fermentation subject

1. FERMENTATIVE
PRODUCTION OF ETHANOL
SUBMITTED TO – Dr. RANJEETA BHARI
SUBMITTED BY- NEHA SINGLA
MSc. ( hon’s) Biotechnology
Roll No. - 18011001

2. CONTENTS-
• History
• Raw materials for ethanol production
• Choice of raw material
• Ethanol fermentation generations
• Pretreatment of different raw materials
• Fermentation- choice of strain
-fermentation kinetics
-Fermentation process
-Recovery and purification of product ethanol
- by product and waste management
• Uses of ethanol

3. HISTORY-
• The preparation of distilled alcohol spirits was first described in 12th
century.
• The preparation of absolute alcohol was first reported in 1796 ( by
repeated distillation over potassium carbonate)
• Extensive industrial use of ethyl alcohol began in the late 1800s with the
growth of synthetic chemical industry
• Industrial alcohol has been valuable as a solvent, germicide , antifreeze ,
fuel and chemical raw material and Use in US grew to 564 million liters per
year in 1941(77% by fermentation)
• During world war 2, US industrial alcohol production was increased 3.8 fold
on a emergency basis to provide alcohol for synthetic rubber ( via
butadiene ) and smokeless powder production.
• Availability after world war 2 of cheap ethylene led to the rapid growth of
the synthetic ethanol industry based on the esterification and hydrolysis of
ethylene in concentrated sulphuric acid – water solution.

4. Raw material
3 classifications by carbohydrate type-
• Saccharine
• Starchy
• Cellulosic
(YEAST can convert simple hexose sugar to ethanol )

5. Choice of raw material-
• Choice of raw material is critical as raw material costs typically make up 55-
77% of the final alcohol selling price.
• Should be available through out
• Require less pretreatment
• Should not complicate downstream process
• Saccharine- require less extensive preparation but expensive to obtain.
• Starchy- often cheaper but require processing to solubilize and convert
starch to fermentable sugars.
• Cellulosic – cheap waste residue but require most extensive and costly
preparation

6. BIOETHANOL PRODUCTION-3 ways
1. First generation bioethanol-
a) Saccharine-(examples-blackstrap molasses , sugarcane juice etc. )
Sugar to ethanol
b) Starchy
Starch to simple sugars then to ethanol
2. Second generation-
a)cellulosic –(examples- leaf litters, grasses , cactus etc.)
Cellulose or hemicellulose to ethanol
3. Third generation-
Algae to sugar to ethanol

7. 1.SACCHARINE MATERIALS-
• 1. SUGARCANE JUICE-
Transportation cost ,direct use of sugar cane is limited to alcohol plants
located within farming districts. Limited to tropical and subtropical climate
In Brazil 43% of alcohol plants process cane directly as raw material.
Juice is recovered by milling . Crushed cane is rinse with hot water to aid
sugar extraction . 85-90% fermentable sugars extracted.
Juice contain 12-16% sucrose sugar
The yield of sugar from cane is typically 125 kg per metric ton .
Solid bagasse residue remain after sugar extraction and this can be used as
boiler fuel .

8. • 2 kg of bagasse with heating value of 19700 KJ per kg are produced
for every kg of sugar.
• Sugar cane wax and aconitic acid can also be recovered as by
products cane crushers are high pressure steam driven using 1 – 1.2
kg steam per kg sugar recovered . Low pressure exhaust steam from
crusher is reused for distillation.

9. Sugar beet juice-
• Sugar beet alcohol industry existed in France in 1940s
• beet sugar – first sliced then followed by sugar extraction in
continuous counter current hot water diffuser- produce 10-15% sugar
solution
• Beet pulp residue is generally used as a low grade cattle roughage .
• Each metric ton of beet produces 140-190 kg of sugar and 70-100 kg
of pulp.

