Methods of industrial production
1
Methods of industrial production
•
Primary & secondary metabolites
– Primary metabolites are produced during the growth phase of the microbe.
Examples: amino acids, nucleotides, fermentation end products, and many
types of enzymes
– Secondary metabolites accumulate during periods of nutrient limitation and
waste buildup. Examples: many antibiotics and mycotoxins
– Primary metabolites are produced during active cell growth, and secondary
metabolites are produced near the onset of stationary phase
2
3
4
Major products
• Antibiotics
– Examples: penicillin & streptomycin
– The yield of both of these antibiotics are optimized by nutrient
limitation (carbon & nitrogen)
• Recombinant DNA products
– Proteins produced from genes introduced into microbes via
recombinant DNA techniques, such as enzymes, peptide hormones,
recombinant vaccines
5
Major products
• Amino acids
– Glutamic acid (monosodium glutamate) is produced by regulatory
mutants of Corynebacterium glutamicum that have a modified Krebs
cycle that can be manipulated to shift -ketoglutarate to glutamate
production
– Lysine is produced by a Corynebacterium glutamicum strain in which
homoserine lactone synthesis is blocked
6
Major products
• Other organic acids
– Acetic acid, citric acid, fumaric acid, gluconic acid, itaconic acid, kojic
acid, lactic acid
• “Speciality” compounds
– A variety of drugs (cholesterol drugs, immunosuppressants, antitumor
drugs), ionophores, enzyme inhibitors, pesticides
• Biopolymers
– Microbial-produced polymers, mostly polysaccharides, useful as
thickening or gelling agents in foods, pharmaceuticals, paints, etc.
7
Major products
• Biosurfactants
– Microbial-produced detergents, such as glycolipids; used in
bioremediation applications such as oil spill cleanups
• Bioconversions
– Using a microbe as a biocatalyst to convert a substrate into a desired
product; for example, in the modification of steroid hormones
8
9
The picture shows the interrelationship of the main
primary metabolic pathway for aromatic amino acid
synthesis
and the secondary metabolic
pathways for a variety of
antibiotics.
10
Productivities of fermentation compared to other processes
11
Chiral Hydroxy Acids from Carbonhydrate Metabolism
• More than 5 million tons of starch are produced per year in the EU from
agriculture products such as maize, wheat, barley and potatoes.
• D-Glucose, the basic building unit of starch, can be formed in situ by the
action of amylase enzymes.
• The glucose can then be converted to a variety of commercially interesting
hydroxy acids.
• The commercially most important is citric acid, which is achiral, but a
variety of chiral hydroxy acids are also produced by carbohydrate
metabolism-
12
Chiral hydroxyacids available from industrial fermentations
13
2-Keto-D-gluconic acid
• 2-Keto-D-gluconic acid is produced by fermentation of glucose with
Acetobacter suboxydans and is an intermediate in the production of
isoascorbic acid (isovitamin C)
14
L-Sorbose production by fermentation of D-Sorbitol
•
About 30 000 tons/a of ascorbic acid are produced by the Reichstein-Grussner
process which dates from 1934. The key step in this process is the microbial
oxidation of D-sorbitol to L-sorbose mediated by Acetobacter suboxydans.
15
Direct fermentation of glucose to 2-keto-L-gulonic acid by a recombinant
strain of Erwinia herbicola
16
Amino acids
• Amino acids are used for a variety of purposes. The food industry requires
L glutamateas a flavour enhancer, or glycine as sweetener in juices, for
instance.
• The pharmaceutical industry requires the amino acids themselves in
infusions - in particular the essential amino acids - or in special dietary
foods.
• And last, but not least, a large market for amino acids is their use as feed
additives. The reason is that typical animal feed, like soybean meal for
pigs, is poor in some essential amino acids, like methionine and lysine.
17
Overview
18
Overview (cont)
19
Classical strain development
• Bacteria do not normally excrete amino acids in significant amounts
because regulatory mechanisms control amino acid synthesis in an
economical way so that the needs of the cell (for protein synthesis)
are exactly matched by the synthetic processes.
• There are no surplus amino acids and only a small pool of them
exists within the cell to meet its immediate needs. Therefore,
mutants have to be generated that over synthesize the respective
amino acid. A great number of amino-acid-producing bacteria have
been derived by mutagenesis and screening programmes.
• This has involved the consecutive application of:
• undirected mutagenesis,
• selection for a specific phenotype, and
• selection of the mutant with the best amino acid accumulation.
