Industrial Biotechnology

1
Industrial Biotechnology

• CHAPTER 8
• Production of Organic Acids and Industrial Alcohol

2
Production of Citric Acid
3
Introduction

• Citric acid is a tribasic acid with the structure

It crystallizes with the large rhombic crystals
containing one molecule of water of
crystallization, which is lost when it is heated
to 130C. At temperatures as high as 175C it is
converted to itaconic acid, aconitic acid, and
other compounds.
4
Uses of Citric Acid

• Uses in the food industry
• Used as acidulant in the manufacture of jellies,
  jams, sweets, and soft drinks.
• It is used for artificial flavoring in various
  foods including soft drinks.
• Sodium citrate is employed in processed cheese
  manufacture.
• Uses in medicine and pharmacy
• Sodium citrate used in blood transfusion and
  bacteriology for the prevention of blood
  clotting.
• The acid is used in efferverscent powers which
  depend for their efferverscence on the CO2
  produced from the reaction between citric acid
  and sodium bicarbonate.
• Since it is almost universally present in living
  things, it is rapidly and completely metabolized
  in the human body and can therefore serve as a
  source of energy.

5
Uses of Citric Acid

• Uses in the cosmetic industry
• It is used in astringent lotions such as
  aftershave lotions because of its low pH.
• Citric acid is used in hair rinses and hair and
  wig setting fluids.
• Miscellaneous uses in industry
• In neutral or low pH conditions the acid has a
  strong tendency to form complexes hence it is
  widely used in electroplating, leather tanning,
  and in the removal of iron clogging the pores of
  the sand face in old oil wells.
• Citric acid has recently formed the basis of
  manufacture of detergents in place of phosphates,
  because the presence of the latter in effluents
  gives rise to eutrophication.

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Biochemical Basis of the Production of Citric Acid

• Since it is an intermediate of Krebs cycle, so
  the acid can be accumulated by using one of the
  following methods
• By mutation giving rise to mutant organisms
  which may only use part of a metabolic pathway,
  or regulatory mutants that is using a mutant
  lacking an enzyme of the cycle.
• By inhibiting the free-flow of the cycle through
  altering the environmental conditions, e.g.
  temperature, pH, medium composition (especially
  the elimination of ions and cofactors considered
  essential for particular enzymes).
• The following are some of such environmental
  conditions which are applied to increase citric
  acid production
• The concentrations of iron, manganese, magnesium,
  zinc, and phosphate must be limited. To ensure
  their removal the medium is treated with
  ferro-cyanide or by ion exchange fresins.
• These metal ions are required as prosthetic
  groups in the following enzymes of the TCA Mn
  or Mg by oxalosuccinic decarboxylase, Fe is
  required for succinic dehydrogenase, while
  phosphate is required for the conversion of GDP
  to GTP

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Citric acid can be caused to accumulate by using
a mutant lacking an enzyme of the cycle or by
inhibiting the flow of the cycle
8
Biochemical Basis of the Production of Citric Acid

• The dehydrogenases, especially isocitrate
  dehydrogenase, are inhibited by anaerobiosis,
  hence limited aeration is done on the
  fermentation so as to increase the yield of
  citric acid.
• Low pH and especially the presence of citric acid
  itself inhibits the TCA and hence encourages the
  production of more citric acid the pH of the
  fermentation must therefore be kept low
  throughout the fermentation by preventing the
  precipitation of the citric acid formed.
• Many of the enzymes of the TCA can be directly
  inhibited by various compounds and this
  phenomenon is exploited to increase citric acid
  production.
• Thus, isocitric dehydrogenase is inhibited by
  ferrocyanide as well as citric acid aconitase is
  inhibited by fluorocitrate and succinic
  dehydrogenase by malonate.
• These at enzyme antagonists may be added to the
  fermentation.

