Appl Microbiol Biotechnol (2005) 68: 598–606
DOI 10.1007/s00253-005-0056-0
MINI-REVIEW
Mette Hedegaard Thomsen
Complex media from processing of agricultural crops
for microbial fermentation
Received: 2 March 2005 / Revised: 3 June 2005 / Accepted: 3 June 2005 / Published online: 5 August 2005
# Springer-Verlag 2005
Abstract This mini-review describes the concept of the
green biorefinery and lists a number of suitable agricultural
by-products, which can be used for production of bioen-
ergy and/or biochemicals. A process, in which one possible
agricultural by-product from the green crop drying in-
dustry, brown juice, is converted to a basic, universal fer-
mentation medium by lactic acid fermentation, is outlined.
The resulting all-round fermentation medium can be used
for the production of many useful fermentation products
when added a carbohydrate source, which could possibly
be another agricultural by-product. Two examples of such
products—polylactic acid and L-lysine—are given. A cost
calculation shows that this fermentation medium can be
produced at a very low cost ≈1.7 Euro cent/kg, when tak-
ing into account that the green crop industry has expenses
amounting to 270,000 Euro/year for disposal of the brown
juice. A newly built lysine factory in Esbjerg, Denmark,
can benefit from this process by buying a low price medium
for the fermentation process instead of more expensive
traditional fermentation liquids such as corn steep liquor.
Introduction
Renewable resources also known as biomass are organic
materials of biological origin and are, by definition, sus-
tainable natural resources. Sustainable implies that the re-
source renews itself at such rate that it will be available for
use by future generations. Generally, renewable resources
are classified as either wastes or dedicated energy crops.
When a waste from one process can be utilized as feedstock
in another process, a more appropriate name is co-product/
by-product. The major virtue of waste materials is their
low cost. By definition, waste materials have little appar-
ent economic value and often can be acquired for little
M. H. Thomsen (*)
Biosystems Department, Risoe National Laboratory,
Roskilde, Denmark
e-mail: mette.hedegaard.thomsen@risoe.dk
Tel.: +45-77-464223
Fax: +45-77-464122
more than the cost of transporting the material from their
point of origin to a processing plant. Increasing costs for
waste disposal and restrictions on land filling with certain
kind of wastes allow some wastes to be acquired at neg-
ative cost. Renewable resources can be converted into either
bioenergy or bio-based products.
The green biorefinery
A wide variety of organic chemicals like amino acids,
enzymes and antibiotics can be produced by fermentation.
Most microorganisms used in commercial fermentations
require six carbon sugars or disaccharides as substrates,
although the microbial world contains organisms that can
breakdown virtually any organic compound. This means
that in fermentation processes, complex raw materials can
be used, as opposed to chemical synthesis where fine chem-
icals are required. Biotechnology offers several other ad-
vantages compared to chemical synthesis, e.g. high product
specificity, low production temperature and low energy con-
sumption. As a result, fermentation is becoming increas-
ingly important in the production of commodity chemicals.
In the green biorefinery, jointly described by the Uni-
versity of Southern Denmark and AgroFerm, different
renewable resources can be converted by means of me-
chanical and biotechnological methods (e.g. fermentation)
into useful materials such as food and feed products and
additives, as well as fermentation medium to be used in the
production of organic chemical compounds, materials and
bioenergy. Common for the production of green pellets,
potato starch, cornstarch and other agricultural products
are that during processing of the crops, a liquid stream
containing part of the crop nutrients is produced.
These liquids (plant juices) are in most countries used as
a fertiliser for crop plants, because they have a valuable
content of nutrients such as potassium and nitrogen. How-
ever, due to environmental problems involved in applying
excess nitrogen, which, if not taken up by plants in the
autumn and winter period, will eventually end up in the
ground water as nitrate, more and more restrictions have
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been introduced regarding the use of these residues as
fertilisers.
The plant juices are regarded as “difficult” waste streams,
but in the concept of the green biorefinery, it is consid-
ered a co-product from which useful products can be pro-
duced. The brown juice, green juice and corn steep liquor
are plant juices rich in protein and elements, and they are
suitable as replacement for yeast extract in fermentation
media.
However, the plant juices often are low in carbohydrate,
and in order to obtain high yields in the green biorefinery,
whether the production is bioenergy or biochemicals, a
carbohydrate source is needed.