20
A genealogy of strains obtained by classical mutagenesis and
screening, showing improved yield and some of the phenotypic characters of
the mutants
21
-Ketoglutarate Family
aKetoglutarate
Glutamate
Glutamine
Proline
Arginine
22
-Ketoglutamate to glutamate/glutamine
Net Reaction:
-Ketoglutarate + NH4+ + NADPH + ATP
L-glutamate + NADP+ + ADP + Pi
23
L-Glutamate
Sketch of main
reactions of C. glutomicum
connected with the citric acid cycle
and of relevance for L-glutamate
production. PyrDH, pyruvate
dehydrogenase; PyrC, pyruvate
carboxylase; PEPC,
phosphoenolpyruvate
carboxylase; GIuDH,
glutamate dehydrogenase;
KetogluDH, ketoglutarate
dehydrogenase.
24
L-Glutamate
• For the biotechnological production of L-glutamate by C. glutamicum, the
intracellularly synthesised amino acid must be released from the cell.
• This requires specific treatments to result in export of the amino acid by a
presumed carrier. A specific carrier must be present since otherwise, in
addition to the charged L glutamate, other metabolites and ions would
also leak from the cell and the cell would not be viable. However, Lglutamate formation is still not fully understood.
• The reason for this is that a wide ringe of treatments lead to the secretion
of glutamate. These include: (i) growth under biotin limitation, (ii)
addition of penicillin, (iii) addition of lysozyme, (iv) addition of
surfactants, (v) use of oleic acid auxotrophs, and (vi) use of glycerol
auxotrophs.
• All these treatments apparently have the cell wall or the lipid membrane
as the target in some way or another.
25
Verbesserung der Sekretion
26
Production process
•
•
•
•
The most relevant factors influencing L-glutamate formation are the
ammonium concentration,
the dissolved 02 concentration
and the pH.
•
Although, in total, a large amount of ammonium is necessary for sugar conversion
to L-glutamate, a high concentration is inhibitory to growth as well to the
production of L-glutamate.
•
Therefore, ammonium is added in a low concentration at the beginning of the
fermentation and is then added continuously during the course of the
fermentation.
27
Production process
•
The oxygen concentration is controlled, since under conditions of insufficient
oxygen, the production of L-glutamate is poor and lactic acid as well as succinic
acid accumulates, whereas with an excess oxygen supply the amount of αketoglutarate as a by-product accumulates.
•
For the actual fermentation, the production strains are grown in fermenters as
large as 500 m3 .
•
After pre-cultivation, the onset of L-glutamate excretion is controlled by the
addition surfactants such as polyoxyethylene sorbitan monopalmitate (Tween 40).
28
Production process
•
Yields of between 60 and 70% L-glutamate, based on the glucose used, have been
reported.
•
At the end of the fermentation the broth contains L-glutamate in the form of its
ammonium salt.
•
In a typical downstream process, the cells are separated and the broth is passed
through a basic anion exchange resin.
•
Glutamate anions will be bound to the resin and ammonia will be released. This
ammonia can be recovered via distillation and reused in the fermentation.
29
Production process
•
Elution is performed with NaOH to form monosodium glutamate (MSG) directly
in the solution and to regenerate the basic anion exchanger.
•
From the eluates, MSG may be crystallised directly followed by further
conditioning steps like decolorisation and sieving to yield a food-grade quality.
30
Biosynthese und Hochleistungsmutanten
31
A scheme of the
material flow in an L-glutamate
production plant.
32
Oxoloacetate Family
Oxaloacetate
Aspartate
Asparagine
Methionine
Lysine
Threonine
33
L-Lysine
• Out of the twenty naturally occurring amino acids, L-Lysine is one of the 9
essential (histidine, isoleucine, leucine, lysine, methionine, phenylalanine,
threonine, tryptophan and valine) and commercially important amino
acids, ecumenically found in naturally occurring proteins of all living
organisms.
•
Its major commercial form is L-Lysine-HCl (L-Lysine monohydrochloride).
• It is mainly used as a feed additive in the animal feed industry, mixed with
various common livestock such as cereals which do not contain sufficient
levels of L-lysine for the livestock's nutritional requirements.
34
L-Lysine
35
Biosynthese und Hochleistungsstämme
36
Lysinfermentation
37
Lysine Production Approaches
Corynebacterium glutamicum is widely used for the biotechnological production
tons of L-glutamate, L-lysine and of several other amino acids.
