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Group 8 SCL 2 presentation(BTC 4205).pptx
1. Productions and applications of amino acids
in livestock and aquaculture
INDUSTRIAL MICROBIOLOGY (BTC4205-1)
GROUP 8
JERRY CALDWELL A/L SIMON WILSOR (203205)
FARIS NULHAQIM BIN MUHAMAD (200663)
RENUGA A/P RAMES (204539)
MANIMEGALAI A/P GANESAMURTHY (202492)
6. LYSINE 2D STRUCTURE
Hydrophilic
Can dissolve in water
L-lysine
L-isomer of lysine(“Lysine”,n.d)
C6H14N2O2
It has two amine groups and one
carboxyl group
7. 3D IMAGE OF LYSINE
Incorporated
biosynthetically into
proteins during
translation
Humans cannot
synthesize it
Proteinogenic
amino acid
Aspartate family
Essential
amino acid
Molecular
weight:146.190
g/mol
Essential amino
acids
biosynthesised
from aspartate
9. Corn stover
Dry acid pretreatment
Biodetoxification
Lysine 33.8
g/L
Aspen plus
modelling and
economic
analysis
C.glutamicum
SIIM B253 for
lysine
producition
L-lysine fermentation from lignocellulose feedstock
10. Strains
C.glutamicum obtained from SIIM
● Traditional homoserine autotrophic
mutant
Amorphotheca resinae ZN1
was isolated
● As biodetoxification
fungus
Raw Materials
Corn stover
● Obtained from Inner Mongolia
Composed of 33.0 % cellulose
● 33.0% cellulose, 26.9%
hemicellulose, 20.8% lignin, and
5.3% ash (Chen et al.,2018)
1 2
Production steps
11. Dry acid pretreatment and
biodetoxification processing
Sulphuric acid added to the reactor
● Solid liquid ratio of 2:1
3
Neutralized with 20%(w/w)
CaOH2
● To a pH value of 5.5
L-lysine fermentation
C. glutamicum B253 cultured at 30 °C
for 48 h on LB agar plates
● 10 g/L tryptone, 5 g/L yeast extract,
5 g/L NaCl, and 17 g/L agar
Simultaneous saccharification and
fermentation (SSF)
4
● Conducted in 5L helical stirring
bioreactors(Chen et al.,2018).
.
Production steps
13. Process model description
Build based on NREL design
● Using Aspen Plus software
Techno-Economic analysis
Calculation of equipment
design and chemical usage
● Aspen Plus
Modelling(Chen et
al.,2018).
7 8
14. Production results and conclusion
Maximum L-lysine of
14.7 g/L with the yield of
0.29 g/g
● This was after the
nutrient addition and
fermenter parameters
were adjusted(Chen et
al.,2018)
Simultaneous saccharification and
fermentation (SSF)
● Efficient way to alleviate the
glucose inhibition on cellulase
activity
The maximum L-lysine titer of
33.8 g/L was obtained using C.
glutamicum B253
● Using SSF at 30 % solid
loading of corn stover
and after dry acid
pretreatment and
biodetoxification
15. APPLICATION OF LYSINE IN LIVESTOCK
INDUSTRY
Lysine is part of the 20 AAs
that serve as building blocks
for protein biosynthesis
Lysine is considered as the
essential dietary AAs since it
needs to be supplied
exogenously
Lysine is usually given as
capsules, tablets, or liquid
mixed with the feedstock
Building blocks
for protein
Cannot be
produced naturally
Used as supplement in
feedstock
WHY LYSINE IS IMPORTANT IN LIVESTOCK INDUSTRY AND HOW IT IS USED?
(ESPECIALLY MONOGASTRIC ANIMALS)
16. BENEFITS OF LYSINE
● Lysine can improve muscle protein
accretion and whole-body growth
of monogastric animals such as
pigs
● Lysine plays a role in the
production of carnitine, which
turns fats into energy and helps
lower cholesterol
17. BENEFITS OF LYSINE
● Lysine assists in calcium absorption
and collagen production.
● Helps in keeping the skin, bones,
cartilage and tissues healthy.