10. HIGH TEST MOLASSES-
-is a concentrated sugar solution, allowing easy shipping and long term
storage
Sugar cane juice or beet juice is partially hydrolysed with dilute acid to
convert sucrose to noncrystalline invert sugar monomers
Then concentrated to 70-78% sugar.
Due to thermal decomposition of sugars during evaporation and to the
presence of small concentration of inhibitors of yeast, these are only
95% fermentable.
Food use generally limits the availability of high test molasses for
industrial purposes

11. BLACK STRAP MOLASSES-
• Noncrystallizable residue byproduct of table sugar.
• Sugar juice is treated with lime at 100 ℃ to neutralise organic acids
• On cooling salts, coagulated albumin, fats and gums are precipitated.
• Clarified juice is then concentrated by multieffect evaporation and
sucrose is precipitated by further evaporation in vacuum pans.
• Approximately 27 kg blackstrap obtained from every metric ton of
cane processed. And 3.5kg or 2.5 l of molasses is required to produce
one litre of ethanol

12. • Upto 17% of blackstrap molasses is nonfermentable reducing
compounds resulting from high temperature destruction of sugars
during evaporation.
• Dehydration and reduction of fructose to 1,3 fructopyranose,
• Glucose and fructose salt catalysed condensation with cane amino
acids – caramel residues
• Sugars also decomposed to hydroxyethyl furfural , acetoin, and formic
acid and levulinic acid

13. Overcoming the problem of decomposition-
• Vacuum sugar evaporation reduces evaporation temperature and
reduce decomposition and increase ethanol yield
• Arroyo process- ammonium sulphate and calcium superphosphate
are added, then heated to 80 ℃ a sludge formed contain calcium
sulphate and organic inhibitors and settles down from the solution.
Then clarified molasses is used for fermentation
• Reich process- concentrated molasses heated to 70-90 ℃ and
sulphuric acid is added and sludge removed and clarified solution is
ready for fermentation.

14. Sweet sorghum-
• Tall grass with high sucrose content in the stems.
• Widely grown as cattle forage.
• In 1942 a test project used 23250 US tons of sweet sorghum for
alcohol production.
• Got attention because –readily cultivated under wide variety of
growth conditions and has sugar more then sugar beets
• Sorghum residue can be used as cattle feed roughage.

15. FRUITS AND JUICES-
• Fruit cannery waste substrate for ethanol production.
• After extraction of juices from fruits, additional sugars can be
extracted from pulp by hot water diffusion.
• Ion exchange processes allow the recovery of sugar from very dilute
waste streams while reducing the BOD of cannery effluents.

16. WHEY-
• Produced by cheese manufacturers and rich in lactose.
• Lactose – glucose and galactose
• not fermentable by most yeast
• Torula cremoris and Candida pseudotropicalis are generally used
• ethanol production is limited with whey ( cattle feed and antibiotic
production)

17. 2.STRACHY RAW MATERIAL-
• Include cereal grains , starch root plants and some cacti
• Cereal grains and starch root plants used for human consumption and
animal feed
• Carbohydrate present in the starchy grains is not directly fermentable
by yeast and these materials require pretreatment that is hydrolysis
of complex sugars into simple sugars .
• Starch is first hydrated and gelatinised by milling and cooking and
then broken down to fermentable sugars by enzymes or weak acids.

18. CEREAL GRAINS-
• Corn, wheat ,rice, barley and grain sorghum
• Cereal grains generally have 50-65% starch .
• Corn is major used for fermentation in US.
• Treatment- first air classified to remove dirt and cracked hulls
-then grounded to allow easy wetting and then gelatinised by cooking.
• Traditional cooking methods replaced by pressure cooking. This lead
to less degradation and also sterilise the mash

20. ENZYMATIC HYDROLYSIS-
• 0.27kg per litre of water and pH adjusted to 5.5 , heated to 135-150
℃ and held for 10-30 minutes. Rapid cooling by pressure blowdown
and then vacuum evaporation reduce the temperature to below 65 ℃
for the addition of enzymes.
• 3 basic type of enzymes-
• Alpha amylase produce dextrose
• Beta amylase-produce maltose
• Glucoamylase –reduce remaining starch

21. BATCH HYDROLYSIS-
• In this pressure cooking is done to gelatinise grains and then cooling
is done on fungus growth is allowed generally Aspergillus .
• Growth is allowed for 24-48 hrs. or maximum for 3 days then fungus
are removed and sugars are extracted out for fermentation.

