTECHNOLOGY RESEARCH COMMUNICATIONS CHARACTERIZATION AND CONTINUOUS PRODUCTION OF ETHANOL 575

FIGURE 5. Continuous production of ethanol using immobilized yeast cells at

different  ow rates

FIGURE 6. Continuous production of ethanol using immobilized

yeast cells at a  xed  ow rate

Data indicated that critical dilution was achieved at

around (270mL h

-1

) having maximum speci c growth,

increase in  ow rate might have led to washing out of

cells resulting in decreased productivity.

Based on the above data, the continuous production

of alcohol using immobilized yeast beads in a CSTR was

continued using at a  ow rate of 190mL h

-1

(residence

time of 26.31h) to access the reusability of immobilized

yeast breads. The data were presented in the graph ( g-

ure 6). The results indicated that production of ethanol

ranged from 6-6.8%, depending on the initial TRS load-

ing.On an average, consistent ethanol production was

observed for more than 16 cycles (maximum cycles

studied). It was observed that the productivity levels

were varied as the fermentation time increased. A visual

observation indicated that a slower rate of fermentation

was observed in the molasses vessel because the medium

is not sterilized.

C ONCLUSION

Effective Fermentation can be made with 1% of Yeast

cell concentration and 12% of initial TRS loading. Plant

and Fungal based Activators were shown to signi -

cantly increase the fermentation Rate. Continuous pro-

duction of Ethanol showed an increase in productivity

up to 270mL h-1 beyond which showed reversed trend.

Continuous production of ethanol at a  xed  ow rate

showed the beads can be reused up to 16 cycles.

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