● Lysine plays a role as a crucial
precursor for de novo synthesis of
glutamate
● It is an excitatory neurotransmitter in
the mammalian CNS.
18. BENEFITS OF LYSINE
● Lysine involves in the synthesis of
cytokines, lymphocytes and the
optimum functioning of the immune
system.
● Lysine also affect the hormone
production and activity in the body.
● Eg: hormone like insulin-like growth
factor (IGF-1).
19. BENEFITS OF LYSINE
● IGF-1 is a hormone in which it functions
primarily to stimulate growth.
● A study conducted on rats showed that IGF-
1 level drops with a low level lysine diet
(Liao et al., 2015).
● Another study conducted on pigs also
showed the same result.
● IGF-1 hormone is influenced by the
concentration of lysine available in which
better lysine concentration helps in
producing more IGF-1 hormone
● This study shows the results of different
concentrations of lysine on body weight of
broilers after 6 weeks (Nasr & Kheiri, 2011).
● It can be concluded that broiler fed with
high lys (120% NRC) gained the most
weight over time.
● High lysine content in the feed is important
for the production of broiler chickens to
produce high quality meat.
20. RECOMMENDED VOLUME FOR LYSINE
CONSUMPTION
- The NRC dietary total lysine
recommendation for starting broiler
chicks from 0 to 21 days of age was
1.2% of the diet in the 1984 edition.
- In the 1994 edition the NRC
lowered this recommendation to
1.1% of the diet.
23. ARGININE 2D STRUCTURE
Hydrophilic
Can dissolve in water
L-arginine
L-isomer of arginine
C6H14N4O2
It is basic and has two functional groups, a
carboxyl group (COOH) and an amine
group (NH2).
24. Type 2
Corynebacterium (Brevibacterium)
N-acetylglutamate-acetylornithine acetyltransferase
catalyzes a transacetylation reaction between
glutamate and N-acetylor-nithine
To form N-acetylglutamate and ornithine.
Type 1
Escherichia coli and Bacillus subtilis
N-acetylglutamate is synthesized from
L-glutamate by N-acetylglutamate
synthase
Inhibited by L-arginine through a
feedback control mechanism.
Metabolic control of L-arginine biosynthesis
N-acetylglutamokinase is inhibited by L-
arginine and ornithine.
25. Fermentation Conditions
Production of L-arginine is strongly
inhibited by the lack of oxygen
Oxygen dissolves in culture broth with a
saturation value as low as 7.5 ppm at
atmospheric pressure
Raw Materials
Starches from tapioca and
corn as carbon source
Hydrolyzed with enzymes; liquid
amylase and glucoamylase to form
liquid glucose.
Ammonium sulfate
L-arginine molecule is 32% nitrogen
needing ample nitrogen source to
produce a large amount of L-arginine.
1 2
pH
Regulated near neutral point
26. Source:
Utagawa, T. (2004). Production of arginine by fermentation. The Journal of nutrition, 134(10), 2854S-
2857S.
Biological Synthesis of L-arginine
27. Fermentation Broth
Filtration
Anion Exchange Resin
Ultrafiltration
Drying
Production of L-arginine from fermentation broth of
Corynebacterium (Brevibacterium)
De-colouration
Cation Exchange Resin
Concentration
Crystalization
Centrifugal separation
Membrane filtration
Minimize the impurities in
the L-arginine crystals
to form the L-
arginine crystals
Active carbon powder
cooled to
10°C
Dried with a dryer to minimize the moisture
concentration at 0.5%
98.5% pure L-arginine crystals obtaines
30. IMMUNOSTIMULATION
● Cell growth and proliferation
● Macrophage stimulation for
secreting cytokines
● Leukocyte immune responses
● Arginine plays a role in the
production of NO, which
responsible for host innate defense
mechanisms.
Metabolites nitric oxide
Biosynthesis of polyamines
31. Fig. 1: Mortality of fingerling channel catfish fed different
levels of L-arginine for two weeks and subsequently
exposed to virulent E. ictaluri.
● This study is to know the impact of arginine
on the immune systems of channel catfish,
and dietary supplementation with arginine
in ability of channel catfish to survive
exposure to Edwardsiella ictaluri
● Increased arginine supplementation
improves the ability of catfish phagocytes
to ingest foreign particles.