22. CONTINUOUS HYDROLYSIS-
• Milled grains slurried with water in a tank then it is passed through
steam jet heater into a cooking tube with a flow residency of 5
minutes and at upto 180 ℃ cooking temperature.
• Flash cooling is used to reduce rapidly the mash temperature and
halt sugar breakdown
• Continuous cooking at lower steam consumption provide more
uniform cooking
• The continuous flow hydrolysis process is used in combination with
continuous cooking . Activated malt solution is added into the stream
of cooked and cooled mash at 63 ℃ in the flow pipe . The mash is
then cooled to fermentation temperature.

24. ROOTS, TUBERS AND CACTI-
• High in carbohydrate content, giving high final yield of ethanol
• Include potatoes , sweet potatoes , artichoke , manioc , sotol.
Potatoes has 15-20 % starch and produce 4700 litre per hectare
Sweet potato -30 % starch and produce 7200 litre per hectare
Potato cost is generally prohibitive for ethanol production.
artichoke tubers and manioc are high in inulin . Used in France for
ethanol production. Prussic acid is high in manioc but his can be broken
down by sun drying.

25. PROCESSING
• Rinsing with water then cutting and then hammer milled to mash
• Other process is same as of grain cooking and hydrolysis.
• Inulase enzyme is required for artichoke and manioc
• In case of potato after cooking , the mash is blown down through
conical vessel bottom into a lower pressure drop tub with violent
boiling resulting steam explosion which disintegrates substrate, thus
eliminating the need for milling.

26. Limitations of first generation biofuels-
• These can also be consumed as human food
• Transportation cost
• Require more land.
• Constricted to specific area

27. 3.Cellulosic raw material-
• Soft wood or woody agricultural residue are made up of hemicellulose ,
cellulose and lignin.
• For 1kg of grain harvested 1 -1.5 kg of straw , cobs , Stover or other residue
generated . About one third of tree is lumber.
• Pretreatment-
• To produce ethanol lignin and hemicellulose must be removed , so that
enzyme can break cellulose into glucose units
• Steam at high pressure will result in separation of hemicellulose and lignin
• Wood is kept at high pressure and temperature for few minutes and this
mechanism will remove hemicellulose (temp-190-230 ℃)

28. • Dilute acid treatment-
With this treatment lignin gets condensed and hemicellulose is
dissolved by the acid to form furfural and hydroxymethyl furfurals.
• Organosolvent pretreatment-
Aqueous ethanol is used to partially hydrolyse cellulose and lignin will
get depolymerised and dissolved .
• Sulphite waste liquor contain sulphur dioxide that has to be removed
by treatment with lime .
Once pretreatment ends cellulytic process has to be started which can
be done by hydrolysis by enzymes or chemical hydrolysis .

29. • Cellulose molecules are broken down into glucose molecules by
cellulase enzymes.
• Cellulases are produced by fungus Trichoderma reesei
• Cellulases are composed of endoglucanases , exoglucanases and beta
gucosidases.
• chemical hydrolysis-
a)Dilute acid at high heat and pressure
b)And concentrated acid at lower
temperature and atmospheric pressure
• Can broke cellulose into glucose
molecules.

30. Hemicellulose fermentation-
• Yeast cannot ferment 5 carbon sugar to ethanol ( xylose present in
hemicellulose)
• Zymomonas mobilis , Pichis stipitis added .
Limitations with cellulose-
• Expensive process ( pretreatment)
• Enzymes expensive.
• Because of high BOD of end product , disposal problems .