To obtain Lysine overproducer, besides the classical method of random
mutagenesis, some metabolic engineering principles were also used for strain
improvement
Some well know targets for lysin overproduction are
– deregulation of lysine production
– Improved carbon precurser supply
– Increased NADPH regeneration
•
Thus, L-lysine producing strains of the gram positive corynebacteria, especially
Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium
lactofermentum, have been used for the last fifty years for the industrial
production of amino acids.
38
L-Lysin fermentation
39
Fermentation medium
• In addition to physical parameters like pH, agitation and aeration rate, air
saturation, temperature, dissolved CO2 and foaming, medium composition
is a very important factor strongly influencing fermentation processes,
often being object of extensive process development and optimization
studies.
• Common fermentation media for L-lysine production contain various
carbon and nitrogen sources, inorganic ions and trace elements (Fe++,
Mn++), amino acids, vitamins (biotin, thiamine-HCl, Nicothinamide) and
numerous complex organic compounds.
• An overexpression of genes is also achieved by optimizing the composition
of the media and the culture technique in addition to physiological and
genetic parameters .
40
CARBON SOURCE
•
Mutants of Corynebacterium and related microorganisms enable the inexpensive
production of amino acids from cheap renewable carbon sources by direct
fermentation.
•
Various carbohydrates are utilized individually or as a mixture for the production of
L-lysine such as glucose, fructose, sucrose, molasses (sucrose, glucose, fructose
etc.), maltose, blackstrap molasses, starch hydrolyzate (glucose, oligosaccharides),
lactose, maltose, starch and starch hydrolysates, cellulose, cellulose hydrolysate,
organic acids such as acetic acid, propionic acid, benzoic acid, formic acid, malic
acid, citric acid and fumaric acid, alcohols such as ethanol, propanol, inositol and
glycerol and certainly hydrocarbons, oils and fats such as soy bean oil, sunflower
oil, groundnut oil and coconut oil as well as fatty acids such as e.g. palmitic acid,
stearic acid and linoleic acid.
41
NITROGEN SOURCE
•
Various sources of nitrogen are utilized individually or as mixtures for the
commercial and pilot scale production of L-lysine, including inorganic compounds
such as gaseous and
•
aqueous ammonia, ammonium salts of inorganic or organic acids such as
ammonium sulfate, ammonium nitrate, ammonium phosphate, ammonium
chloride, ammonium acetate and ammonium carbonate.
•
Alternatively, natural nitrogen containing organic materials like soybeanhydrolyzate, soyprotein HCl-hydrolyzate (total nitrogen of about 7%), soybean
meal, soybean cake hydrolysate, corn steep liquor, casein hydrolysate, yeast
extract, meat extract, malt extract, urea, peptones and amino acids may also be
utilized.
42
INFLUENCE OF OXYGEN
• L-lysine fermentation is an aerobic process demanding large amounts of
oxygen and strongly influenced by the air saturation in bioreactor.
• Lactic acid is formed as a byproduct under anaerobic conditions, which is
reconsumed after the establishment of aerobic conditions.
43
pH
• The pH is a very important factor strongly influencing microbial
fermentations.
• Basic compounds such as sodium hydroxide, potassium hydroxide,
ammonium hydroxide, calcium carbonate, urea, ammonia and gaseous
ammonia, or inorganic acid compounds such as phosphoric or sulfuric acid
and organic acids are utilized for controlling pH in L-lysine cultures at a pH
ranging from 5 to 9.
44
The industrial production of antibiotics begins with screening for
antibiotic producers .
45
46
47
Biosynthetic pathways for
penicillin and cephalosporin.
48
Enzyme processes in the production of β-Lactam antibiotics
• Penicillins and chephalosporins belong to the class of β-lactam antibiotics
that are formed from the common precursor tripeptide isopenicillin N.
• The β-lactam structure is formed by ring-closure reactions between Cys
and Val,where (S)-Val is isomerized to ( R )-Val.
• The β-lactam precursors of all penicillins and cephalosporins are produced
by fermentation in fermentors of up 1000 m3.
• The concentration of the products in the medium on completion of
fermentation that takes between five and seven days, is up to 100 g/L
penicillin and 20 g/L cephalosporin C.
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Penicilline
50
Cephalosporine
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Biosynthese und Genstruktur
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Major antibiotics of clinical significance include the -lactam antibiotics
penicillin and cephalosporin and the tetracyclines .
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Syntheseweg
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Syntheseschritte
55
Suitable Reactors and pH-controlling Buffers
• The main reason for developing the enzyme process was to reduce the
amount of problematic wast produced in the chemical process. This was
successful, and wast production was reduced typically from 31 to 0,3 tons
per ton 7-ACA synthesized.
