Biotechnological Innovations and Alternative Uses for the

Sugar waste-stream, Molasses.

Report Prepared by: Kevin ByrneCaitríona DalyYvonne Anders

Subject: Waste Management.Lecturer: Dr. Michael Broaders.Date of submission: 16/12/’05

Contents.

Chapter Page Number

Introduction………………………………………………………………….3Production of MolassesTypes of MolassesBoston Molasses Flood (History)

Primary Uses of Molasses……………………………………………….12Silage Preservation

Biotechnological Uses of Molasses…………………………………….17

Bioethanol……………………………………………………………..17Production ProcessSuitable MicroorganismsUses of Bioethanol

SCP……………………………………………………………………26Production ProcessCase Study – Indonesia Repetila III

Citric Acid……………………………………………………………..34

Bioremediation Using Molasses………………………………………..39

Conclusion………………………………………………………………….42

References………………………………………………………………….43

2

Introduction.

Molasses is a liquid sugar extracted from the juice of the sugar cane or sugar

beets. In making refined table sugar, the sugar cane is harvested, mashed and

the raw juices are boiled to extract the sugar grains for further processing. The

syrup is placed into a very large pan for boiling, the last stage. In the pan even

more water is boiled off until conditions are right for sugar crystals to grow. You

may have done something like this at school but probably not with sugar because

it is difficult to get the crystals to grow well. In the factory the workers usually

have to throw in some sugar dust to initiate crystal formation. Once the crystals

have grown the resulting mixture of crystals and mother liquor is spun in

centrifuges to separate the two, rather like washing is spin dried. The crystals are

then given a final dry with hot air before being stored ready for despatch. (1) The

remaining syrup is called first molasses. First molasses is then thinned with water

and re-boiled so that more raw sugar can be extracted. The leftover syrup from

the second boiling is second molasses, and so on. After three or more boilings,

molasses is called blackstrap, which has a bitter taste, and is used primarily for

cattle feed and industrial purposes.

Molasses is sold in a variety of grades. The highest grade is made from clarified

and reduced pure sugarcane juice, before the sugar extraction process begins.

The Grandma Molasses Company sells this as their “gold label molasses.” Their

“green label” is the first molasses described above. Either high grade or first

molasses is appropriate for cooking. Molasses from sugar beets has a bitter

taste, and is not sold for consumption.

Some manufacturers will make sugar and molasses from sugar cane which is

picked unripe and then ripened through exposure to sulphur. In that case,

molasses will be labelled “sulphured.” Although cheaper, try not to buy sulphured

molasses – the sulphur taste is especially retained in the syrup.

3

Sugar Beet (Beta Vulgaris) is a member of the order Chaenopodeacae. It has

been developed by genetic selection from the commonly found wild plant sea

beet, strains of the species with large root size and high sugar content being

developed over time.

Fig1.1. World Map of Sugar cane and Beet Production

Sugar Beet Molasses

Molasses from the sugar beet is different from cane molasses. Only the syrup left

from the final crystallisation stage is called molasses; intermediate syrups are

referred to as high green and low green. It is about 50% sugar by dry weight,

predominantly sucrose but also containing significant amounts of glucose and

fructose. The non-sugar content includes many salts such as calcium, potassium,

oxalate and chloride. As such, it is unpalatable and is mainly used as an additive

to animal feed or as a fermentation feedstock. It is possible to extract additional

sugar from beet molasses through a process known as molasses desugarisation.

The processing starts by slicing the beets into thin chips. This process increases

4

the surface area of the beet to make it easier to extract the sugar. The extraction

takes place in a diffuser where the beet is kept in contact with hot water for about

an hour. The diffuser is a large horizontal or vertical agitated tank in which the

beets slices slowly work their way from one end to the other and the water is

moved in the opposite direction. This technique exploits industrial scale

chromatography to separate sucrose from non sugar components. The technique

is only economically viable in areas where the price of sugar is supported above

the world market. The non-sucrose elements in the beet sugar production

process are called molassegenic because they take equal amounts of sucrose

with them to the molasses stage where it cannot normally be economically

extracted.

Fig.1.2 The Sugar Beet Plant

There are three major types of molasses: unsulphured, sulphured and

blackstrap. There are also three major grades of molasses: first molasses,

second molasses, and blackstrap molasses. The Unsulphured molasses is the

finest quality. It is made from the juice of sun-ripened cane and the juice is

clarified and concentrated. Sulphured molasses is made from green (not yellow)

sugar cane and is treated with sulphur fumes during the sugar extraction

process. Each season, the sugar cane plant is harvested and stripped of its

5

leaves. Its juice is then extracted from the canes (usually by crushing or

mashing), boiled until it has reached the appropriate consistency, and processed

to extract the sugar. The results of this first boiling and processing is first

molasses, which has the highest sugar content because comparatively little

sugar has been extracted from the juice. Second molasses is created from a

second boiling and sugar extraction, and has a slight bitter tinge to its taste.

Further rounds of processing and boiling yield the dark blackstrap molasses,

which is the most nutritionally valuable, and thus often sold as a health

supplement, as well as being used in the manufacture of cattle feed, and for

other industrial uses.

Fig. 1.3 The Sugar Cane Plant

6

Harvesting

Harvesting is carried out using special sugar Beet harvesters. These machines

lift the plants from the ground, remove the tops and clean and convey the roots to

a storage hopper or directly to a trailer being driven alongside. Sugar Beet yields

on average, 40 tonnes of roots and 25-30 tonnes of tops per ha.

Fig. 1.4 Section through a Beet Chopper Harvestor

Typical yields of Sugar Cane are generally around 40-50 tonnes of Cane per

hectare. Harvesting the Sugar Cane crop is different to the harvest of Sugar Beet

crops. The crop is harvested straight off the surface whereas Sugar Beet is

plucked straight out of the ground. The biggest challenge for the industry has

been to become internationally competitive against low-cost countries like Brazil,

rationalisation has seen changes to sugar marketing and hundreds of farmers

7

have left the industry. But at least cane growers have a positive outlook to

undertake further reform. (3)

Fig 1.5. A crop of Sugar Cane being Harvested

PaymentFarmers are payed by Sugar factories on the basis of (a) weight of washed roots

and (b) sugar content. The price per tonne is based on a sugar content of 16%.

Bonuses and reductions are made for sugar contents above or below this value.

HistoryMolasses has been imported into the United States from the Caribbean Islands

since the time of the early colonists. It was the most popular sweetener used until

the late 19th century since it was much more affordable than refined sugar, which

was very expensive at that time.

8

Blackstrap molasses gained in popularity in the mid-20th century with the advent

of the health food movement. Today, the largest producers of molasses are India,

Brazil, Taiwan, Thailand, the Philippines and the United States.