32. ALGAE- 3rd generation
• Advantages-
• Don’t require land
• No fertilizer is required
• Significant carbohydrate content and high ethanol yield
• Can grow in every season and everywhere.
2 types-
• Microalgae - high amount of lipid , protein and carbohydrate mainly
starch
• Macroalgae- laminaran and mannitol

33. FERMENTATION-
• Fermentation equipment forms 10-25% of the total fixed capital.
• Raw material is aerated prior to inoculation as aeration is required by
yeast for the formation of sterol.
• Organism for ethanol production-
• Yeast are the organism used for large scale ethanol production.
• Yeast produce ethanol with high yield less by products.
• Clostridium thermosaccharolyticum and other thermophilic bacteria
Pachysolen tannophilus yeast are under study for use in fermenting
pentose sugars which are non fermentable by ordinary yeast.

34. Yeast strain selection-
• Yeast strains are generally chosen from among Saccharomyces cerevisiae ,
S .ellypsoides , S. carlsbergenesis for whey fermentation, Torula cremoris
and Candida pseudotropicalis is used.
Yeast ideal characteristics for fermentation-
• 1. high growth and fermentation rate.
• 2.high ethanol yield
• 3. ethanol and glucose tolerance.
• 4.osmotolerance
• 5.low pH fermentation optimum
• 6. high temperature fermentation optimum
• 7.Crab tree effect.

35. Fermentation kinetics-
• In the anaerobic pathway glucose is converted into ethanol and
carbon dioxide and some amount of energy is released.
• By this pathway 1 gram of glucose yield 0.511 g of ethanol.
• Secondary reactions consume some amount of glucose to produce
ethanol only 95% by theoretical.
• Aerobic respiration sugar is converted to
carbon dioxide and no ethanol will be
formed hence must be avoided.

36. EFFECT OF SUGAR CONCENTRATION-
• Primary reactant in yeast metabolism.
• At very low conc. The yeast is starved and productivity decreases.
• At higher concentration a saturation limit is reached . So sugar conc.
Can be maximum upto 150g per litre
• More than this will lead to catabolite inhibition of enzymes in the
fermentative pathway

37. Effect of ethanol and oxygen-
• High ethanol conc. Is toxic
• 110g ethanol per litre can halt the process although another sake
yeast can tolerate 160g ethanol per litre at low temperatures.
• Ethanol inhibition is directly related to inhibition and denaturation of
important glycolytic enzymes as well as modification to cell
membrane.
• High oxygen conc. Lead to aerobic metabolism which will produce no
alcohol, but low amount of alcohol is required for synthesis of
polyunsaturated fats and lipids required in membranes.

38. EFFECT OF pH and TEMPERATURE-
• Fermentation is sensitive to pH
• Mostly range is kept between 4 to 6
• High tolerance is desirable feature and most distillery yeast have
optima between 30-35 ℃ .
• Alcohol tolerance is improved at low temperature

39. Additional nutrient requirements-
• Glucose supplemented with NH4CL, MgSO4, CaCl2 and yeast extract.
• Yeast extract is water soluble and contain all growth factors
• Blackstrap molasses is supplemented with ammonium sulphate and
sulphuric acid .
• High test molasses and cane or beet juice are supplemented with
ammonium sulphate and sodium phosphate
• Sulphite liquor is neutralised with lime and supplemented with
ammonium hydroxide or urea.

40. Secondary component inhibition-
• Yeast growth inhibited by product or by non metabolised feed
components
• Acetate and lactate are most important inhibitory fermentation by
products
• Presence of high salt concentration will encourage production of
undesirable by product such as glycerol.
• Backsetting is not normally practised in waste sulphite liquor plants

41. Fermentation process-
• Conventional batch fermentation-
• It begins with the production of an active yeast inoculum. This can be
either by the conventional serial growth method or by the rapid
semiaerobic method.
• Aseptic techniques are used throughout.
• In serial growth culture , a pure culture inoculum from an agar slant is
used to seed a laboratory shake flask at the peak of growth this
culture is used to seed a succeeding culture . This is repeated
generally 3 stages .this inoculum is grown on a medium similar to the
final fermentation mash.