Lignocellulosic material such as agricultural waste, e.g.
wheat straw, represents an abundant renewable raw mate-
rial source. In the lignocellulosic biomass, hemicellulose
and cellulose are infiltrated with the stiffening bonding
material lignin. The two polysaccharides are not directly
available for bioconversion; a pretreatment is needed to
overcome the physical barrier of lignin and make sugars
available for the microorganisms. A wet oxidation process
has been developed, using water, oxygen pressure and
elevated temperature, for fractionation of wheat straw at
alkaline conditions. By this treatment, most of the hemi-
cellulose is solubilised mainly as oligomers and poly-
mers, and the cellulose is recovered in the solid fraction.
The fibre fraction as well as the solubilised hemicellulose
can be hydrolysed by enzymes and used as a carbohydrate
source in the green biorefinery (Bjerre et al. 1996; Schmidt
and Thomsen 1998).
Starch is another agricultural carbohydrate source. Wheat
has certain advantages over other carbohydrate sources.
Cereals, being low in moisture, are more energy inten-
sive and have the advantage that they can be stored and
transported easily. The sugar is in the form of starch, and
in addition, cereal grains contain nutrients that can be
separated easily from the grain and sold as lucrative by-
products: bran, gluten and A-starch. Gluten is the most
profitable of these by-products—a protein used in the bak-
ing industry. Starch can be saccharified to fermentable
sugars by either acid hydrolysis or by enzymatic hydrolysis.
Agricultural by-products
In this mini-review, a number of agricultural by-products
suitable for use in the green biorefinery and other fer-
mentation industry are described (Table 1).
Brown and green juice
The green crop drying industry produces fodder pellets by
drying crops such as perennial rye grass (Lolium perenne),
Italian rye grass (Lolium multiflorum), clover grass and
alfalfa (Koegel and Bruhn 1977). In order to reduce the

Table 1 Agricultural by-products, their main components and potential industrial use
Agricultural Valuable components Climate zone Example on References
by-product industrial use

Brown juice Protein/elements Temperate Lactic acid/L-lysine Andersen and Kiel 2000; Thomsen
et al. 2004
Green juice Protein/elements Temperate Lactic acid/L-lysine Andersen and Kiel 1997; Andersen and
Kiel 2000
Corn steep liquor Protein/elements Temperate/dry Ethanol Amartey and Jeffries 1994; Stock et al. 1995
Potato waste water Carbohydrate (starch)/ Temperate/dry Biomass Mishra et al. 2004
protein
Molasses Sucrose All Lactic acid Wee et al. 2004
Straw (wheat straw) Cellulose/hemicellulose Temperate Ethanol Bjerre et al. 1996; Thygesen et al. 2003
Corn stover Cellulose/hemicellulose Temperate/dry Ethanol Varga et al. 2003
Corn cob Cellulose/hemicellulose Temperate/dry Xylitol/ethanol Tada et al. 2004; Rivas et al. 2004
Rice straw Cellulose/hemicellulose Dry/tropical Cellulase/ethanol Kaur et al. 1998
Rice hull Cellulose/hemicellulose Dry/tropical Ethanol Schultz et al. 1984
Wine shoots Cellulose/hemicellulose Temperate/dry Lactic acid Bustos et al. 2004
Sugar cane bagasse Cellulose/hemicellulose Tropical Ethanol Gong et al. 1993
Sorghum bagasse Cellulose/hemicellulose Temperate/dry/ Ethanol Gnansounou et al. 2005
tropical
Olive cake Cellulose/hemicellulose/ Dry Lipase/biogas Cordova et al. 1998; Al-Masri 2001
protein
Citrus waste Carbohydrate/protein Dry/tropical Pectinase Silva et al. 2002; Tripodo et al. 2004
Banana waste Cellulose/hemicellulose Tropical Enzymes for Krishna 1999; Shah et al. 2005
lignocellulose
Peanut shell Cellulose/hemicellulose Tropical Tetracycline Asagbra et al. 2005
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energy consumption in the drying process, part of the
water content in the grass is normally removed from the
green crops before drying in a drum dryer, resulting in
significant productions of green plant juice (green juice).