Recently the E.U commissioner for agriculture Peter Mandelson has been trying

to force reforms of Sugar Beet production in Europe. At present our minister for

Agriculture, Minister Coughlan has been negotiating with the 11 States which are

currently opposed to the reforms. It was the same principle that applied to the

Milk Quota across countries like Canada but now in the sugar case, Europe has

taken a much tougher line with its sugar producers than Canada has with its dairy

farmers. Canada simply reduced the quota but maintained the price. Europe is

effectively forcing whole regions out of sugar production and bringing its price

down much nearer to that of sugar cane. For the rest of the tillage sector, Europe

is already at or below world prices according to an article in the December 3 rd

edition of the Farmers Journal.

The EU are now looking for more efficient and cost effective ways of producing

sugar which in time will wipe out the whole Beet industry in Ireland and Europe,

and may eventually lead to global wipeout of the whole Sugar Beet industry.

9

Boston Molasses Flood.

Other catastrophes destroyed more houses and killed more people. 21

dead people (choked or crushed) and one million dollar damage to property- that

is peanuts in the registration of world- shaking adversities. But, in so far as

aesthetics are concerned, it can hardly be outdone: Bostons big molasses flood.

It happened on the 15th of January 1919. A big steel tank (15 m high and 27m

diameter) standing in the harbor of Boston.

Almost 9 million litre raw molasses were waiting to be decanted.

Around lunchtime a deep flicking bang was hear- the bottom plate of the

molasses tank burst.

Steel anchors left their anchorages, more plates got blown off and a huge

plethora of brown, tenacious molasses poured out from the tank.

The molasses made its way into town. It ran through the streets at an estimated

35 MPH (60 km/h) A six meter high molasses wave wallowed through the harbor

of Boston towards a residential area.

Things that were in the way of the wave, people, railway wagons, horse carts got

cleared away.

The stream rooted out foundations of wood houses and pushed them away.

Doors and windows of more stable built houses got pushed in, basements and

floors got filled up metres high with the sticky pulp. Who was not able to run away

fast enough got sucked into the pulp.

Passersby, who helped the unlucky stuck people, got sucked to the “molasses

covered victims” themselves and got roped in the stream. Who wanted to wipe off

the molasses on the body by hands made everything worst.

Also, the local fire brigade did not have any miracle cure against the molasses

ready, they got stuck like everybody else.

After three hours the stream stopped moving.

10

Those who could be rescued from the waist-high molasses, had to be cut out of

the solidified clothes outright.

Any hopelessly covered horses got shoot dead.

The clean up took months. The whole harbor smelled after molasses and fouling

cadavers for months.

Molasses had to be pumped out from houses. Walls and streets got hosed down

from molasses.

After recovering from the fright people asked themselves how it came that the

accident occurred? First of all people thought the molasses could have exploded

due to natural reasons.

After six years of in depth analysis experts had an answer- highly unpleasant for

the company that was producing the tank. For the building of the tank too thin

steel plates were apparently used, that was less expensive. Nobody ever verified

the plans or even the tank itself, and nobody ever heard from double walled

security tanks since then. REF 29

Figure 2.The Boston Molasses Disaster

11

Silage Preservation - (Biochemical Processes Involved)When freshly cut green vegetation is made into a heap, it continues to respire until all the

oxygen available is used up. Respiration involves oxidation of carbohydrates to carbon

dioxide which means a loss of food value in the vegetation. Thus, as much of the air as

possible should be squeezed out of the vegetation. This is done by rolling the silage heap

with a tractor. A polythene sheet of appropriate size is pulled tightly over the heap of

silage to complete the sealing. When the oxygen is used up and the environment

anaerobic, bacteria present on the vegetation begin to cause fermentation, which is the

conversion of carbohydrate to organic acids (Lactic Acid). This continues until the pH

falls to a certain level at which all microbial activity is inhibited and the material is

“Pickled” or preserved. This is known as controlled fermentation through anaerobic

respiration. (2)

When the concentration of carbohydrates in the vegetation is high, the bacteria

which control the fermentation process are Lacto-bacillus and Streptococcus

species. The acid produced is Lactic Acid. Lactic Acid Silage is palatable to stock,

highly nutritious and can be kept safely for years provided air is excluded from it.

Packing forage quickly and tightly helps to eliminate residual oxygen. The low pH

stops plant enzymatic activity and further microbial metabolism which preserves

the forage as silage assuming that oxygen is not allowed to penetrate the mass

as Breen and Mullen state in their 1992 edition of Agricultural Science. (2)

If, on the other hand, the concentration of carbohydrates is low, fermentation is

controlled by clostridium which leads to the formation of Butyric Acid. Butyric Acid

silage is unpalatable to stock, less nutritious than lactic acid silage and far less

stable, lasting only a few months.

Advantages of Including Molasses In Animal Feeds:

12

It is frequently claimed that small amounts of molasses in a roughage-based diet

stimulate rumen fermentation. The most appropriate role for small amounts of

molasses in ruminant diets is as a vehicle for other nutrients (e.g. urea and

minerals). A drought feeding strategy based on the use of liquid molasses

supplements containing from 8 to 10 percent urea is now an established practice

in Australia (Nicol et al., 1984) and has been introduced sucessfully in Africa

(Preston and Leng, 1986). (11)

The incorporation of urea and other nutrients in molasses-based (multi-

nutritional) blocks promises to be an even more attractive technology, especially

for smallholder-village farmers, for supplementation of locally available crop

residues which are of low digestibility and also deficient in fermentable nitrogen

(Leng and Preston, 1984; Sansoucy et al., 1986). (11)

Molasses can improve the taste of a food simply by having traces in the solution.

It reduces the dust element in the food as well because of its stickiness. Dust

may also result in feed wastage. Literature reports have shown that molasses

practically eliminated 10% all dust and eliminated 30% fine particles. It is used

for pelleting feeds for poultry and farm animals. The cost of molasses is

outweighed in the long run when you look at the returns and profits.

13

Nutritional Uses of Molasses In The DietMolasses may be fed to livestock in several ways such as molassed meal,

molasses blocks, and liquid form to provide energy directly or be used as a

carrier for non-protein nitrogen, vitamins and minerals as well as medicinal

compounds.

Molasses Blocks- While the formulations were being tested for setting and

hardness, they were also fed to animals. When placed in feed troughs, animals

had a tendency to move the blocks about and at times to bite off big chunks.

Treacle Cakes- Treacle is a British term, a generic word for any syrup made in

the process of refining sugar cane and it can range from very light to very dark.

However, when used in cooking, treacle has come to mean the equivalent of dark

molasses.