9
Fermentation for Citric Acid Production

• For a long time the production of citric acid has
  been based on the use of molasses and various
  strains of Aspergillus niger and occasionally
  Asp. wenti.
• Production by Penicillium is available, in
  practice are not used because of low
  productivity.
• Recently yeasts, especially Candida spp.
  (including Candida quillermondi) have been used
  to produce the acid from sugar.
• Japanese workers described a method to produce
  the acid by paraffins by bacteria and yeasts.
  Among the bacteria were Arthrobacter paraffineus
  and corynebacteria the yeasts include Candida
  lipolytica and Candida oleiphila.
• Fermentation with molasses and other sugar
  sources can be either surface or submerged.
  Fermentation with paraffins however is submerged.
• (a) Surface fermentation Surface fermentation
  using Aspergillus niger may be done on rice bran
  as is the case in Japan, or in liquid solution in
  flat aluminium or stainless steel pans.
• Special strains of Asp. niger which can produce
  citric acid despite the high content of trace
  metals in rice bran are used. The citric acid is
  extracted from the bran by leaching and is then
  precipitated from the resulting solution as
  calcium citrate.

10
Fermentation for Citric Acid Production

• (b) Submerged fermentation As in all other
  processes where citric acid is made the
  fermentation the fermentor is made of
  acid-resistant materials such as stainless steel.
  
• The carbohydrate sources are molasses
  decationized by ion exchange, sucrose or glucose.
  MgSO4, 7H2O and KH2PO4 at about 1 and 0.05-2
  respectively are added (in submerged fermentation
  phosphate restriction is not necessary).
• The pH is never allowed higher than 3.5.
• Copper is used at up to 500 ppm as an antagonist
  of the enzyme aconitase which requires iron.
• 1-5 of methanol, isopranol or ethanol when
  added to fermentations containing unpurified
  materials increase the yield the yields are
  reduced in media with purified materials.
• As high aeration is deleterious to citric acid
  production, mechanical agitation is not necessary
  and air may be bubbled through. Anti-form is
  added.
• The fungus occurs as a uniform dispersal of
  pellets in the medium.
• The fermentation lasts for five to fourteen days.

11
Extraction

• The broth is filtered until clear.
• Calcium citrate is precipitated by the addition
  of magnesium-free Ca(OH)2.
• Since magnesium is more soluble than calcium,
  some acid may be lost in the solution as
  magnesium citrate if magnesium is added.
• Calcium citrate is filtered and the filter cake
  is treated with sulfuric acid to precipitate the
  calcium.
• The dilute solution containing citric acid is
  purified by treatment with activated carbon and
  passing through iron exchange beds.
• The purified dilute acid is evaporated to yield
  crystals of citric acid.
• Further purification may be required to meet
  pharmaceutical stipulations.

12
Production of Lactic Acid
13
Properties and chemical reactions of lactic acid

• (i) Lactic acid is a three carbon organic acid
  one terminal carbon atom is part of an acid or
  carboxyl group the other terminal carbon atom is
  part of a methyl or hydrocarbon group and a
  central carbon atom having an alcohol carbon
  group. Lactic acid exists in two optically active
  isomeric forms.
• (ii) Lactic acid is soluble in water and water
  miscible organic solvents but insoluble in other
  organic solvents.
• (iii) It exhibits low volatility.
• (iv) The various reactions characteristic of an
  alcohol which lactic acid (or it esters or
  amides) may undergo are xanthation with carbon
  bisulphide, esterification with organic acids and
  dehdrogenation or oxygenation to form pyruvic
  acid or its derivatives.
• (v) The acid reactions of lactic acid are those
  that form salts and undergo esterification with
  various alcohols.
• (v) Liquid chromatography and its various
  techniques can be used for quantitative analysis
  and separation of its optical isomers

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Properties and chemical reactions of lactic acid