• Due to product inhibition, a CSTR is unfavorable, as much more enzyme is
required for a given space time yield than for a packed-bed reactor.
• The latter is not suitable in the deamination step as it is difficult to supply
the necessary oxygen, and the pH will change along the lenght of the
reactor.
56
Comparison of the old (chemical) and new (enzyme)processfor the hydrolysis
of penicillin G
57
The graph shows the kinetics of the penicillin
fermentation with Penicillium chrysogenum.
58
Penicillin production
•
Penicillin is a secondary metabolite of fungus ''Penicillium'' that is produced
when growth of the fungus is inhibited by stress. It is not produced during active
growth. Production is also limited by feedback in the synthesis pathway of
penicillin.
•
The Penicillium cells are grown using a technique called fed-batch culture, in
which the cells are constantly subject to stress and will produce plenty of
penicillin.
•
The carbon sources that are available are also important: Glucose inhibits
penicillin, whereas lactose does not. The pH and the levels of nitrogen, lysine,
phosphate, and oxygen of the batches must be controlled automatically.
59
Penicillin variants
• The term “ penicillin" is often used in the generic sense to refer to one of
the narrow-spectrum penicillins, in particular, benzylpenicillin ( penicillin
G).
• Other types include:
• Phenoxymethylpenicillin
• Procaine benzylpenicillin
• Benzathine benzylpenicillin
• Benzylpenicillin potassium
• Benzylpenicillin sodium nevertheless, penicillinis still the most common
cause of severe allergic drug reactions.
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Induced mutation of
Penicillium chrysogenum
Breeding of strain NRRL-B25 for production of penicillin
Strain
Mutagen
NRRL-B25
Yield (U/ml)
Time
250
1943
NRRL-X1612
X-rays
500
1943
NRRL-Q176
UV light
850
1945
NRRL-WIS-47-1564
UV light
850
1947
---
---
50000
1977
61
A “classical” genetic technique
for strain breeding
Physical agents
Mutagenic agents
e.g. X-rays, g-rays, UV
Chemical mutagens
e.g. base analogs, nitrous acid,
alkylating, arylating agents
62
Penicillin
production
improvement:
a classical
approach
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Stamm-Verbesserung
64
Classical mutagenesis
• Classical mutagenesis of microorganisms comprises exposing to
ultraviolet light irradiation (UV), X-ray irradiation, radiation irradiation
(e.g. exposing to UV radiation at 30oC for about180 sec) and chemical
mutagen treatment (e.g. 250 or 500 mg/l N-methyl-N'-nitro-Nnitrosoguanidine (NTG)
• at room temperature, 30 or 32°C for 10-30 min (e.g. in tris/maleic buffer
of pH 6.0; cells are washed with 0.1M tris buffer of pH 7.2)
• followed by mutant selection conducting replication on selective minimal
agar plate mediaor LB agar plates.
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66
UV Damage & photoreactivation of DNA
UVL
VL
Photolyase
67
Tetracycline
68
69
Tetracycline
70
Fermentation und Aufarbeitung von Chlortetracyclin
71
Polyketid Antibiotika
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Citric acid
•
Citric acid is the most important organic acid produced in tonnage by
fermentation. Global production of citric acid in 2004 was about 1.4 million tonnes
estimated by Business Communications Co. (BCC).
•
The citric acid market has been under pressure for more than two years and
continues to oscillate with prices falling from $2/kg to $0.70–$0.80/kg.
•
About 64 % of U.S. citric acid usage in 2004 was for foods and beverages, 22 % for
detergents and cleaning products and 10 % for pharmaceutical and nutritional
products. About 2 % went into cosmetics and toiletries. Around 2 % were used in
different applications. The actual price of citric acid is about $1 to $1.3 per kilo.
In general, citric acid is commercially produced by submerged microbial
fermentation of molasses; the fermentation process using Aspergillus niger is still
the main source of citric acid worldwide.
•
73
Microorganisms
•
A large number of microorganisms including fungi and bacteria such as
Arthrobacter paraffinens, Bacillus licheniformis and Corynebacterium ssp.,
Aspergillus niger, A.aculeatus, A. carbonarius, A. awamori, A. foetidus, A.
fonsecaeus,A. phoenicis and Penicillium janthinellum; and yeasts such as Candida
tropicalis, C. oleophila, C. guilliermondii, C.citroformans, Hansenula anamola and
Yarrowia lipolytica have been employed for citric acid production .
•
Most of them, however, are not able to produce commercially acceptable yields
due to the fact that citric acid is a metabolite of energy metabolism and its
accumulation rises in appreciable amounts only under conditions of drastic
imbalances.