Molasses- A renewable Fuel FeedstockWith the inevitable depletion of the world's petroleum supply, there has been an

increasing worldwide interest in alternative, non-petroleum-based sources of

energy. A growing source of transportation fuel worldwide is fermentation derived

bio-ethanol. As well as reducing green-house gases, bio-ethanol produces less

harmful emissions during combustion than its fossil fuel equivalents. Bio-ethanol

is produced from a wide variety of raw materials – including cornstarch, sugar

cane and lingo-cellulosic waste materials. The main cost element in bio-ethanol

production is the feedstock. The use of alternative types of biomass as a source

of carbohydrates for fermentation to ethanol has been studied by many

investigators worldwide. Potential cheap feedstocks include lingo-cellulosic

biomass from urban and industrial waste and from wood/agricultural residues.

Researchers at the Institute of Technology Carlow have investigated the

conversion of a variety of waste materials to ethanol – including grasses, cereal

straws, newspaper and waste office paper. This research has now been

expanded to include sugar processing waste.

14

1.34 million tonnes of sugar beet are grown in Ireland each year by 3,700

growers based mainly in the southeast of the country. The sugar beet is

processed at two plants, one in Carlow and one in Mallow, which yields 200,000

tonnes of sugar. The main by-products of sugar processing are molasses and

sugar beet pulp. (8) The Carlow factory produces 25,000 tonnes of molasses and

80,000 tonnes of sugar beet pulp annually. Molasses and pulp are currently used

as animal feed. However, as pulp and the molasses contain substantial amounts

of carbohydrates, they have potential as a raw material for the production of fuel

ethanol or bio-ethanol. Molasses contains 50% sucrose, with smaller amounts of

fructose and glucose. Sugar beet pulp contains approximately 30% cellulose,

30% hemi-cellulose, and 30% pectin.

The research at Carlow is concentrating on the enzymatic hydrolysis of the sugar

beet pulp to its constituent monomeric sugars – which include glucose, fructose,

mannose, galactose, xylose and arabinose. While technology for fermentation of

single sugars (particularly glucose) is very well developed, the fermentation of

complex mixtures of sugars which include pentoses is not. The use of

unconventional fermenting microorganisms to maximize ethanol production is

being investigated. The ultimate aim is to work towards the development of new

microbial strains with improved properties for the conversion of such waste-

derived sugar hydrolysates to ethanol.

Legislation:

The Molasses Act of 1773 was a tariff passed by England to try to discourage the

colonists from trading with areas of the West Indies that were not under British

rule. This legislation is thought to be one of the events that catalyzed pre-

revolutionary colonial dissent and unrest. The Act was passed by Parliament at

the request of planters of the British West Indies placing a heavy duty on all

sugar, molasses, and rum imported into the American colonies from non-British

islands in the Caribbean. The British West Indies feared that American trade with

15

other islands would destroy the British sugar industry. The Molasses Act proved

ineffective owing to colonial smuggling and Parliament's failure to enforce it.

The Sugar Act of 1764 was a measure passed by Parliament placing heavy

duties on sugar, textiles, coffee, indigo, and wine imported to the American

colonies from foreign countries to help pay the costs of keeping British troops in

America. It lowered the duty on molasses to discourage the smuggling that had

followed the Molasses Act of 1733, but raised the duty on refined sugar and other

foreign goods. Unlike the Molasses Act, the Sugar Act was strictly enforced.

Colonists saw it as unnecessary taxation, and it was one of the causes of the

American Revolution.

16

Production Of Ethanol using Biotechnology.

While bioethanol production is well documented and has moved from a

laboratory scale to commercial production. The use of a molasses substrate is

still in research stage. However there have been many recent advances in this

particular field and also certain issues that have pushed for an increase in

bioethanol production (regardless of its origins). The main issue concerning

bioethanol production has been the steady, inevitable, decrease in the supply of

fossil fuels. An accepted alternative to fossil fuels are biofuels. Within the scope

of “biofuels” is the use of bioethanol as a fuel. The EU Biofuels Directive

(2003/30/EC) has also caused increased awareness of this problem and also

calls for alternatives to be found. It calls on member states to include minimum

levels of biofuel or other renewable in their transport fuel supplies and sets non-

mandatory targets;

• 31/12/05 – 2% biofuels in overall transport fuels.

• 31/12/10 – 5.75% biofuels in overall transport fuels.

According to a recent report by Sustainable Energy Ireland (SEI), Ireland could

meet approximately 23% of the 2010 target without relying on imports and

assuming no major changes to agricultural practices and landuse. This would

mean that of the transport fuels being used, biofuels would account for 1.3225%.

This is a very low proportion considering the availability of the necessary raw

materials for bioethanol. Molasses, an agricultural waste, could be utilised to

produce bioethanol.

17

Production Process.Bioethanol is produced by the fermentation of beet or sugar cane molasses by a

suitable microorganism in a fermentor. The ethanol formed in the fermentor is

recovered by operating a primary distillation step in a circulation circuit with the

fermentor. A stream of fermentation liquor with an ethanol content of 3-7% by

weight and a content of fermentable material less than 2% by weight is

continuously withdrawn from the fermentor. The aforementioned stream is

separated in a centrifugal separation step into a microorganism enriched stream

and a liquid stream. This microorganism-rich stream is then recirculated to the

fermentor, into an essentially microorganism-free stream. The primary distillation

step is repeated, i.e. it is separated into an ethanol enriched top stream and a

residual liquid stream, a part of which is recirculated to the fermentor and the

remaining part is supplied to a secondary distillation step. The secondary

distillation step involves stripping off remaining ethanol and forming a

concentrated ethanol free stillage. The stillage is discharged. The ethanol-

containing vapour stream is collected and, along with the first fermentation

stream, is condensed to yield liquid ethanol. In a long time fermentation of

molasses at steady-state concentration in the range 10-18% by weight dry solids

content in the fermentor, an ethanol productivity of 8-15 kg ethanol per hour and

1m3/hr fermentor liquid is generally achieved [20]. The productivity will also

depend on the type of microorganism used in the process.

18

Suitable Microorganisms.Bioethanol is ethanol that has been produced by a certain type of

microorganism. Microorganisms that can produce ethanol are known as

ethanologenic organisms. Ethanologenic microorganisms include; Aspergillus

niger (mould/higher fungi), Kluyveromyces marxianus (yeast),

Schizosaccharomyces pombe (yeast), Saccharomyces cerevisiae (yeast),

Clostridia spp. (bacteria) and Zymomonas mobilis (bacteria). There is great

diversity among them, i.e. yeasts, moulds, bacteria. Saccharomyces cerevisiae is

the most commonly used organism for bioethanol production, hence, more is

known about its specific qualities and requirements. Bioethanol production is of

interest as the microorganisms used can utilise many types of substrate in their

growth and ethanol production. Bioethanol can be produced from a wide variety

of raw materials including cornstarch, sugar cane, sugar beet and lignocellulose

waste materials. As waste streams from various industries, e.g. sugar refining,

can be utilised and thus turned into a further resource, and also reducing waste,

the bioethanol production process is of great importance. The use of alternative

types of biomass as a source of carbohydrates for fermentation to ethanol has

been studied by many investigators worldwide.