● Indicating that dietary arginine
supplementation is also an efficient way to
boost fish immunocompetence and disease
resistance, reducing the need to treat
culture systems during epizootics
J. Alejandro Buentello &
Delbert M. Gatlin III (2001)
32. Growth Enhancer
Regulates Hormone
Production
● Arginine acting as a secretagogue that activates
growth hormone biosynthesis and exhibit
circulating levels that directly correlate with the rate
of fish growth
33. RECOMMENDED VOLUME FOR ARGININE
CONSUMPTION
Dietary arginine level at 2.81% could
optimize anti-ammonia-nitrogen stress
ability of juvenile yellow catfish and a level
of 3.23% arginine seemed to depress the
growth performance of fish and decreased
their tolerance to the ammonia-nitrogen
stress under current study.
Table 1. Formulation and proximate composition
of experimental diets (air-dry basis, %).
36. Application of other amino acid
in feedstock and aquaculture
Amino acid Feedstock application
Proline Synthesis of polyamines (Wu, G. et
al.,2011)
Threonine Reduced Salmonella and Escherichia
coli (E. coli) colonies,
Amino acid Aquaculture application
Histidine Protein synthesis, Tissue
formation(Khan, M. A. ,n.d.)
Tryptophan Stress responses, and antioxidant and
immune systems
37. REFERENCES
● Buentello, J. A., & Gatlin III, D. M. (2001). Effects of elevated dietary arginine on resistance of channel
catfish to exposure to Edwardsiella ictaluri. Journal of Aquatic Animal Health, 13(3), 194-201.
● Chen, Z., et.al . (2018, September 20). A preliminary study on L-lysine fermentation from
lignocellulose feedstock and techno-economic evaluation. Bioresource Technology. Retrieved January
24, 2022, from https://www.sciencedirect.com/science/article/pii/S0960852418313518
● Lysine. National Center for Biotechnology Information. PubChem Compound Database. Retrieved
January 24, 2022, from https://pubchem.ncbi.nlm.nih.gov/compound/Lysine#section=3D-Conformer
● Khan, M. A. (n.d.). Histidine requirement of cultivable fish species: A Review. Retrieved January 29,
2022, from https://juniperpublishers.com/ofoaj/pdf/OFOAJ.MS.ID.555746.pdf
● Hoseini, S. M., Ahmad Khan, M., Yousefi, M., & Costas, B. (2020). Roles of arginine in fish nutrition and
health: insights for future researches. Reviews in Aquaculture, 12(4), 2091-2108.
● Javad Nasr & Farshid Kheiri (2011) Effect of different lysine levels on Arian broiler performances,
Italian Journal of Animal Science, 10:3, DOI: 10.4081/ijas.2011.e32
● Nandkishor Jha(n.d).Commercial Applications of Amino Acids: 3 Applications
https://www.biologydiscussion.com/amino-acids/commercial-applications-of-amino-acids-3-
applications/10334
38. REFERENCES
● Hoseini, S. M. (2017, October 25). Physiological roles of tryptophan in teleosts:
Current knowledge and perspectives for future studies. Wiley Online Library.
Retrieved January 29, 2022, from
https://onlinelibrary.wiley.com/doi/10.1111/raq.12223
● Tang, Q. (2021, July 28). Physiological functions of threonine in animals: Beyond
nutrition metabolism. Nutrients. Retrieved January 29, 2022, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8399342/
● Utagawa, T. (2004). Production of arginine by fermentation. The Journal of
nutrition, 134(10), 2854S-2857S.
● Xu, J.,et al. (2014, May 31). Metabolic Engineering Corynebacterium glutamicum
for the L-lysine production by increasing the flux into L-lysine biosynthetic
pathway - amino acids. SpringerLink.Retrieved January 24, 2022, from
https://link.springer.com/article/10.1007/s00726-014-1768-1
● Wu, G. et al,(2011, April). Proline and hydroxyproline metabolism: Implications
for animal and human nutrition. Amino acids. Retrieved January 29, 2022, from
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3773366/
41. Which microoorganism efficiently biosynthesise L-arginine
amino acid for the use of aquaculture industry?