At some green pellets factories, the crops are steam-heat-
ed in a cooker to a temperature of about 80°C before
pressing. This process results in coagulation of a large
proportion of the protein, as well as the damage to the
plant cells and the residue from these factories are known
as brown juice. Approximately 200,000 m3 of brown juice
is produced in Denmark each year, and of this, approx-
imately half is evaporated and used in the production of
pellets. Both green and brown juice has a dry matter (DM)
content of approximately 6%, and the green juice contains
13% carbohydrate (in DM) and 35% protein (in DM), and
the brown juice contains 33% carbohydrate (in DM) and
26% protein (in DM) (Andersen and Kiel 2000).
Corn steep liquor
Corn steep liquor is a by-product from the corn wet milling
industry. The corn wet milling industry separates the corn
into several fractions: starch, germ, fibres, and gluten.
Steeping of the corn before the wet milling process is
essential for high yields and high starch quality. The
cleaned corn is filled into a battery of large steeping tanks,
where the corn is soaked in hot water for up to 50 h. The
steeping is actually a controlled fermentation (with pri-
marily Lactobacillus), and the addition of 1,000–2,000 ppm
of sulphur dioxide helps to control that fermentation. Dur-
ing steeping, solubles are extracted and the kernel softens.
The steep water is drained from the kernels and evaporat-
ed to approximately 50% DM. Corn steep liquor contains
approximately 47% protein and is a valuable nutrient
source in the fermentation industry (Stock et al. 1995).
Potato waste water
The potato processing industry uses large volumes of wa-
ter during operations such as washing, peeling and blanch-
ing during production of potato chips, slices and shredded
potatoes. The effluent generated in these processes are
characterised by high content of carbohydrates (starch)
(19.5 g/l) and also some protein (2.9 g/l) (Mishra et al.
2004). These starch-rich wastes can cause environmental
problems if discharged in nature.
Molasses
Molasses is a by-product of sugar manufacturing. The
sugar is extracted from the beets (in the case of beet sug-
ar production) with hot water (70°C), and the pulp and
liquid are separated. This liquid is purified with calcium
carbonate, and the purified liquid is cooked in two steps
in order to remove water and to form sugar crystals. The
thick liquid with crystals is centrifuged, and sugar crystals
and molasses (a thick brown liquid) are separated. The
molasses is used as animal feed and in fermentation in-
dustry, and it generally consists of 50% of sucrose and
some amount of protein (6–9%) and elements (Wee et al.
2004; Stock et al. 1995).
Wheat straw
Wheat straw is an abundant by-product from farming, and
large amounts are produced in many countries. In a small
country as Denmark alone, approximately 6 million tons
are produced. Of these 6 million tons, 35% is excess straw
not used for burning, animal feed or bedding. Wheat straw
has a content of approximately 40% cellulose and 30%
hemicellulose (Bjerre et al. 1996; Thygesen et al. 2003).
Corn stover and cob
Corn stover and cob are by-products from cultivation of
corn. The stover is the stem and leaves, and the cob is the
part of the plant, where the corns are attached. These
materials are very abundant in large regions of the world
and the production is increasing, e.g. in Spain, the pro-
duction shifted from 1,848 tons in 1970 to 3,898 tons in
2000. Corn stover contains approximately 50% cellulose
and 28% hemicellulose (Varga et al. 2003), and the cob
contains 34% cellulose in DM and 39% hemicellulose in
DM (Rivas et al. 2004).
Rice straw and hull
Among the easily available agricultural by-products, rice
straw is the most abundant, with 82 million metric tons
annually available in India, and the estimated amount of
rice straw in Asia in 1993 was 483 million tons. Rice straw
contains approximately 40% cellulose and 18% hemicel-
lulose (Kaur et al. 1998; Liu et al. 1999). The rice hull is
another waste from rice production. Rice hull is also a
lignocellulosic material and contains 26% cellulose and
13.5% hemicellulose (Schultz et al. 1984).
Wine shoots
Pruning/trimming of vine stocks produces large amounts
of lignocellulosic residues. These wastes are usually burned
in the field, causing environmental problems as well as
risk to human health owing to the formation of toxic com-
pounds from lignin combustion. Wine-trimming wastes
contain approximately 34% cellulose and 19% hemicellu-
lose (Bustos et al. 2004).

Sugar cane bagasse
Sugar cane bagasse is the fibrous residue obtained after
extraction of sugar from sugar cane. Sugar cane bagasse
contains significant amounts of cellulose (40% of DM) and
hemicellulose (21%) (Gong et al. 1993; Schultz et al. 1984).