The choice of substrate used is an important deciding factor in bioethanol

production. It is generally accepted that the greatest cost involved in bioethanol is

that of the substrate. By using waste materials e.g. molasses, as the substrate

material this cost can be significantly reduced. The main requirement of the

substrate is that it is; carbohydrate-rich, contains nutrients, easily broken down by

microorganisms. Molasses meets all of these requirements. It is a carbohydrate

and sugar rich compound. It is made up of 50% sugars and …..

The production of bioethanol using molasses is a simple two-step process. The

molasses must first undergo enzymatic hydrolysis to break it down into its

constituent monomeric sugars. The sugars that make up 50% of molasses are:

sucrose (50-60%), glucose (25-35%), fructose (20-30%), and mannose,

galactose, xylose and arabinose (1-5%). Once the molasses has been broken

down, the microorganisms can then ferment the various monomeric sugars to

19

ethanol. Depending on the microorganism being used, the conditions for ethanol

production will vary.

Of the ethanologenic species already mentioned, extensive research has

been done on the utilisation of molasses substrates by K. marxianus

[14,15,16,17] and, the use of high glucose-content substrates by Z. mobilis

[18,19].

Kluyveromyces marxianus is a thermotolerant ethanol-producing yeast.

Studies have been conducted using it to produce ethanol from a molasses

substrate [14,15,16,17]. In these laboratory assessments, a strain of K.

marxianus known as IMB3 was used. Many factors that might affect the

efficiency and growth of K. marxianus IMB3 such as; substrate concentration,

type of immobilisation, pre-treatment; were investigated by S. Gough et al.

Fermentation of the molasses occurred at 45oC. The cells were immobilised in

either calcium alginate gel or poly(vinyl alcohol) cryogel (PVAC) beads. During

the first investigation, molasses originating in Guatemala, Honduras, Senegal,

Guyana and the Philippines was used [14]. The molasses used in this

assessment was produced from the sugar cane industry. The molasses was

diluted to yield a sugar concentration of 140g/L.The dilution factor would vary for

each molasses due to the nature of its sugar content. Fermentations were then

carried out as outlined above. During the first 24 hours, the maximum ethanol

concentrations obtained ranged from 43-57g/L with optimum production on the

molasses from Honduras. Ethanol production, during subsequent re-feeding of

the fermentations at 24 hour intervals over a 120-hour period, decreased steadily

to concentrations ranging from 20-36g/L. It was found that ethanol productivity

remained highest in fermentations containing the molasses from Guyana. When

each set of fermentations was re-fed again at 120 hours and allowed to continue

for 48 hours, the ethanol production was found to increase again with maximum

concentrations ranging from 25-52g/L. However, it was also found that increasing

the time between re-feeding at this stage had a detrimental effect on the

functionality of the biocatalyst (K. marxianus IMB3) [14]. This study shows the

20

varying efficiencies of molasses taken from different areas. Ethanol production in

this manner, using molasses, is proven to be quite efficient.

Further assessments conducted by S. Gough et al found that the

efficiencies of the continuous flow bioreactor ranged from 31-76% [15]. A

maximum ethanol concentration was reached after 10 hours of fermentation

using molasses diluted to yield a sugar concentration of 140g/L [15]. A residence

time within the bioreactor of 0.66hours was deemed to be most efficient when

dealing with a fixed molasses sugar concentration of 140g/lL [15].

Immobilisation Max. Ethanol Yield(First cycle)

Max. Yield reached:

Average Yield over prolonged period

Calcium alginate gel 57g/L

(80%Ty)

72hrs 21g/L

PVAC beads 52-53g/L

(73%Ty)

72hrs 45g/L

Ty – Theoretical Yield.Table1 : Efficiency of Different Immobilisation Media using K. marxianus fermentation at 45oC on molasses substrate.

A study investigating the most suitable immobilisation media for K.

marxianus IMB3 found that a PVAC-based immobilised system may provide a

more practical alternative to alginate for the production of ethanol in continuous

or semi-continuous fermentation systems [16]. Table 1 above shows the findings

of this study. It illustrates that although calcium alginate gel will give a higher

initial yield (80%Ty), on an ongoing basis, the use of PVAC beads will give a

better return.

A second microorganism that has been found to be suitable for growth on

molasses substrate is Z. mobilis. It is a gram-negative bacterium, and has been

considered as an alternative organism in large-scale fuel ethanol production. It

grows anaerobically and so does not require the controlled addition of oxygen to

21

maintain viability at high cell concentrations. Much of the research relating to Z.

mobilis has arisen from the brewing industry as alternatives to the traditional

yeast strains, e.g. Saccharomyces uvarum, formerly carlsbergerensis, were

sought. Detailed comparative research of Z. mobilis and commercial yeast was

undertaken by Jacek Nowak of the Agricultural University of Poznań, Institute of

Food Technology of Plant Origin, Poznań, Poland [19].

Traditionally, ethanol has been produced in batch fermentation with yeast

strains that can- not tolerate high concentration of ethanol. This necessitated the

strain improvement program for obtaining alcohol-tolerant strains for fermentation

process. Comparative laboratory- and pilot-scale studies on kinetics of batch

fermentation of Z. mobilis versus a variety of yeasts have indicated the suitability

of Z. mobilis over yeasts. It was found that Z. mobilis had many advantages over

the traditional brewing yeasts. Its advantages included:

s higher sugar uptake and ethanol yield,

s lower biomass production,

s higher ethanol tolerance,

s does not require controlled addition of oxygen during the fermentation,

and

s amenability to genetic manipulations.

Z. mobilis is a very suitable organism. It converts glucose almost

stoichiometrically to ethanol and CO2, grows more rapidly than the yeasts and

demonstrates highest productivity during continuous fermentation. Significantly

higher specific rates of sugar uptake and ethanol production were found

compared to those found for yeasts. In the comparative studies dried Polish

commercial yeast Saccharomyces cerevisiae Bc-16a and D2 as well as bacterial

culture Z. mobilis CCM 3881 and CCM 3883 from Czech Culture Collection were

used [19]. On higher glucose content (250g/dcm3), it was found that the

bacterium, Z. mobilis, produced a higher yield than the yeast cultures.

