Ethanol Production

Ethanol produced as a component of alcoholic fermentation

Used as chemical feed stock and fuel supplement

World wide- 4 million tons of ethanol produced annually [ 80% is by fermentation]

Used as solvent

Used to produce ethylene and other petrochemicalsIntroduction

Biosynthesis of EthanolProduced by fermentation f sugar rich products with help of yeast- Saccharomyces cerevisiae or some times Kluyveromyces fragilis

Bacteria also used ( widely used one is Zymomonas mobilis)

S.cerevisiae suitable for fermentation of hexosesK.fragilis- suitable source is lactoseCandida sps. suitable source is pentose

Most microbes requires readily available sugar compounds for fermentation ( eg. Sugar cane ,sugar beet juices, Molasses)

Carbohydrate Hydrolysis Glucose

Glycolysis

Pyruvate Pyruvate decarboxylase, Mg2+

Acetaldehyde + CO2

Alcohol dehydrogenase

Ethanol

Ethanol Biosynthesis Pathway

Schematic representation of Ethanol productionSterilizationRaw Materials (Starch,Cellulose,Molasse))Pretreatment of substratesFermentationSeparationDistillationEthanol(94.5% w/v)

Ethanol ProductionEthanol is produced in 3 steps-1. Preparation of substrate2. Fermentation3. DistillationPreparation of substrate 3 types of substrate are used a) Starch containing roots , tubers, or grainsb) Molasses or juice from sugar cane or sugar beetc) Wood or agricultural waste or wood wasteRoot starch is derived from Manihot esculenta (Root contains 20-30% of starch and 1 to 2 % protein)S.cerevisiae donot produce amylase; so roots are first ground, squeezed and dried. Then starch is liquified by boiling under pressure and hydrolyzed enzymaically

Glucose production glucoamylase and alpha amylase are added after boiling and cooling process

1 tone of starch app. 1 liter of alpha amylase and 3.5 L of glucoamylase are used

Molasses byproduct of sugar ,commonly used

Wood- Is not yet used in commercial production

Sulfite waste liquor- source of sugar formed during production of paper from conifers

2. Fermentation processBoth Batch and continuous fermentation was employed for ethanol production.

When compared to batch , continuous fermentation is more useful for ethanol production

Industrial production of alcohol is carried out in large fermentaers upto a size 5 lakh liters

Inoculum volume is around 4 % of the fermentor

pH around 4-4.5

Initial temperature is kept between 21-26 C, During fermentation temp. rise to around 30 C

Cooling devices are necessary to maintain temp. less than 27C

Duration is 2 -3days, but actual time period depends in raw materials used and incubation temp.

After fermentation , spent medium contains ethanol in the range of 6-9% by volume. This represents 90-95 % of substrate isconverted in to ethanol

3. DistillationEthanol is recovered from fermented medium by distillation

Cell mass is separated initially by centrifugation or sedimentation

1st Distillation 80 % of pure ethanol is obtained

Repeated Distillation- 99.4% or more ethanol is achieved

Final dessication is by chemical dessicants and by absorbtion

Ethanol 92.4% - used as solvent

99.2% - used as fuel

Methods to increase ProductivityProcess should be carried out in large scale by continuous fermentation

Continuous operation with cell recycle- results in faster conversion of substrates to ethanol

Maximum productivity with glucose as carbon source

82 g/L/h by yeast

120g/L/h by Z. mobilis

Use of immobilized cells ( high cell concentration in fluidized bed bioreactor)

If Molasses is used as substrate

Batch system yield 2g/L/h of ethanol

Continuous system yield 3.35g/L/h of ethanol

Immobilized cells with continuos system yeild 28.6g/l/h of ethanol

Solvents - Acetone , Butanol & Glycerol

Acetone Butanol ProductionAcetone used as solvent for fats ,oils, resins, waxes - used as gelatinizing agent for nitrocellulose - used in dyeing industry

Butanol- used in production of detergent and brake fluids - used as solvents for fats, waxes and resins

Butanol is better fuel additive than ethanol due to its low vapour pressure, low solubility with water, complete solubility with diesel fuel

Acetone, Butanol, Butyric acid & isopropanol are produced by Clostridium sps.- by fermenting starch, molasses, sucrose, pentoses and wood hydrolysate

Products will vary and it depends o strain and fermentation conditionsAcetone-butanol fermentation C. acetobutylicumButanol-Isopropanol fermentation- C. butylicumButyric acid-Acetic acid fementation-C. butyricum Clostridium acetobutylicum strict anaerobe, hydrolyze gelatinized starch in to glucose & maltose

Production processCane Molasses ( Raw Material)

C. acetobutylicum

Low inoculum is inoculated into production fermenters (1:3000) Fermentation (pH 5.8-6.0),Temp.34 C, Fermenter Volume is 90 m3 - batch reactor

After sterilization of fermentor, it is gassed with CO2

Before and after inoculation, the medium contents are also stirred with CO2

This is to ensure that it will support the growth of the organismTo remove any residual O2 present in the fermenterTo maintain anaerobic condition

Fermentation takes 36 hours to completeFirst Phase (18hrs) pH decreases to 5.2 {formation of acids}

Second phase (18hrs) - pH increases { formation of A/B}

Third phase pH value remain constant 5.8{growth and solvent production stop}

Continuous distillation & fractionation

Product recovered

* Contamination by phages & Lactobacilli is a common problem

Several ways to improve solvent yieldAddition of Acetic & Butyric acids

High agitation (300rpm) during acid phase followed by low agitation (25 rpm) during solvent phase

Simultaneous removal of inhibitory products (A/B) by extraction and adsorption

Continuous or fed batch operations improve solvent productivities

Use of immobilized cultures

Gene manipulations of C.acetobutylicum

Glycerol Production

Glycerol is formed by yeast along with ethanol during alcoholic fermentation

Usually glycerol is formed in low amount . By modifying the fermentation balance high amount of glycerol is produced

Acetaldehyde is the intermediary product in ethanol fermentation

When sodium sulfite is added, it blocks alcohol production and diverts the pathway for large scale production of glycerol.

Sodium sulfite reacts with CO2 in the medium and gets converted to Sodium bisulfite

Then combines with acetaldehyde to form acetaldehyde sulfite complex and blocks alcohol production

NADH2 formed during glycolysis usually reduces acetaldehyde to ethanol

Due to unavailability of acetaldehyde ,NADH2 reduces Dihydroxyacetone phosphate to form glycerol 3- phosphate then glycerol

Glycerol fermentation carried out in 1000m3 size of fermenters

Fermentation duration within 2 to 3 days

Osmotolerant yeast are used

Commonly used strains S. rouxii & Torulopsis magnoliae

Used in cosmetics & pharmaceutical industries

Glycerol Production Using AlgaeDunaliella salina is halophilic algae commonly found in salty lakes.

While growing in hypersaline conditions ,this algae synthesizes glycerol within the cells to balance the high osmotic pressure in the surroundings (due to salts)

So Higher the salt concentration in the surroundings results in high intracellular glycerol production.

Extraction of glycerol from algaeCollect the algal cells and place it in low salt environmental condition.

Sudden shift in high salt to low salt condition will excrete the intracellular glycerol into the surrounding medium

This new fermentation process is developed in Israel

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