Sorghum bagasse
Sweet sorghum is a crop in the grass family that is con-
sidered to be one of the most drought-resistant agricultural
crops. Sweet sorghum is mostly used in the production of
syrups, brown sugar and forage. The residual plant matter
from syrup and brown sugar production the sorghum ba-
gasse contains significant amounts of cellulose (37% of
DM) and hemicellulose (26% of DM) (Gnansounou et al.
2005).
Olive cake
Olive cake is a by-product generated by olive oil extrac-
tion. Olive oil cake can be used as fuel, but is mostly
discarded as waste in the environment. Olive cake contains
approximately 10% cellulose in DM, 16% hemicellulose
in DM and 5% protein in DM (Cordova et al. 1998).
Citrus waste
In Italy alone, 1,376,600 tons of citrus fruits were used in
the production of juices, essential oils and other products
during 2001–2002. As a result of the processing of citrus
fruits, a large amount of residue is produced consisting of
washing waters, peel, membranes, seeds and pulp. The
pulp is rich in sugar and fibres, and contains some protein
(Tripodo et al. 2004). The peel (orange bagasse) contains
fibres (11.8% of DM), reducing sugars (9% of DM) and
protein (6.4%) (Silva et al. 2002).
Banana waste
Banana is one of the most consumed fruits in the world.
Each hectare of banana crop generates nearly 220 tons of
plant residue that consists mainly of lignocellulose mate-
rial. In, e.g. India, where 4.796·105 ha of banana is cul-
tivated, farmers discard banana waste into rivers, lake and
on roads, causing serious environmental problems. The
main residuals of banana crops are leaves and pseudostem,
both containing high levels of lignocellulose. Banana fruit
stalks contain approximately 57% total sugar of which 27%
601
is starch and approximately 24% is cellulose (Krishna and
Chandrasekaran 1996; Krishna 1999; Shah et al. 2005).
Peanut shell
Residue from peanut (groundnut) is abundant in Nigeria,
where the annual production of peanut in 1996 was
7,608 tons. The peanut shell has a carbohydrate content of
approximately 17%, a protein content of 7% and a fibre
content of 70% (Asagbra et al. 2005).
Preservation of plant juices
From a theoretical point of view, brown juice and other
plant juices are well suited as fermentation media, as they
contain all the necessary growth factors such as amino
acids, vitamins and minerals (Thomsen et al. submitted for
publication). But in practice. it is difficult to handle due to
the transportation and storage difficulties of these sub-
strates. If the substrates have to be stored or transported
before use in an industrial process, it is necessary to pre-
treat them in some way. Traditional heat sterilisation at
121°C for about 30 min reduces the quality of the juice as a
fermentation medium due to formation of inhibitors from,
e.g. Maillard reactions (Andersen and Kiel 1999). How-
ever, a process has been developed in which plant juices
can be converted to an all-round, storable and stable fer-
mentation medium by means of lactic acid fermentation.
The fresh non-sterilised juice is inoculated with a suit-
able lactic acid bacteria, Lactobacillus salivarius subsp.
salivarius DSM 20555, which has been shown to be the
best strain (Thomsen et al. 2004), thereby converting the
crop sugars into lactic acid. It has been shown that in fresh
brown juice, all sugars, even fructans, are converted to
lactic acid (by the help of plant and microbial enzymes)
lowering pH and giving a good preservation of the juice
(Andersen and Kiel 2000). In order to effectively preserve
the juice, fermentation is continued until the pH drops to
below 4.5, and preferably to below 4.0 (Andersen and Kiel
1999). The resulting lactic acid fermented juice is a stable
product that can be easily stored under anaerobic condi-
tions (Andersen and Kiel 1997) for further use as a fer-
mentation medium either as it is or in concentrated form.
This substrate can be used as fermentation broth (by ad-
dition of a carbon source) in a number of different fer-
mentation processes producing valuable products such as
organic acids, amino acids, feed additives, enzymes, pro-
teins, peptides or a fungus or bacteria (Andersen and Kiel
1999). In the following, a couple of examples of products
based on acidified brown juice medium will be given, and
finally, the price of this acidified brown juice medium will
be evaluated.