22

Strain Sugars used

(g/ dcm3)

Sugar utilization

(%)

Ethanol

(% w/v)

Ethanol yield

(% of

theoretical)

Biomass (g/dcm3

fermentation broth)

3881 83.82 ± 0.04 98.01 ± 0.05 4.01 ± 0.15 93.60 ± 3.29 1.0585 ± 0.0870

3883 83.25 ± 0.19 97.35 ± 0.21 4.03 ± 0.08 94.08 ± 1.64 1.0560 ± 0.0840

D2 83.96 ± 0.24 98.17 ± 0.27 4.03 ± 0.08 93.91 ± 2.62 2.6105 ± 0.0965

Bc-16a 83.55 ± 0.23 97.70 ± 0.26 4.03 ± 0.08 94.70 ± 1.65 2.0885 ± 0.2895

Table 2. Comparison of glucose (85.5 g/dcm3) batch fermentation by Z. mobilis (3881, 3883) and S. cerevisiae (D2, Bc-16a) (temp.30°C, 48 h, starting pH 5.2). Mean ± S.D.[19]

Table 2 shows the results of the comparative study conducted by J.

Nowak. The study shows that Z. mobilis is very suitable for growth on a glucose-

rich substrate. Molasses, which is made up of 50% sugars, would indeed be a

glucose-rich substrate that could be used in this particular process. The only

limitation of Z. mobilis compared to the yeast is that its utilizable substrate range

is restricted to glucose, fructose, and sucrose [18]. However, as Z. mobilis is

amenable to genetic manipulation, it is conceivable that alterations to Z. mobilis

could broaden its substrate range.

23

The Uses of Bioethanol.

Bioethanol has the same chemical make-up and characteristics as ethanol

that has been produced by a chemical reaction e.g. Hydrogenation of ethane.

Bioethanol therefore is suitable for all of the uses currently employed by ethanol.

It can be used as a solvent, for which there is huge demand. Ethanol has

many industrial uses due to its relatively high affinity both for water and a great

range of organic compounds. It can be used in any extraction processes which

require either a water or organic-solvent. It may also be added to solvent-based

paints, lacquers, inks, household cleaning products, and, external

pharmaceuticals (rubbing alcohol). The cosmetics industry uses alcohols in

many different products. The most often used alcohol in cosmetics is ethanol. It is

used in products such as toners and astringents, because of its solvent and

antibacterial properties.

One of the main uses that have recently been highlighted is that of

Biofuels. It is non-petroleum-based alternative source of energy. Molasses

fermentation-derived Bioethanol is very suitable for use as a biofuel. Biofuels

reduce green-house gases and produce less harmful emissions. Biofuels can be

used in addition to, or as an alternative to petrol. It is suitable for use in petrol

engines without the need for any alterations. The use of bioethanol as a fuel to

power motor vehicles has already become a reality. At the 2004 Le Mans

Endurance Series, the Team Nasamax car was run completely on bioethanol

fuel. The bioethanol used had been produced from sugar beet and potatoes. Any

doubts over the performance of a car run on vegetables were dispelled as the car

reached speeds of 200mph (321kph). The fuel contains 98% ethanol and 2%

added chemicals. The chemicals are added in such a way as to make it difficult

and expensive to filter them out. The chemicals are added purely to make the

fuel inedible, to prevent people from drinking it. By making it inedible, the liquid

can only be used as a fuel, this leads to reducing the tax that must be paid on it.

A drinkable fuel would incur much higher tax!

24

According to SEI [21], Bioethanol produced from sugar beet would cost

€0.74/L at the petrol station, excl VAT and duty charges. At todays present price

of fuel, Bioethanol seems to be a more environmentally-friendly and economic

choice.

25

Single Cell Protein Production.

Single Cell Protein (SCP) is a biomass based on protein extracts derived

from microorganisms (usually filamentous fungi) grown in large quantities for

either human or animal consumption. It is a mass of mushroom-like growth

compressed into a product. It is used as a protein supplement or as meat

alternative. SCP is currently being produced for human consumption by a

number of food companies. It is generally marketed as a vegetarian ready-made

meal, or as a healthy option meal e.g. Quorn™, Pekilo™, Symba™.

SCP is produced for a few reasons. The first is that it provides an alternative

protein source. It is suitable for both human and animal consumption. It can be a

useful additive to animal feedstuffs. In this case, it proves its real worth. By using

microorganisms, a high quality protein can be produced as animal feed. It

requires considerably less land area to produce an equivalent amount of protein

derived from a feed crop such as grain. The use of microorganisms to produce

food on a large scale can help to alleviate many of the problems facing

sustainable intensive agriculture.

SCP is currently produced in large quantities in Russia where meat shortages are

a problem. Russian biotechnology has also enabled the country to gain

agricultural self-sufficiency and managed without grain imported from the US. By

the 1980s the annual SCP production in the former USSR was 1million metric

tonnes.

26

Production Process.

Many of the manufacturing processes use wastes, which could not

otherwise be used as food, as the substrate for the microorganism being used.

There are many microorganisms that may be used. The choice will depend on

which is most suitable for the substrate in question. Of the SCP products

mentioned earlier, Pekilo™ (based in Finland) uses paper and timber wastes,

and the organism Paecilomyces vacioti, while Symba™ (a Swiss-Swedish

collaboration) uses potato processing wastes, and the organisms Candida utilis

and Endomycopsis fibuliger. Quorn™ contains mycoprotein which is derived from

flour production wastes using the fungus Fusarium garminearum. For the use of

a molasses substrate, Candida utilis, Rhodotorula gracilis, Hansenula seeturnus

are all suitable microorganisms.

Of the microorganisms researched, C. utilis is the most versatile, commonly

used and productive.

The production process involves the fermentation of the substrate by the

microorganism followed by the recovering of the cells. It can be represented as

follows:

Yeast/fungi + 1.68CH2O + NH3 +0.68O2 → 10{CH17O0.5N0.19ash} cells + 0.17CO2 + 1.14H2O + 80,000 calories.

An inoculum of the microorganism, C. utilis, is prepared. This is the primary

fermenting yeast. A molasses/nutrient solution is made up.

It is fermented under the following conditions:

20% substrateTemperature – 30C ±0.5Agitation Rate – 325-490 rpmFermentation Time – 10hours

A batch process is most suitable as there is a need to regulate the production

conditions to suit the quality of raw materials. The efficiency of the process will

largely depend on the organism.

27

Microorganism Productivity (crude protein)

Candida utilis 35%

Rhodotorula gracilis 26.7%

Hansenula seeturnus 15.5%

Table 3. Productivity of particular microorganisms in terms of crude protein produced.

28

Case Study – Indonesia Repetila III (1979-1984) [22].