42. • An inoculum 3-4 times concentrated in yeast can be produced by
semiaerobic method.
• Yeast are grown in an aerated and semi work fermenter operated in
fed batch mode. A large portion of the previous batch is retained to
provide an inoculum.
• A high nutrient medium is added and optimum conditions are given
then sterile air is sparged at a rate of one – eighth volume of air per
fermenter volume per minute.
• Aerobic metabolism will stimulate and will form high cell density .
This high cell density can be used as inoculum .
• After fermentation the product is feed to distillation. The fermenters
are then cleaned and sterilised for another cycle.

43. Continuous fermentation-
• In continuous fermentation fresh medium flows into the fermenter
continuously and a part of it is withdrawn from fermenter at the same
flow rate of the inlet flow.
• Continuous fermentation can be performed in staged which will give
better results than single stage fermentation.
• In single stage fermentation 52 g ethanol per litre
• In 2 stage fermentation- 73gper litre
• In 3 stage fermentation- 99gper litre

44. In two stage fermentation-
• In 1st stage sugar is partially utilised and there is no production of
ethanol, hence no inhibition.
• In 2nd stage complete utilisation of sugar take place and there is
ethanol fermentation . When the fermentation is near completion
temperature is raised to mark the complete utilisation of sugar.

45. TOWER FERMENTATION-
• Fermenter arrangement for rapid alcohol production .
• Consists of cylindrical tower typically 2 m in diameter and 15 m tall
and is topped by a larger diameter settling zone fitted with baffles.
• Sticky flocculant yeast with high settling rate is used
• Prehydrolysed sugar wort is pumped into the base of the tower and
reaction proceed as alcohol rises up through a dense yeast plug .
• Yeast will settle back against the flow and very high cell densities are
achieved . Temperature is regulated by cooling jackets at low brewing
temperature (16 -23 C )

46. • Major drawback is long time required for set up.
• Two to three weeks are required to build up the desired high cell
density and achieve stable operation

47. Distillation and Recovery of product-
• Cell biomass is separated by centrifugation or sedimentation.
• Yeast form flocs which can be separated by filtration. Then the filtrate
is distilled to separate ethanol from other products.
• Distillation depends on differences in boiling points of components of
liquids .
• Mixture is added into distilled pot-head portion is discarded which is
having acetaldehydes.
• Heart part is having 70-75% ethanol
• Tail part is also discarded or can be used for other purposes and
constitute of pentanols and propanol (high boiling liquids)

49. • With the condenser the vapor is cooled and it liquefies which is
collected in flask which is further purified by vacuum rectification at
13.3 KPa pressure.
• During distillation there is formation of azeotropic mixtures which can
be separated by adding benzene
The residual water and solids that remain after distillation called
stillage is then centrifuged to separate solid and liquids.
The thin stillage is evaporated to remove water and the syrup is
blended with distillers and dried to produce animal feed called distillers
dried grains with solubles. (DDGS)

50. By products or waste uses-
• Grains can be used as cattle feed
• CO2 produced can be used for formation of dry ice.
• Stillage with low feed value can be used for anaerobic digestion
• Acetaldehyde can be separated for other purposes.
• The low pressure steam can be used for process of heating of
distillery processes.

51. Ethanol uses-
• Ethanol can be used as a fuel with mixing with petrol
• It can be used as antiseptic , germicide.
• It can be used as solvent.
• It can be used for formation of rubber.
• Common ingredient in many cosmetics and beauty products.

52. REFERENCES-
• Comprehensive biotechnology- Murray Moo Young

53. THANK
YOU