•
A. niger has remained the organism of choice for commercial production because
it produces more citric acid per time unit.
74
Strains
•
Among the mentioned strains, the fungus A. niger has remained the organism of
choice for commercial production because it produces more citric acid per time
unit.
•
The problem in the production of citric acid for yeasts is the simultaneous
formation of isocitric acid.
•
The main advantages of using A. niger are its ease of handling, its ability to
ferment a variety of cheap raw materials, and high yields.
•
Industrial strains which produce commercial citric acid are not freely available and
only a few can be obtained from international culture collections.
75
Citric acid production
• The industrial citric acid production can be carried in three different ways:
by
• submerged fermentation,
• surface fermentation and
• solid-state fermentation or »Koji« process.
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Submerged fermentation
•
The submerged technique is widely used for citric acid production. It is estimated
that about 80 % of world production is obtained by submerged fermentation .
•
This fermentation process employed in large scale requires more sophisticated
installations and rigorous control.
•
On the other hand, it presents several advantages such as higher productivity and
yields, lower labour costs, lower contamination risk and labour consumption.
•
Submerged fermentation can be carried out in batch, fed batch or continuous
systems, although the batch mode is more frequently used.
•
Normally, citric fermentation is concluded in 5 to 12 days, depending on the
process conditions.
77
Surface fermentation
• Liquid surface culture is the classic citric production process and was
the first industrial manufacture; submerged fermentation was
developed only after that.
•
Surface fermentation is still used in industries of small and medium
scale because it requires less effort in operation, installation and
energy cost.
• The process is carried out in fermentation chambers where a great
number of trays is arranged in shelves. The culture solution is held in
shallow trays with capacity of 0.4 to 1.2 m3 and the fungus develops
as a mycelial mat on the surface of the medium.
78
Surface fermentation
•
The trays are made of high purity aluminium, special grade steel or polyethylene,
however steel trays supply better yields of citric acid .
•
The fermentation chambers are provided with an effective air circulation, which
passes over the surface in order to control humidity and temperature by
evaporative cooling.
79
Solid state fermentation
•
Solid-state fermentation, also known by »Koji« process, was first developed in
Japan where abundant raw materials such as fruit wastes and mainly rice bran are
available. It is the simplest method for citric acid production and it has been an
alternative method for using agro-industrial residues.
•
Solid-state culture is characterized by the development of microorganisms in a
low-water activity environment on an insoluble material that acts both as
physical support and source of nutrients.
•
Some similarities are observed with the surface process since the fungus also
develops on material surface.
The substrate is solid and it is moistened to about 70 % moisture, depending on
the substrate absorption capacity. The initial pH of the material is normally
adjusted to 4.5–6.0 and the temperature ofincubation is about 28–30 °C.
•
80
Product recovery
•
The recovery of citric acid from fermented broth is generally performed through
three procedures: precipitation, extraction and adsorption (mainly using ion
exchange resins). The first method is the most frequently used and it is applicable
in all types of processes. The second one requires a fermented broth with little
impurities.
•
In both of the methods there is the need to remove the fermented broth, micelles
of the fungus, and materials in suspension by filtration .
•
Precipitation method is the classical method and it is performed by the addition of
calcium oxide hydrate (milk of lime).
The acid is transformed into tri-calcium citrate tetrahydrate, which is lightly
soluble. The precipitate is recovered by filtration, treated with sulphuric acid
forming calcium sulphate (gypsum), which is filtered off.
•
81
Product recovery
•
Mother liquor of citric acid solution is treated with active carbon and passed
through cation and anion exchangers.
•
Finally, the liquor is concentrated in vacuum crystallizers at 20–25 °C, forming citric
acid monohydrate . Anhydrous citric acid is obtained at crystallization temperature
higher than 36.5 °C. The crystals are separatedThe crystals are separated by
centrifugation and the dry stage is conducted at a temperature bellow 36.5 °C for
monohydrate product and above this for anhydrous product.
•
Generally, a bed flowing dryer is used. Two kinds of wastes are generated through
precipitation technique: the microorganism residue contains proteins, amino acids,
inorganic matter, sugar, colloid, pigment, biotin, etc., and the other is calcium
sulphate.
•
The first one can be dried and used as forage or supplied to forage factory and the
second can be supplied to cement factories.
82
Lists amino acids used in the food industry.
83
Clean rooms
84
Airborne classification in EU GMP
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Federal Standard 209D Class Limits
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ISO14644-1 Standard
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