This Case Study is based on a proposal for the use of mini-

fermentation technology to produce single-cell protein from molasses. The

reason for this development plan was that agriculture in Indonesia was

suffering due to lack of land area and also problems with food transport and

distribution. The Repetila III was a five year development plan put in place by

the United Nations and the United Nations University. It covers the time

period 1979-1984. It was the third development plan proposed by the UN.

Indonesia is made up of 13,367 islands, with a total land area of

1,907,950 km2. The transportation and distribution of food is a major difficulty.

The population of Indonesia in 1978 was about 140 million, and the net

population growth rate is about 2.2 per cent per year. About 80 per cent of the

population lives in rural areas and represents mostly low income groups.

70 per cent of the population lives in Java and Madura, which make up only 7

per cent of the total land area. Kalimantan, Sulawesi, and Sumatra make up

28, 10, and 25 per cent of the total land area, respectively, and are used as

transmigration areas for people from Java. The intensity of agricultural land

use in Java and Madura is about 0.07 hectares per person.

In Repelita lll, the protein supply and demand pattern is a problem

because of the population growth rate. This increases the requirement for protein

and better-quality foods in general. As a consequence, "better-quality foods"

implies increased quantities of animal protein. On the supply side, plant protein is

not sufficient to supply total requirements, although the opening-up of new

transmigration areas has been adding to food crop production. One way to

improve the supply of animal protein for human consumption is to increase the

production of animal feedstuffs. Animal feed production at present is based on

fish waste and plant protein sources, but because of their relatively high cost it is

necessary to seek others. The new sources must (a) have a high nutritional

29

value, (b) not be competitive with food for human consumption, (c) be

economically feasible, and (d) be locally available.

As a solution to this, SCP was found to be very suitable. It is possible to

introduce single-cell protein (SCP) for animal feeding. Its production will use

renewable resources and waste sources such as molasses. SCP can minimize

the use of fish waste, soybean cake, peanut cake, etc. for animal feeds. This has

been shown in poultry feeding trials. SCP also has higher protein content, 40-

80% dry weight crude protein, compared to traditional feedstuffs. Molasses is

available year-round due to the sugar industries present in Indonesia.

Suitability of SCP

s High nutritional values Does not compete with food for human consumptions Is economically feasibles Is locally availables Requires comparatively small land areas Uses waste material (molasses) from other local production processes

(sugar cane refinement)

The current status of sugar production in Indonesian factories is increasing

not only in quality but also in quantity. In the past ten years, 56 sugar cane

factories have processed 12 million tons of cane per year into 1.4 million tons of

cane sugar and 480,000 tons of molasses. In Repelita 111, the government has

launched a mini-technology for sugar cane factories that are spread throughout

such islands as Sumatra, Kalimantan, and Sulawesi. Three mini-technologies for

sugar cane factories have already been set up in Aceh, West Sumatra, and

Kalimantan. The capacity of each factory is 2,000 tons per year. The target of this

plan is to establish about 200 mini sugar factories. The private sector plans to

erect seven mini sugar factories outside Java. One of the aims of the mini sugar

factories is to create a model in order to encourage the private sector to erect

more factories of a similar kind. It is clear that the higher the total cane sugar

30

production, the higher the total availability of molasses. The production of SCP

from molasses by using mini-fermentation technology is relevant to rural

development and particularly to increasing per capita income. Some

considerations in the selection of molasses as a raw material are (i) its year-

round availability, contributing to the development of medium and small scale

industries throughout Indonesia, (ii) its potential for helping maintain the efforts of

low income farmers and decreasing unemployment in rural communities, and (iii)

the encouragement these factors may be expected to give to an increase in the

spontaneous and regular flow of transmigrants from Java to other islands.

The objectives of the project are:

to study the properties of micro-organisms that are not pathogenic or toxic and have high protein and carbohydrate contents, a rapid growth rate, etc.;

to study on the laboratory research scale optimum conditions of fermentation, product recovery, safety, improvement of products, etc.;

to study kinetic analysis of SCP fermentation; to study and evaluate the pilot plant for SCP production from molasses; to scale up SCP production from the pilot-plant to the commercial scale; to carry out field trials of this SCP with broilers, layers, pigs, cattle, fish,

etc.

In preliminary research activities on the production of SCP, the scale of

operation should be considered first, as this will be influenced by the capacity of

the pilot plant and commercial scale in the future. The end-product of

fermentation technology will be mini-fermentation technology, in terms of simple

procedure, simple equipment, and low cost. The capacity of commercial scale

production is planned to be about 1,000 tons per year, using molasses as a raw

material substrate. By comparison, mini sugar factories in Indonesia were

designed for a capacity of 2,000 tons of sugar per year. According to this

information, the following is the sequence of capacity at each stage:

31

a. The capacity of the commercial plant will be 1,000 tons of SCP per year, 3.3 tons or (3,300 kg) per day.

b. The capacity of the pilot plant will be 100 tons of SCP per year, or 334 kg per day.

c. The capacity of laboratory activities related to the scaling-up process will be 34 kg of SCP per day. This is possible by using six fermentors (4- to 8-litre capacity each).

Laboratory research was conducted on bioconversion of molasses to SCP. It

was found that 2% molasses should be used as a substrate with a nutrient

solution added. This substrate is then fermented with Fleischmann's active dry

yeast in a special fermentor with a capacity of about 4 to 8 litres; its working

capacity is 4 litres. The inoculum used should be 20% substrate and the

temperature is 30° ± 0.5°C. Other parameters, such as pH, can be adjusted from

a control panel. The recommended fermentation time is ten hours. The number of

cells per litre and the oxygen absorption rate during fermentation can be

calculated. The specific growth rate can be calculated using this equation:

X = X0Ekt

Where:X = total cells at t hour, k = specific growth rateX0 = total cells at tt = fermentation time

At an agitation rate of 325 rpm, the total number of cells will increase

exponentially and will attain equilibrium after eight hours of fermentation. At the

higher rate of 490 rpm, the total number of cells will attain equilibrium after seven

hours of fermentation. This might be the result of the autocatalysis of cells.

Further laboratory development will take place after the joint proposal on feed

from agricultural and agro-industrial wastes has been approved.

32

Summary of Repetila III

The demand for and supply of protein are not in balance, nor is the supply

adequate for Indonesia's total population of about 140 million (1978), especially

when considering the net population growth rate of 2.2 per cent per year and the

difficulties of transportation in the archipelago.

To improve the supply of animal protein for human consumption it is

necessary to increase the production of animal feedstuffs. SCP from molasses

can replace some of the usual protein sources in feedstuffs. The availability of

molasses is at least 480,000 tons per year, and this is increasing because mini

sugar factories are operating on the islands outside Java. These experiments are

to study the optimum conditions of fermentation and to set up a pilot plant and

field trials for SCP production. A pilot plant will be set up to process 100 tons per

year, the duration of the project will be three years, and the cost of the

programmes for research and development and a pilot plant will be US$900,000

and US$1,500,000, respectively. The plant will be located in Bandung.