• Technical grade lactic acid is used as an
  acidulant in vegetable and leather tanning
  industries.
• Various textile finishing operations and acid
  dyeing of food require low cost technical grade
  lactic acid to compete with cheaper inorganic
  acid.
• Lactic acid is being used in many small scale
  applications like pH adjustment, hardening baths
  for cellophanes used in food packaging,
  terminating agent for phenol formaldehyde resins,
  alkyl resin modifier, solder flux, lithographic
  and textile printing developers, adhesive
  formulations, electroplating and
  electro-polishing baths, detergent builders.
• Lactic acid has many pharmaceutical and cosmetic
  applications and formulations in topical
  ointments, lotions, anti acne solutions,
  humectants, parenteral solutions and dialysis
  applications, and anti carries agents.
• Calcium lactate can be used for calcium
  deficiency therapy, and as an anti caries agent.
• Its biodegradable polymer has medical
  applications as sutures, orthopedic implants,
  controlled drug release, etc.

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Properties and chemical reactions of lactic acid

• Polymers of lactic acids are biodegradable
  thermoplastics. These polymers are transparent
  and their degradation can be controlled by
  adjusting the composition, and the molecular
  weight.
• Their properties approach those of petroleum
  derived plastics. Lactic acid esters like
  ethyl/butyl lactate can be used as green
  solvents.
• They are high boiling, non-toxic and degradable
  components. Poly L-lactic acid with low degree of
  polymerization can help in controlled release or
  degradable mulch films for large-scale
  agricultural applications.
• Lactic acid was among the earliest materials to
  be produced commercially by fermentation and the
  first organic acid to be produced by
  fermentation.
• Chemical processing has offered and continues to
  offer stiff competition to fermentation lactic
  acid.
• Very few firms around the world produce it
  fermentatively, but this could change when the
  hydrocarbon-based raw material, lactonitrile,
  used in the chemical preparation becomes too
  expensive because of the increase in petroleum
  prices.

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Properties and chemical reactions of lactic acid

• Lactic acid exists in two forms, the D-form and
  the L-form. When the symbols () or (-) are used,
  they refer to the optical rotation of the acid in
  a refractometer.
• However optical rotation in lactic acid is
  difficult to determine because the pure acid has
  low optical properties.
• The acid also spontaneously polymerizes in
  aqueous solutions furthermore, salts, esters,
  and polymers have rotational properties opposite
  to that of the pure acid from which they are
  derived. All this makes it difficult to use
  optical rotation for characterizing lactic acid.
• Many organisms produce either the D-or the L-form
  of the acid. However, a few organisms such as
  Lactobacillus plantarum produce both. When both
  the D- and L- form of lactic acid are mixed it is
  a racemic mixture.
• The DL form which is optically inactive is the
  form in which lactic acid is commercially
  marketed.

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19
Uses of lactic acid

• (i) It is used in the baking industry. Originally
  fermentation lactic acid was produced to replace
  tartarates in baking powder with calcium lactate.
  Later it was used to produce calcium stearyl 2-
  lactylate, a bread additive.
• (ii) In medicine it is sometimes used to
  introduce calcium in to the body in the form of
  calcium lactate, in diseases of calcium
  deficiency.
• (iii) Esters of lactic acid are also used in the
  food industry as emulsifiers.
• (iv) Lactic acid is used in the manufacture of
  rye bread.
• (v) It is used in the manufacture of plastics.
• (vi) Lactic acid is used as acidulant/ flavoring/
  pH buffering agent or inhibitor of bacterial
  spoilage in a wide variety of processed foods. It
  has the advantage, in contrast to other food
  acids in having a mild acidic taste.
• (vii) It is non-volatile odorless and is
  classified as GRAS (generally regarded as safe)
  by the FDA.
• (viii) It is a very good preservative and
  pickling agent. Addition of lactic acid aqueous
  solution to the packaging of poultry and fish
  increases their shelf life.

