ethanol fermentation

ETHANOL FERMENTATIONMOKSHA CHIB 13FET1003

INTRODUCTION

• Ethanol which is widely used as a biofuel as well as an alcoholic beverage is increasingly being consumed globally

• Ethanol is being increasingly produced by fermentation

• Currently, about 80% of fuel-grade production in US comes from fermentation & current ethanol production level is the equivalent of about 65,000 barrels per day of imported oil

RAW MATERIAL Ethanol can be derived from either sugars, starchy materials or lignocelluloses Main feedstocks for ethanol production include sugarcane, sugar beet, corn,

wheat, cassava• Sugarcane (molasses & juice)• Cane sugar (clarified concentrated syrup)• Sugar beet• Beet sugar ( diffusion juice & beet molasses)

Sugars• Corn• Wheat• Sweet sorghum• Cassava

Starchy Materials

• Sugarcane bagasse• Corn stover• Cereal straws

Lignocellulosic materials

FEEDSTOCK CONDITIONING & PRETREATMENT

Some substances in the solutions can have inhibitory effect on the fermentation by microorganisms since the used cultivation media are complex. Their composition is not completely defined as it varies due to factors like techniques used, climate, type of employed fertilizers, water availability etc. Dilution- Molasses must be diluted to below 25° brix as yeast start to ferment quickly at this

concentration Sedimentation- is performed to prevent any incrustation in the pipelines or distillation

towers due to ash content in molasses greater than 10%. Special chelating agents can also be employed to remove the solids causing incrustation

Addition of org & inorganic compounds- This is done to offset the negative effect of salts which in turn increase the osmotic pressure. Yeast strains resistant to salts are also developed

Microfiltration- To remove the impurities that stick to the surface of the biocatalyst when immobilized cells are used

• Nitrogen source: Urea is the most suitable. Gaseous ammonium increases the pH of the medium & ammonium sulfate can lead to incrustation

• Phosphorous source:

Diammonium phosphate is used

• Some hydrolytic enzymes can also be added to convert biopolymers & non fermentable substances in the molasses to monosaccharides or amino acids

BIOCHEMICAL PATHWAY

pH : 4-4.5Temperature: 30°C

MICROORGANISMS INVOLVED

Saccharomyces cerevisiae Convert hexose into pyruvate by Glycolysis

which is finally reduced to ethanol generating 2 moles of ATPs under anaerobic conditions

Can tolerate high concentrations of ethanol up to 150g/L

Ethanol production is coupled with yeast cell growth, which means yeast must be produced as a co-product

By products like glycerol, organic acids are also produced

Without the continuous consumption of the ATPs by the growth of yeast cells, the glycolytic metabolism will be interrupted immediately because of the intracellular accumulation of ATP, which in turn inhibits phosphofructokinase (PFK)

Microorganisms utilizing both hexoses & pentoses show diauxic growth. They first utilize hexoses which is followed by consumption of pentoses.


Zymomonas mobilis Anaerobe, gram-negative bacteria which produces ethanol via

ED pathway converting 1 mol of hexose into 2 mol of ethanol, but releasing only 1 mol of ATP

Lower cell yield due to lower energy yield of bacterium, increasing the amount of ethanol from the substrate (97%)

High ethanol tolerance (100g/L) & higher optimum production temp.

Drawbacks Highly specific substrate spectrum : glucose, fructose & sucrose

decreasing ethanol yield It’s biomass is not acceptable to be used as animal feed, which

generates a problem for biomass disposal Continuous ethanol fermentation is oscillatory which can

increase the average residual sugar but decrease the ethanol yield


Features of microorganisms in ethanol fermentation: Due to their small size, microbial cells present a very high surface/volume ratio,

which makes possible the active input of many substances to the cytoplasm. Due to the presence of a resistant cell wall, the microorganisms can build up

many substances in high concentrations which ensures faster fermentation rate & higher division cell rate

The intense metabolism permits the development of continuous fermentation processes at an industrial level since the cell growth rate offsets the rate at which cells are removed from the bioreactor with the effluent

Have the ability to ‘predigest’ the available food sources. Thus, they release both the end products and the intermediate metabolites

PROCESS FLOW

Classical fermentation can be achieved in three steps:

• During the first 12-24 h, yeast cells multiply rapidly aerobically by consuming oxygen present in the mash

• In the middle phase (12-48h), alcohol production occurs with postsaccharification of oligosaccharides & multiplication of yeast falls off , accompanied by release of heat and rise in temp to 40°C

• Decrease in alcohol formation along with insignificant yeast growth at the final stage ( 48-72h)

BATCH FERMENTATION- MELLE BOINOT PROCESS

Weighing & sterilization

Adjustment of pH using

H2SO4 & brix to 14-22

Fermentation Decantation

& centrifugatio

n

Molasses or sugarcane juice

Yeast Propagation

Fermenter wort Wine

Yeast reutilization

Yeast reuse results in a decrease in new growth with more sugar available for ethanol production & an increase in the yield from 2 to 7%. Traditional yields – 1-3 g/L High concentration of yeasts (44g) & high supplementation of yeast extract (28g/L)at low ethanol content (60g/L)if employed in glucose based medium, ethanol productivity is as high as 21g/L

The stillage represents one of the distillation product streams during the subsequent ethanol recovery step that contains a significant amount of water and a much reduced amount of ethanol. The addition of stillage to the culture broth can lead to lower water consumption & reduction of stillage volume to be treated.

Operating procedures for fermentation include

• Washing and disinfection of the fermenter• Filling up of the fermenter with the culture

medium and sterilization of such medium• Inoculation of microbial cells• Fermentation• Unloading of bioreactor content at the end of

the cultivation process.

CONTINUOUS FERMENTATION

To ensure system homogeneity & reduce concentration gradients in culture broth, CSTR is employed. • Reduced construction costs of

bioreactors• Lower requirements of maintenance and

operation• Better control of process• Higher productivities• Cultivation of yeasts under anaerobic

condition s for a long time diminish their ability to produce ethanol

• Aeration is important which can enhance cell concentration, cell yield from glucose & yeast viability

• Diminution of product inhibition effect

FERMENTATION USING IMMOBILIZED CELLS

Attachment of cells onto a support in a defined space Cells do not leave the bioreactor & continuous fermentation is implemented Substrates are transformed into products in the biocatalyst (cells + support ) bed Products abandon the system in cell free effluent system leading to easier product

recovery Microbial cells are immobilized by entrapping within them porous, solid supports like

calcium alginate, carageenan and are adsorbed on the surface of materials like woodchips, bricks with a large surface area

2% Na Alginate solution, 2% CaCl2 solution

Strain : S.cerevisiaeSterilization: 100°C, 30 minViscosity : 1000-2000 cps

METABOLIC ENGINEERING

S. cerevisiae cannot utilize lactose directly, whereas the yeast Kluyveromyces lactis can utilize lactose but cannot perform an efficient alcohol fermentation

To develop an efficient lactose fermenting yeast, the β-galactosidase gene from K. lactis, along with the cloned lactose permease gene, was introduced into S. cerevisiae, leading to the fermentation of lactose.

ethanol fermentation

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