33

Citric Acid Production using MolassesStructural formula of Citric acid:

Chemical formula of Citric acid: C6H8O7, or alternatively:

CH2(COOH)•COH(COOH)•CH2(COOH)

Synonyms for Citric acid:2-hydroxy-1,2,3-propanetricarboxylic acid

Citric acid in natureCitric acid is ubiquitous in nature and exists as an intermediate in the citric acid

cycle when carbohydrates are oxidized to carbon dioxide.

Citric acid- characteristicsCitric acid is solid at room temperature, melts at 153ºC and decomposes at

higher temperatures into other products (Rajoka et al. 1998).

It is responsible for the tart taste of various fruits in which it occurs, i.e. lemons,

limes, figs, oranges, pineapples, pears and goose-berries. Citric acid can be

recovered from its calcium salt by adding sulfuric acid (Anon, 1975). It is non-

toxic and easily oxidized in the human body. Because of its high solubility,

palatability and low toxicity, it can be used in food, biochemical and

pharmaceutical industries. These uses have placed greater stress on increased

citric acid production and search for more efficient fermentation process.

34

Citric acid demandThe worldwide demand of citric acid is about 6.0 x 105 tons per year and it is

bound to increase day by day (Ali et al. 2001).

Citric acid production The production of citric acid by Aspergillus niger is one of the most

commercially utilized examples of fungal overflow metabolism. Many

microorganisms such as fungi and bacteria can produce citric acid. The

various fungi, which have been found to accumulate citric acid in their

culture media, include strains of

o Aspergillus niger,

o awamori,

o Penicillium restrictum,

o Trichoderma viride,

o Mucor piriformis and

o Yarrowia lipolytica

(Arzumanov et al. 2000).

But Aspergillus niger remained the organism of choice for the production of citric

acid.

Although the surface culture process is still being used, most of the newly built

plants have adopted the submerged fermentation process. Kamal et al. (1999)

reported the effect of oxygen transfer rate on the citric acid production by

Aspergillus niger under submerged fermentation. In submerged fermentor, either

purified compressed air or oxygen along with agitation is used (Moreira et al.

1996).

Why is molasses used for the production of Citric acid?

Molasses is a desirable raw material for citric acid fermentation because of its

availability and relatively low price.

35

Cultural conditions for Citric acid production:

Incubation temperature plays an important role in the production of citric acid.

Temperatures between 25-30ºC are usually employed for culturing of

Aspergillus niger but temperature above 35ºC are inhibitory to citric acid

formation because of the increased the production of by-product acids and

also inhibition of culture development. Sanjay and Sharma, 1994 reported

that citric acid production by Aspergillus niger is sensitive to the initial pH of

the fermentation medium. The maximum production of citric acid (6.5%) can

be obtained at pH of around 5 in molasses medium. The appropriate pH is

important for the progress and successful termination of fermentation.

Arzumanov et al. 2000 reported that the citric acid produced by Aspergillus

niger is extremely sensitive to trace metals present in molasses. The trace

metals such as iron, zinc, copper, manganese present a critical problem in

submerged fermentation. The organisms need major elements such as

carbon, nitrogen, phosphorus and sulphur in addition to various trace

elements for growth and citric acid production.

Cultural conditions for citric acid production by fungi vary from strain to strain and

also depend on the type of process. The optimisation of cultural conditions is the

key for high and consistent yields of metabolites like citric acid.

Strains of Aspergillus niger:

In a present study, the mutant strain of Aspergillus niger GCMC-7 supported

maximum production of citric acid (106.65 g/l) without supplements which is

substantial.

What kind of molasses is used for the experimental citric acid production?

Clarified molasses

Clarified molasses- Example in Pakistan:

36

Cane molasses was clarified. After neutralization with calcium hydroxide solution

(1.55%), the sugar concentration was maintained at different required levels. The

clarified cane-molasses was kept under dark in a sterilized UV-chamber.

REF28

1919 - Mass Production of Citric Acid Pfizer:

For decades, citric acid was Pfizer's most popular product. Until 1880, most of

the raw material for citric acid was imported from Italy, but political instability,

unpredictable weather, and World War I led Pfizer to pursue other supply

sources.

A new era dawned when Dr. James Currie joined Pfizer in 1917. As a

government food chemist, Currie had been studying fermentation in cheese-

making and had discovered that one of the by-products was citric acid. At Pfizer,

Currie began a series of fermentation experiments using sugar and bread mold

and was able to produce small amounts of crude citric acid, but manufacturing

large quantities of the substance was quite another matter.

Working in secrecy, Currie and his assistant, Jasper Kane, gradually developed a

process known as SUCIAC — Sugar Under Conversion Into Acid Citric. The

company gambled on the process, taking a calculated risk in turning over its still-

profitable borax and boric acid production facilities to SUCIAC. In time, SUCIAC

production began to outperform conventional extraction from citrus products, and

by 1929, Pfizer no longer needed any imported citrus product at all. Kane went

on to develop a new deep-tank fermentation method using molasses rather than

refined sugar as raw material. No one yet knew the implications, but it was this

process that ultimately unlocked the secret for large-scale production of penicillin.

History biosynthesis of Citric acid:

37

Until 1930’s, citric acid was derived from lemons and the production monopoly

belonged to Italy. In mid-30’s, the Czech Republic undertook production of

citric acid by microbiological method from raw stuff containing sugar.

In 1948, Latvian scientists pioneered the production of citric acid from molasses,

spin-off of sugar production, in the former USSR.

Riga Citric Acid Plant elaborated an economic technology for yielding crystalline

citric acid and did serious work on obtaining highly productive Aspergillus niger

strains – R3, R5, R6.

The scheme of Citric acid production :

Implementation:

Licences for the developed technology and selected Aspergillus niger

producers were sold to foreign companies in Bulgaria, France, Slovenia,

Slovakia, Turkey and India.

Several plants were set up in Russia, Belarus and Armenia on the grounds

of citric acid technology developed in Riga.

Riga Citric Acid Plant is the only producer of citric acid in the Baltic States.

REF27

38

Bioremediation Using Molasses.

Case study (Oldenburg, Germany)On the site of an ordnance factory located in Oldenburg the bioremediation of a

former water body for the infiltration of surface water took place.

This water body was contaminated by explosives specially TNT.

TNT

TNT stands for 2,4,6 trinitrotoluene.

TNT was the most often used military explosive during the two worl wars.

Former military sites contaminated with explosives especially TNT cover large

areas.

TNT is toxic and a mutagen.