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Uses of lactic acid

• (ix) The esters of lactic acid are used as
  emulsifying agents in baking foods
  (stearoyl-2-lactylate, glyceryl lactostearate,
  glyceryl lactopalmitate). The manufacture of
  these emulsifiers requires heat stable lactic
  acid, hence only the synthetic or the heat stable
  fermentation grades can be used for this
  application.
• (x) Lactic acid has many pharmaceutical and
  cosmetic applications and formulations in topical
  ointments, lotions, anti acne solutions,
  humectants, parenteral solutions and dialysis
  applications, for anti carries agent.
• (xi) Calcium lactate can be used for calcium
  deficiency therapy and as anti caries agent.
• (xii) Its biodegradable polymer has medical
  applications as sutures, orthopaedic implants,
  controlled drug release, etc.
• (xiii) Polymers of lactic acids are biodegradable
  thermoplastics. These polymers are transparent
  and their degradation can be controlled by
  adjusting the composition, and the molecular
  weight. Their properties approach those of
  petroleum derived plastics.
• (xiv) Lactic acid esters like ethyl/butyl lactate
  can be used as environment-friendly solvents.
  They are high boiling, non-toxic and degradable
  components.
• (xv) Poly L-lactic acid with low degree of
  polymerization can help in controlled release or
  degradable mulch films for large-scale
  agricultural applications.

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Fermentation for lactic acid

• The organisms which produce adequate amounts and
  are therefore used in industry are the
  homofermentative lactic acid bacteria,
  Lactobacillus spp., especially L. delbruckii.
• In recent times Rhizopus oryzae has been used.
  Both organisms produce the L- form of the acid,
  but Rhizopus fermentation has the advantage of
  being much shorter in duration further, the
  isolation of the acid is much easier when the
  fungus is used.
• Lactic acid is very corrosive and the fermentor,
  which is usually between 25,000 and 110,000
  liters in capacity is made of wood. Alternatively
  special stainless steel (type 316) may be used.
• They are sterilized by steaming before the
  introduction of the broth as contamination with
  thermophilic clostridia yielding butanol and
  butyric acid is common. Such contamination
  drastically reduces the value of the product.

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Fermentation for lactic acid

• During the step-wise preparation of the inoculum,
  which forms about 5 of the total beer, calcium
  carbonate is added to the medium to maintain the
  pH at around 5.5-6.5.
• The carbon source used in the broth has varied
  widely and have included whey, sugars in potato
  and corn hydrolysates, sulfite liquour, and
  molasses.
• However, because of the problems of recovery for
  high quality lactic acid, purified sugar and a
  minimum of other nutrients are used.
• Lactobacillus requires the addition of vitamins
  and growth factors for growth.
• These requirements along with that of nitrogen
  are often met with ground vegetable materials
  such as ground malt sprouts or malt rootlets.
• To aid recovery the initial sugar content of the
  broth is not more than 12 to enable its
  exhaustion at the end of 72 hours.
• Fermentation with Lactobacillus delbruckii is
  usually for 5 to 10 days whereas with Rhizopus
  oryzae, it is about two days.

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Fermentation for lactic acid

• Although lactic fermentation is anaerobic, the
  organisms involved are facultative and while air
  is excluded as much as possible, complete
  anaerobiosis is not necessary.
• The temperature of the fermentation is high in
  comparison with other fermentation, and is around
  45C.
• Contamination is therefore not a problem, except
  by thermophilic clostridia.

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Extraction

• Recovery is the main problem in fermentative
  lactic acid production.
• Lactic acid is crystallized with great difficulty
  and in low yield. The purest forms are usually
  colorless syrups which readily absorb water.
• At the end of the fermentation when the sugar
  content is about 0.1, the spent medium is pumped
  into settling tanks.
• Calcium hydroxide at pH 10 is mixed in and the
  mixture is allowed to settle. The clear calcium
  lactate is decanted off and combined with the
  filtrate from the slurry.
• It is then treated with sodium sulfide,
  decolorized by adsorption with activated
  charcoal, acidified to pH 6.2 with lactic acid
  and filtered.
• The calcium lactate liquor may then be spray-dried

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Extraction

• For technical grade lactic acid the calcium is
  precipitated as CaSO4.2H2O which is filtered off.
  
