Okafor N. Modern Industrial Microbiology and Biotechnology

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deficiency therapy, and as an anti caries agent. Its biodegradable polymer has medical

applications as sutures, orthopedic implants, controlled drug release, etc. 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.

Table 20.1 Physical properties of ethyl alcohol

Boiling point 78.2

Explosive limit in air, vol % 43-19.0

Freezing point 114.1

Specific gravity at 20/20

C 0.7905

Surface tension at 20

C dynes/cm 22.3

Vapor pressure at 20

C mg/HG 44

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.

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.

20.1.6.2 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.

Production of Organic Acids and Industrial Alcohol !%

(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.

(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.

Table 20.2 Physical properties of lactic acid

Appearance Yellow to colorless crystals or syrupy 50% liquid

Melting point 16.8°C

Relative density 1.249 at 15°C

Boiling point 122° @ 15 millimeter

Flash point 110°C

Solubility Soluble in water, alcohol, furfurol

Slightly soluble in ether

Insoluble in chloroform, petroleum ether, and carbon

disulfide

20.1.6.3 Fermentation for lactic acid

Although many organisms can produce lactic acid, the amount so produced is small: 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

!% Modern Industrial Microbiology and Biotechnology

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.

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.

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 45°C. Contamination is therefore not a problem, except by thermophilic

clostridia.

20.1.6.4 Extraction

The main problem in lactic acid production is not fermentation but the recovery of the

acid. 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 beer 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.

For technical grade lactic acid the calcium is precipitated as CaSO

.2H

O 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.

Production of Organic Acids and Industrial Alcohol !%!

20.2 INDUSTRIAL ALCOHOL PRODUCTION

Ethyl alcohol, CH

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.

20.2.1 Properties of Ethanol

Some of the physical properties of ethanol are given in Table 20.1

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:

Cu, Ag

OH CH

CHO + H

(ii) Halogenation: Halides of hydrogen, phosphorous and other compounds react with

ethanol to replace the – OH group with a halogen:

3CH

OH + PCl

3CH

Cl + P (OH)

(iii) Reaction with metals: Ethanol reacts with sodium, potassium and calcium to give the

alcoholates (alkoxides) of these metals:

2CH

OH + 2Na 2CH

ONa + H

(iv) Haloform Reaction: Hypohalides will react with ethanol to yield first acetaldehyde

and finally the haloform reaction:

OH + NaOCl CH

CHO + NaCl + H

CHO + 2NaOCl CCl

CHO + 3NaOH

CCl

CHO + NaOH CHCl

+ HCOONa

Chloroform

(v) Esters: Ethanol reacts with organic and inorganic acids to give esters:

OH + HCl CH

Cl + H20

Ethylchoride

(vi) Ethers: Ethanol may be dehydrated to give ethers:

Catalyst

2CH

OH CH

OCH

+ H

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

:CH

HSO

(ethyl hydrogen sulfate)

:CH

(ethyl amine)

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20.2.2 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.

20.2.3 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 dispened 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.

20.2.4 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. The production of alcohol from ethylene is

discussed in Chapter 13.

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. 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.

Production of Organic Acids and Industrial Alcohol !%#

20.2.4.1 Substrates

The various substrates which may be used for ethanol production have been discussed in

Chapter 2. It is clear that the substrate used will vary among countries. Thus, 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.

20.2.4.2 Fermentation

The conditions of fermentation for alcohol production are similar to those already

described for whisky or rum production. Alcohol-resistant yeasts, strains of

Saccharomyces cerevisiae are used, and nutrients such as nitrogen and phosphate lacking

in the broth are added.

20.2.4.3 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.

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 64.6°C. 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%.

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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Fig. 20.4 Flow Diagram of Conventional Alcohol Production

YEAST

CULTURE

MOLASSES OR

CANE JUICE

PREPARATION

OF INOCULUM

WEIGHING

BRIX

ADJUSTMENT

TO 22

WORT

FERMENTATION

RECUPERATION OF YEAST

CENTRIFUGATION

FERMENTED

WORT

(7–8%V/V)

CENTRIFUGED MEDIUM

BENZENE

DECANTATION

RECYCLE

(TO RECTIFICATION)

(RECUPERATION

OF BENZENE)

ABSOLUTE

ALCOHOL

(DEHYDRATION)

(RECTIFICATION)(DISTILLATION)

STILLAGE

ALCOHOL

(40% - 50% V/V)

ALCOHOL

(96% V/V)

WATER

FUSEL

OIL

Production of Organic Acids and Industrial Alcohol !%%

20.2.5 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.

(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 off-

season 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.

(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.

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.

!%& Modern Industrial Microbiology and Biotechnology

Fig. 20.5 Alcohol Production by Yeast and

Zymomonas mobilis

Production of Organic Acids and Industrial Alcohol !%'