Bioremidiation process

The entire clean up took place in three steps:

1. Removal of the highest contaminated soil.

2. Application of the process of bioremediation In- situ among the addition of

molasses and reactive iron.

The addition of iron can enhance the effectiveness of the method.

(Having used that process the TNT concentrations decreased over 90% after two

years.)

3. Planting of the area for phytoremidiation and long term safeguard.

(Phytoremediation is the process of using plants for pollution clean-up of

contaminated soils or water. REF23

Advantages of using molassesAdvantages of using molasses for bioremediation of with explosives

contaminated soil and groundwater are:

Molasses is a very effective substrate for the biological decontamination of

TNT.

39

Armament-related contaminants, which are contaminated with explosives

(especially TNT) strech across wide areas. The decontamination of soil

and groundwater using traditional methods is due to that reason very

costly. An option for those sites is the bioremediation using molasses.

Microorganisms and bioremidiationTNT and other explosives can be biological transformed, immobilised and broken

down by “in situ microorganism” if they are stimulated by nutrients.

Molasses contains next to water around 50% sugar. REF24

Molasses is a nutrient supplier for microorganisms. REF25

REF26

Air Force Centre for Environmental Excellence (AFCEE)The AFCEE of the United States has programs and initiatives supporting

enhanced in situ bioremediation.

Through the Enhanced in Situ Anaerobic Bioremediation Iniative the AFCEE

Technology Transfer has generated several successful forms of the technology

of enhanced in situ bioremediation including:

edible oil injection,

molasses injection ,

hydrogen injection,

passive in situ bioreactors,

biogeochemical investigations and

biobarriers.

40

Figure 4. AFCEE Technology Transfer Enhanced in Situ Anaerobic Bioremediation Field Sites

Molasses Injection:Typical delivery techniques: injection wells

Form of application: dissolved in water

Frequency of injection: continous to monthly

41

Conclusion.Molasses, although considered as a waste, can be used in a wide number of

areas. The results of these various studies show that molasses can be used as a

substrate for bioethanol production, SCP and citric acid.

As the demand for bioethanol will inevitably increase, molasses will become

more and more appealing as a value-added product. It will no longer be

considered as a waste but as a further resource.

42

References

1. http://www.sucrose.com/lcane.html a. Submitted – May 1992b. Searched - 11-11-05

2. Agricultural science – John Breen & George Mullen (1992) 3. The Farmers Journal – Weekly Edition

a. Submitted – Dec 2005b. Searched - 11-11-05

4. http://www.whfoods.com/genpage.php?tname=foodspice&dbid=118 a. Submitted – Feb 2001b. Searched - 11-11-05

5. http://journeytoforever.org/biofuel_library/ethanol_motherearth/meCh3.html 6. Submitted – July 20037. Searched - 11-11-058. http://www.unu.edu/unupress/food/UNU06/cap_8.htm

a. Submitted – May 1997b. Searched - 11-11-05

9. http://www.premiermolasses.ie/about.htm a. Submitted – Sep 2002b. Searched - 11-11-05

10. http://www.iccept.ic.ac.uk/research/projects/SOPAC/PDFs/Energy%20Fiji.pdf a. Submitted – Feb 2002b. Searched - 11-11-05

11. http://www.cstars.ucdavis.edu/projects/ag-21/yield/caneyield.html a. Submitted – March 1999b. Searched - 11-11-05

12. http://cecommerce.uwex.edu/pdfs/A3544.PDF a. Submitted – August 1998b. Searched - 13-11-05

13. http://www.fao.org/docrep/003/s8850e/S8850E19.htma. Submitted – July 2000b. Searched - 12-12-05

14.Bioprocess and Biosystems Engineering. Vol. 16, No.6. (389-392). “Production of ethanol from molasses at at 45oC using alginate-immobilised Kluyveromyces marxianus IMB3.” S. Gough, D. Brady, P. Nigam, R. Marchant, A.P. McHale.

15. Bioprocess and Biosystems Engineering. Vol. 19, No.1. (33-36). “Continuous ethanol production from molasses at 45oC using alginate-immobilised K. marxianus IMB3 in a continuous-flow bioreactor.” S. Gough, A.P. McHale.

16. Bioprocess and Biosystems Engineering. Vol. 19, No.2. (87-90). “Production of Ethanol from molasses at 45oC using K.marxianus IMB3 immobilized in calcium alginate gels and poly(vinyl alcohol) cryogel.” S.Gough, N.Barron, A.L.Zubov, V.I.Lozinsky, A.P.McHale.

17. Bioprocess and Biosystems Engineering. Vol. 19, No.3. (217-219). “Ethanol production at 45oC by K. marxianus IMB3 during growth on molasses pre-treated

43

with Amberlite® and non-living biomass.” E.A. Farrell, M. Bustard, S. Gough, G. McMullan, P. Singh, A.P. McHale.

18. “Ethanol fermentation technology – Zymomonas mobiles.” P. Gunasekaran and K. Chandra Raj. Department of Microbial Technology, School of Biological Sciences, Madurai Kamaraj University, Madurai 625 021, India.

19. Electronic Journal of Polish Agricultural Universities – Food Science and Technology. Vol. 4, Issue 2. “Comparison of Polish Industrial Distillery Yeast with Ethanol-producting Bacteria, Zymomonas mobilis.” Jacek Nowak 2001.

20. “Process for the production of ethanol through molasses fermentation” United States Patent 4886751 (www.freepatentsonline.com)

21. “Biofuels currently the only feasible alternative to petrol and diesel” Sustainable Energy Ireland, Report 16th December 2004. (www.sei.ie)

22. Dr. Ign. Suharto (National Institute for Chemistry, Indonesian Institute of Sciences, Bandung, Indonesia), Dr S. Redyowati B. (Faculty of Engineering, University of Gadjah Mada, Yogyakarta, Indonesia). “Mini-fermentation Technology to produce Single Cell Protein from Moalsses” (Repetila III 1979-1984)

23. REF1: www.csa.com/hottopics/gmfood/gloss.php

24. REF2: www.cma.de/wissen_99254.php

25. REF3: www.gleich-lesen.de/Oldenbourg%20Industrieverlag%20/%20Vulkan-Verlag/GWF%20Wasser%20Abwase/content/pages24627.html

26. REF4: www.msrmagazin.de/__C1256BC100324BD1.nsf/previewFS?OpenPage&A&ArticleIDsorted/~A2EFA6FA00148D65C1256FB0004474A6

27. REF5: http://izgudrojumi.lza.lv/eng/izgudrojumi/citronskabe.asp

28. REF6: Biotechnology Research Laboratories, Centre of Excellence in Molecular Biology, Department of Botany Lahore, Pakistan

29. REF7: www.bronline.de/wissenbildung/kalenderblatt/2001/01/kb20010115.html

44

45