• It is 44-45 total acidity. Food grade acid has a
  total acidity of about 50.
• It is made from the fermentation of higher grade
  sugar and bleached with activated carbon.
• Metals especially iron and copper are removed by
  treatment with ferrocyanide.
• It is then filtered.
• Plastic grade is obtained by esterification with
  methanol after concentration.
• High-grade lactic acid is made by various
  methods steam distillation under high vacuum,
  solvent extraction etc.

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INDUSTRIAL ALCOHOL PRODUCTION
27
Introduction

• Ethyl alcohol, CH3 CH2 OH (synonyms ethanol,
  methyl carbinol, grain alcohol, molasses alcohol,
  grain neutral spirits, cologne spirit, wine
  spirit), is a colorless, neutral, mobile
  flammable liquid with a molecular weight of
  46.47, a boiling point of 78.3 and a sharp
  burning taste.
• Although known from antiquity as the intoxicating
  component of alcoholic beverages, its formula was
  worked out in 1808.
• It is rarely found in nature, being found only
  in the unripe seeds of Heracleum giganteun and H.
  spondylium.

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Properties of Ethanol

• Ethyl alcohol undergoes a wide range of
  reactions, which makes it useful as a raw
  material in the chemical industry.
• Some of the reactions are as follow
• (i) Oxidation Ethanol may be oxidized to
  acetaldehyde by oxidation with copper or silver
  as a catalyst
• (ii) Halogenation Halides of hydrogen,
  phosphorous and other compounds react with
  ethanol to replace the OH group with a halogen

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Properties of Ethanol

• (iv) Haloform Reaction Hypohalides will react
  with ethanol to yield first acetaldehyde and
  finally the haloform reaction
• (v) Esters Ethanol reacts with organic and
  inorganic acids to give esters
• (vi) Ethers Ethanol may be dehydrated to give
  ethers

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Properties of Ethanol

• (vii) Alkylation Ethanol alkylates (adds
  alkyl-group to) a large number of compounds

31
Uses of Ethanol

• (i) Use as a chemical feed stock In the chemical
  industry, ethanol is an intermediate in many
  chemical processes because of its great
  reactivity as shown above. It is thus a very
  important chemical feed stock.
• (ii) Solvent use Ethanol is widely used in
  industry as a solvent for dyes, oils, waxes,
  explosives, cosmetics etc.
• (iii) General utility Alcohol is used as a
  disinfectant in hospitals, for cleaning and
  lighting in the home, and in the laboratory
  second only to water as a solvent.
• (iv) Fuel Ethanol is mixed with petrol or
  gasoline up to 10 and known as gasohol and used
  in automobiles.

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Denatured Alcohol

• All over the world and even in ancient times,
  governments have derived revenue from potable
  alcohol. For this reason when alcohol is used in
  large quantities it is denatured or rendered
  unpleasant to drink.
• The base of denatured alcohol is usually 95
  alcohol with 5 water for domestic burning or
  hospital use denatured alcohol is dispended as
  methylated spirit, which contains a 10 solution
  of methanol, pyridine and coloring material.
• For industrial purpose methanol is used as the
  denaturant.
• In the United States alcohol may be completely
  denatured (C.D.A. completely denatured alcohol)
  when it cannot be used orally because of a foul
  taste or four smelling additives.
• It may be specially denatured (S.D.A. specially
  denatured alcohol) when it can still be used for
  special purposes such as vinegar manufacture
  without being suitable for consumption.

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Manufacture of Ethanol

• Ethanol may be produced by either synthetic
  chemical method or by fermentation.
• Fermentation was until about 1930 the main means
  of alcohol production.
• In 1939, for example 75 of the ethanol produced
  in the US was by fermentation, in 1968 over 90
  was made by synthesis from ethylene.
• Due to the increase in price of crude petroleum,
  the source of ethylene used for alcohol
  production, attention has turned worldwide to the
  production of alcohol by fermentation.
• Fermentation alcohol has the potential to replace
  two important needs currently satisfied by
  petroleum, namely the provision of fuel and that
  of feedstock in the chemical industry.
• The production of gasohol (gasoline alcohol
  blend) appears to have received more attention
  than alcohol use as a feed stock.
• Nevertheless, the latter will also surely assume
  more importance if petroleum price continues to
  ride.