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Production of PLA from brown juice
Polylactic acid
Polylactic acid (PLA) is a polymer made by polymerisation
of lactic acid. PLA shows great potential as material for
food packaging due to its mechanical properties. Due to the
moisture and gas barrier and the ability to produce flexible
water-resistant films, PLA is suitable for packaging of
respiring fruit and vegetables and for liquid food applica-
tions, e.g. juice. PLA can be used as a pure product or it can
be used in combination with other polymers (Shogren
1997). It may contain natural extracts/components, e.g.
lignin and waxes, acting as preservatives or antioxidants
preventing oxidation-sensitive products from deteriorating
(Petersen et al. 1999). Other uses for PLA are surgical
sutures, implantable medical devices, pharmaceutical con-
trolled drug delivery systems, fibres and yarns for manu-
facturing of clothing, etc. In order for PLA to be able to
compete with conventional packaging materials, it has to
be produced from cheap raw materials and feasible pro-
cesses. The typical fermentative route to lactic acid starts
with some sort of substrate pretreatment, either just addi-
tion of essential growth components, e.g. suitable mineral
and proteinaceous nutrients, or in addition, mechanical
treatment combined with chemical/enzymatic hydrolysis.
Nutritionally lactic acid bacteria are extremely fastidious
due to their limited ability to synthesize B vitamins and
amino acids (Chopin 1993); a medium that will support
their growth must contain a fermentable carbohydrate and
many growth factors such as amino acids, peptides, nuclei
acid derivatives and vitamins. Brown juice is a cheap me-
dium, and it contains all nutrients necessary for lactic acid
fermentation (Andersen and Kiel 2000; Thomsen et al.
submitted for publication).
PLA from brown juice
If the acidified brown juice medium is to be used for actual
lactic acid fermentation, the fresh brown juice is typical-
ly supplemented with additional carbohydrate, so that the
initial acidification step for preservation of the juice is
followed by additional lactic acid fermentation. That is, in
order to profitably utilize brown juice in the production of
PLA, a carbohydrate source must be added with the pur-
pose of increasing the lactic acid yield (Andersen and Kiel
1999). The carbohydrate source could be another waste
product from the agricultural industry such as molasses
from beet sugar production, a lignocellulosic by-product as
the ones described in Table 1, or wheat starch.
The purification process
The commercial success of PLA hinges on the purity (heat
stable grade) and the cost of the fermentation-produced
lactic acid. Therefore, one of the major challenges in lactic
acid production is to reduce the cost of the acid recovery
and purification, which could amount to almost 50% of the
final product cost (Evangelista et al. 1994). For the prep-
aration of lactic acid polymers, it is advantageous to use
aqueous solutions of lactic acid at about 90% by weight
(Mantovani et al. 1992). Several methods for purification
of lactic acid from fermentation broth have been described,
such as gypsum precipitation (Datta 1995), extraction
using a trialkyl amine in an organic solvent as employed
by Cargill (Bizzari et al. 1999), ion-exchange (Mantovani
et al. 1992; Evangelista et al. 1994; McKetta and
Cunningham 1983), solvent extraction (Jeneman 1931;
McKetta and Cunningham 1983; Bailey et al. 1986;
Kascak et al. 1996) and membrane processes such as
electrodialysis (Czytko et al. 1987; Hongo et al. 1983;
Madzingaidzo et al. 2002). A process has been developed
whereby lactic acid is neutralised with piperazine; an amine
that combined with two molecules of lactic acid makes
piperazinium dilactate. The piperazinium dilactate can be
converted into dilactid (a building block in production of
polylactate) without the production of undesired by-prod-
ucts. Ultrafiltration, nanofiltration and electrodialysis are
used for purification of the lactide from the fermentation
broth. In this process, no problems with fouling of the ul-
trafiltration membrane was experienced, but still, some
optimising is needed on the nanofiltration and electrodi-
alysis processes (Kamm et al. 2000). At the Technical
University of Denmark, a process has been developed, in
which lactic acid is continuously removed and purified
from the fermentation broth using various membrane pro-
cesses (Donnan dialysis, electrodialysis with bipolar mem-
branes and electrodialysis). In this process, problems with
fouling of the membranes when using a complex medium
such as brown juice was minimised or avoided (Garde et al.
2000).
Production of L-lysine from brown juice
L-Lysine
L-Lysine is an essential amino acid in animal nutrition.
Many plant products used for livestock feed, e.g. wheat
and corn, are deficient in L-lysine, and thus, it must be
added as a supplement. The bulk of L-lysine production
throughout the world depends on direct fermentation of
carbohydrates by auxotrophic and regulatory mutants of
Corynebacteria and Brevibacteria spp. in batch culture
(Kiss and Stephanopoulos 1991).