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Manufacture of Ethanol

• Governments the world over have set up programs
  designed to conserve petroleum and to seek other
  energy sources.
• One of the most widely publicized programs
  designed to utilize a new source of energy is the
  Brazilian National Ethanol Program. Set-up in
  1975, the first phase of this program aims at
  extending gasoline by blending it with ethanol to
  the extent of 20 by volume.
• The United States government also introduced the
  gasoline programme based on corn fermentation in
  1980 following the embargo on grain sales to the
  then Soviet Union.

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Substrates

• The substrate used will vary among countries.
• In Brazil sugar cane, already widely grown in the
  country, is the major source of fermentation
  alcohol, while it is planned to use cassava and
  sweet sorghum.
• In the United States enormous quantities of corn
  and other cereals are grown and these are the
  obvious substrates.
• Cassava grows in many tropical countries and
  since it is high yielding it is an important
  source in tropical countries where sugar cane is
  not grown.
• It is recognized that two important conditions
  must be met before fermentation alcohol can play
  a major role in the economy either as gasohol or
  as a chemical feedstock.
• First, the production of the crop to be used must
  be available to produce the crop without
  extensive and excessive deforestation.
• Secondly, the substrate should not compete with
  human food.

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Fermentation

• The sterilized fermentable sugars are pumped or
  allowed to flow by gravity into fermentation
  tanks and yeast is inoculated or pitched in at
  a rate of 7-15 x 106 yeast cells/ml, usually
  collected from a previous process.
• These broths are inoculated with up to 5 (v/v)
  of thick yeast broth.
• Although yeast is re-used there is still a need
  for regular inocula.
• In general the inocula are made of selected
  alcohol-tolerant yeast strains usually Sacch.
  cerevisiae grown aerobically with agitation and
  in a molasses base.
• Progressively larger volumes of culture may be
  developed before the desired volume is attained.
• When the nitrogen content of the medium is
  insufficient nitrogen is added usually in the
  form of an ammonium salt.
• As in all alcohol fermentations the heat
  released must be reduced by cooling and
  temperatures are generally not permitted to
  exceed 35-37C.
• The pH is usually in the range 4.5-4.7, when the
  buffering capacity of the medium is high.
• Higher pH values tend to lead to higher glycerol
  formation.
• When the buffering capacity is lower, the initial
  pH is 5.5 but this usually falls to about 3.5.
  During the fermentation contaminations can have

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Distillation

• After fermentation the fermented liquor or beer
  contains alcohol as well as low boiling point
  volatile compounds such as acetaldeydes, esters
  and the higher boiling, fusel oils.
• The alcohol is obtained by several operations.
• First, steam is passed through the beer which is
  said to be steam-stripped.
• The result is a dilute alcohol solution which
  still contains part of the undesirable volatile
  compounds.
• Secondly, the dilute alcohol solution is passed
  into the center of a multi-plate aldehyde column
  in which the following fractions are separated
  esters and aldehydes, fusel oil, water, and an
  ethanol solution containing about 25 ethanol.
• Thirdly, the dilute alcohol solution is passed
  into a rectifying column where a constant boiling
  mixture, an azeotrope, distils off at 95.6
  alcohol concentration.

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Distillation

• To obtain 200 proof alcohol, such as is used in
  gasohol blending, the 96.58 alcohol is obtained
  by azeotropic distillation.
• The principle of this method is to add an organic
  solvent which will form a ternary
  (three-membered) azeotrope with most of the
  water, but with only a small proportion of the
  alcohol.
• Benzene, carbon tetrachloride, chloroform, and
  cyclohezane may be used, but in practice, benzene
  is used.
• Azeotropes usually have lower boiling point than
  their individual components and that of
  benzene-ethanol-water is 64.6C.
• On condensation, it separates into two layers.
• The upper layer, which has about 84 of the
  condensate, has the following percentage
  composition benzene 85, ethanol 18, water 1.
• The heavier, lower portion, constituting 16 of
  the condensate, has the following composition
  benzene 11, ethanol 53, and water 36.