L-Lysine is added to feed in amounts of 0.2 to 1% and
reduces the livestock’s consumption of nitrogen-rich feed
such as soybean, fish protein, and flesh and bone meal.
Furthermore, L-lysine improves nitrogen’s biological avail-
ability in the animal, thereby diminishing the agricultural
nitrogen emission to the environment (Ruklisha et al.
2001).
The world marked for L-lysine is about 600,000 tons
a year. In Europe, approximately 95,000 tons a year is
consumed, and the market is up going. The L-lysine prod-
ucts produced to day are based on industrial fermentation

of sugar and hydrolysed starch and are marketed in the
form of a lysine–HCl purified by ion exchange. A few big
manufacturers dominate the world market, and about 35 to
40% of the L-lysine consumed in Europe today is imported
(Christensen 1998).
A Danish L-lysine production—from brown juice
The Danish marked for L-lysine is approximately 14,000
tons a year. Up till now, there have been no Danish pro-
ducers of L-lysine. The company AgroFerm now initiates
a Danish production of L-lysine; the new L-lysine factory
is planned to produce approximately 12,500 tons of L-
lysine a year based on the acidified brown juice medium.
In the lysine factory, the acidified brown juice is sterilised
in a continuous steriliser and led to a sterile, aerated stirred
tank reactor (STR) reactor (sterilisation of the brown juice
is now possible because the sugars are converted to lactic
acid, so no damage by Maillard reactions between carbo-
hydrates and amino acids can take place). The fermentors
are inoculated with a culture of Corynebacterium glutami-
cum and carried out as fed batch fermentation. A sterile
carbohydrate solution is added continuously to the fermen-
tation tank, after the sugar in the initial medium has been
utilized. pH of the medium is controlled by addition of am-
monia. The fermentation continues until a certain L-lysine
concentration is reached. pH in the media is dropped to 4.0
by adding sulphuric acid, and the final liquid product with
25% L-lysine is achieved after vacuum evaporation of the
whole media containing all remaining nutrients and bio-
mass. The result is production of a new valuable product
without formation of new waste streams.
Andersen and Kiel (1999) has shown that the acidified
brown juice resulting from the initial lactic acid fermen-
Fig. 1 The lactic acid fermentation of brown juice in the green crop drying plant as it was simulated in Super Pro Designer (Thomsen 2004)
603
tation as described above is a perfect substrate for amino
acid producing microorganisms, which are able to utilize
the available organic acids and amino acids as building
blocks and as energy source for production of both cell
mass and the desired amino acids such as L-lysine. It
has been shown that the use of the acidified brown juice
makes possible a very high microorganism growth rate as
well as a very high yield and productivity.
In particular, it has been found that the biomass yield is
increased in the lactic acid fermented juice, as the lactic
acid bacteria are able to utilize a higher proportion of the
organic acids and sugar present in the juice than is the case
for the Corynebacteria alone. Also, the lactic acid bacteria
hydrolyse some of the proteins in the juice making them
available for the Corynebacteria in the subsequent amino
acid fermentation. The net result is that the lactic acid
fermentation converts compounds that are otherwise non-
fermentable for the Corynebacterium to compounds, which
the Corynebacteria are able to ferment. The overall yield
is therefore greatly increased. The acidified brown juice
medium is a complete medium with a high content of free
amino acids, which is highly useful for microorganisms
able to utilize lactic acid as a carbon source such as
Corynebacteria, which are able to utilize the produced
lactic acid for both cell mass and amino acid production
(Andersen and Kiel 1999).
Price of the brown juice medium
Based on chemical analyses of the juice, laboratory as well
as large pilot scale fermentation experiments, and simula-
tion in a design and simulation program called Super Pro
Designer, a plant for production of 10,000 tons of acidified,
concentrated (25% DM) brown juice a year has been
604
designed (Thomsen et al. 2004). The price of the acidified
brown juice medium was determined from investment
costs, interests and the total operating cost, which was
determined by simulation of the process in Super Pro
Designer (Fig. 1).