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Distillation

• In practice, the condensate is not allowed to
  separate out, but the arrangement of plates
  within the columns enable separation of the
  alcohol. Four columns are usually used.
• The first and second columns remove aldehydes and
  fusel oils, respectively, while the last two
  towers are for the concentration of the alcohol.
• A flow diagram of conventional absolute alcohol
  production from molasses is given in Fig. 20.4

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Distillation
41
Some Developments in Alcohol Production

• Due to the current interest in the potential of
  ethanol as a fuel and a chemical feedstock,
  research aimed at improving the conventional
  method of production has been undertaken, and
  more will, most certainly, be undertaken. Some of
  the techniques aimed at improving productivity
  are the following
• (i) Developments of new strains of yeast of
  Saccharomyces uvarum able to ferment sugar
  rapidly, to tolerate high alcohol concentrations,
  flocculate rapidly, and whose regulatory system
  permits it to produce alcohol during growth.
• (ii) The use of continuous fermentation with
  recycle using the rapidly flocculating yeasts.
• (iii) Continuous vacuum fermentation in which
  alcohol is continuously evaporated under low
  pressure from the fermentation broth.
• (iv) The use of immobilized Saccharomyces
  cerevisiae in a packed column, instead of in a
  conventional stirred tank fermentor. Higher
  productivity consequent on a higher cell
  concentration was said to be the advantage.

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Some Developments in Alcohol Production

• (v) In the Ex-ferm process sugar cane chips are
  fermented directly with a yeast without first
  expressing the cane juice.
• The chips may be dried and used in the offseason
  period of cane production.
• It is claimed that there is no need to add
  nutrients as would be the case with molasses,
  since these are derived from the cane itself.
• A more complete extraction of the sugar,
  resulting in a 10 increase in alcohol yield, is
  also claimed.
• (vi) The use of Zymomonas mobilis, a
  Gram-negative bacterium which is found in some
  tropical alcoholic beverages, rather than yeast
  is advocated.
• The advantages claimed for the use of Zymomonas
  are the following
• (a) Higher specific rates of glucose uptake and
  ethanol production than reported for yeasts.
• Up to 300 more ethanol is claimed for Zymomonas
  than for yeasts in continuous fermentation with
  all recycle.

43
Some Developments in Alcohol Production

• (b) Higher ethanol yields and lower biomass than
  with yeasts.
• This deduction is based on Fig. 20.5 where,
  although the same quantity of alcohol is produced
  by the two organisms in 30-40 hours, the biomass
  of Zymomonas required for this level of
  production is much less than with yeast.
• The lower biomass appears to be due to the lower
  energy available for growth.
• Zymomonas utilized glucose by the
  Enthner-Duodoroff pathway (Fig. 5.4) which yields
  one mole of ATP/mole glucose, whereas yeasts
  utilize glucose anaerobically via the glycolytic
  pathway (Fig. 5.1) to give two ATP/mole glucose.
  Its use does not appear to have gained general
  acceptance.

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Some Developments in Alcohol Production

• (c) Ethanol tolerance is at least as high or even
  higher up to 16 (v/v) in some strains of the
  bacterium than with yeast.
• (d) Zymomonas also tolerates high glocuse
  concentration and many cultures grow in sugar
  solutions of up to 40 (w/v) glucose which should
  lead to high ethanol production.
• (e) Zymomonas grows anaerobically and, unlike
  yeasts, does not require the controlled addition
  of oxygen for viability at the high cell
  concentrations used in cell recycle.
• (f) The many techniques for genetic engineering
  already worked out in bacteria can be easily
  applied to Zymomonas for greater productivity.

45
Some Developments in Alcohol Production
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