The acidification process
The acidification process should be simple, robust, cost
efficient, have low energy consumption and be run in the
green crop factory by personnel not educated in microbi-
ology. The hot (60–70°C) brown juice from the green crop
drying plant is at the first step cooled to fermentation
temperature. Only the best quality of the juice is used in
the acidification process, that is, juice with a pH higher
that 5.5, whereas juice with a pH below 5.5 is returned
to the pellet factory in a concentrated form. Fermentation
is performed in a 100 m2 continuous stirred tank reactor
(CSTR) inoculated with a strain of L. salivarius and run
at high dilution rate (0.5–1.0) (Thomsen et al. 2004). The
acidified brown juice with pH between 4.0 and 4.5 is led
to a sedimentation tank, from where the supernatant is led
to a storage/buffer tank before evaporation to 25% DM.
The sediment is led to a sediment buffer tank. The con-
centrated acidified brown juice is stored until transport to
the lysine factory. Using this concept, it is possible to sup-
ply the lysine factory with stabilized high quality brown
juice all year round and produce about 50,000 tons of liq-
uid lysine feed concentrate a year (Fig. 1) (Thomsen et al.
2004).
Cost evaluations
Following unit operations is used in the production of the
acidified brown juice medium: cooling tower, fermenta-
tion tank, sedimentation tank, buffer tank, evaporator and
a storage tank (Fig. 1). The total capital investment of
this simple process has been found to be approximately
10,000,000 DDK or 1,300,000 Euro (Thomsen 2004).
Price of cooling tower and evaporator was neglected be-
cause these unit operations are already present at the green
crop drying plant (and the capacity of this equipment is
sufficient to also be used in the acidification process).
It was assumed that the capital investment comes from
loan (annuity loan), and that the investment is written off
in a period of 10 years at an interest rate of 5% per year.
The annual costs of such a loan will be approximately
1,255,000 DDK or 170,000 Euro. The annual operating
costs is determined as cost of raw materials, power and
utilities, maintenance, operating supplies, insurance and
transportation of the brown juice to the fermentation fac-
tory. The cost of the brown juice was set to a negative value
of 2,000,000 DDK or approximately 270,000 Euro, be-
cause the green crop drying industry has expenses amount-
ing to approximately 20 DDK/m3 fresh brown juice for
disposal. About 100,000 tons will be spread on the fields
every year, giving an annual expense of 2,000,000 DDK
a year. The remaining part of the brown juice will be re-
cycled and evaporated in the production of green pellets.
The price of power and utilities was determined by
simulation in Super Pro Designer and maintenance set as
4% of the fixed capital investment, operating supplies as
15% of maintenance and insurance as 1% of the fixed
capital investment. Transportation costs of the brown juice
of course depend on how far away from the green crop
drying plant the fermentation factory is situated, but in the
case of the L-lysine factory AgroFerm in Esbjerg, Den-
mark, transportation of the brown juice amounts to a cost
of 50 DDK/m3 brown juice, that is, 500,000 DDK or
approximately 70,000 Euro a year (the distance is approx-
imately 80 km).
Summing it up, the price the L-lysine factory will have
to pay for the acidified brown juice medium (25% DM)
will be approximately 1,150,000 DDK or 156,000 Euro a
year (10,000 m3) or 1.7 Euro cent/kg (Thomsen 2004).
Discussion
The actual price of the acidified brown juice medium
might be even lower because of the protein (value) added to
the green pellet production. It is assumed that the flow of
brown juice is halved after sedimentation. In reality, it will
be more because brown juice with a pH below 5.5 will be
led directly to a sediment buffer tank, where also the sed-
iment from the sedimentation tank is led. After evapora-
tion, the concentrated brown juice is used in the production
of fodder pellets. The estimated amount (by simulation) of
sediment led back to the production of fodder pellets is
approximately 10,000 tons DM/year. The sediment is rich
in precipitated protein and will increase the protein content
(and value) of the green pellets.
The price of the acidified brown juice medium can be
compared to corn steep liquor, which is also a waste prod-
uct (Agricultural by-products section, Table 1) often used
as fermentation medium. The price of corn steep liquor
(25% DM) in Denmark is 1.5 DDK/kg or 20 Euro cent/kg.
From this comparison, it is evident that converting the
fresh brown juice to a universal stable fermentation me-
dium by lactic acid fermentation is a feasible process. The
lysine factory benefits from this process by buying a low
price medium for the fermentation process instead of more
expensive traditionally fermentation liquids such as corn
steep liquor, and at the same time, pollution of the ground
water by this nutrient-rich liquid in the autumn period is
avoided.
Acknowledgements Margrethe Andersen and Pauli Kiel (Agro-
Ferm) are acknowledged for their extensive research leading to the
development of this process and for input to this mini-review.

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