Type 1 Type 2
Escherichia coli and Bacillus subtilis Corynebacterium (Brevibacterium)
A B
42. Which microoorganism efficiently biosynthesise L-arginine
amino acid for the use of aquaculture industry?
Type 2
Corynebacterium (Brevibacterium)
B
SInce L-arginine is biosynthesized from the
precursor L-glutamic acid through ornithine
and citrulline, the use of strains with a high
capability for producing L-glutamic acid such
as Corynebacterium (Brevibacterium) is
desirable. It is well known in the production
of L-glutamic acid on a commercial scale
Editor's Notes
Arginine is one of the essential amino acids used in aquaculture industries aside from glutamic acid. For today, we will emphasize on the production of L-arginine using microbiological synthesis. Most arginine with the L-configuration has been produced by the direct-fermentation method from natural carbon sources.
As of now, we will talk about the production of amino acid in aqualculture industry as these dietary amino acids needed for growth and maintenance of aquatic species being cultured. In fish, shrimp and other aquatic species, the role of amino acids in the diet is to work as the basic unit for protein synthesis.
Arginine is often considered to be the most hydrophilic of the 20 natural amino acids. It is a nonessential amino acids that can be produced by the human body. Its side chain contains a large guanidinium moiety that has the capacity for up to six hydrogen bonds as shown in the figure. L-arginine is an L-alpha-amino acid that is the L-isomer of arginine. This is the chemical formula of arginine. It is basic and has two functional groups, a carboxyl group (COOH) and an amine group (NH2).It is also from glutamine family amino acid which plays role as a Escherichia coli and Cornyebacterium metabolite.
There are two types of metabolic control mechanisms in microorganisms. In type 1, which includes Escherichia coli and Bacillus subtilis, N-acetylglutamate is synthesized from L-glutamate by N-acetylglutamate synthase, and this enzyme is strongly inhibited by L-arginine through a feedback control mechanism. In type 2, which includes Corynebacterium (Brevibacterium), N-acetylglutamate-acetylornithine acetyltransferase, which is not inhibited by L-arginine, catalyzes a transacetylation reaction between glutamate and N-acetylor-nithine to form N-acetylglutamate and ornithine. However, N-acetylglutamokinase is inhibited by L-arginine in type 2. Briefly, to produce L-arginine efficiently by fermentation, it is necessary to breed strains with a strong biosynthetic pathway to L-arginine. Since it is biosynthesized from the precursor L-glutamic acid through ornithine and citrulline, the use of strains with a high capability for producing L-glutamic acid is desirable. And so the Corynebacterium (Brevibacterium) enters the picture as a better alternative, which is well known in the production of L-glutamic acid, was selected as a starting strain for the breeding of an L-arginine producer and has been used on a commercial scale.
Glucose from tapioca or corn is one of the best carbon sources for L-arginine production. Because 1 molecule of L-arginine contains 4 nitrogen atoms, ammonia is an essential raw material for supplying nitrogen. Starches from tapioca and corn are the main raw materials for amino acid fermentation. Sugar and sugar syrup are also used. Starches are first hydrolyzed with enzymes such as liquid amylase and glucoamylase to form liquid glucose. After filtration, the glucose solution is 95% dextrose. Another important raw material is ammonium sulfate. The L-arginine molecule is 32% nitrogen, the highest nitrogen content of any amino acid. This means that an ample nitrogen source must be supplied to produce a large amount of Larginine. Regarding the fermentation conditions, as for other amino acids, L-arginine fermentation is controlled by regulating pH near the neutral point. Due to its high oxygen requirement, L-arginine production is seriously impaired without sufficient oxygen. The production of L-arginine is strongly inhibited by the lack of oxygen, because oxygen is one of the raw materials for L-arginine. Oxygen dissolves in culture broth with a saturation value as low as 7.5 ppm at atmospheric pressure, so air must constantly be introduced into the fermentation vessel. Lactic acid accumulates in a culture where oxygen is extremely deficient. In aerobic amino acid fermentation, oxygen is one of the raw materials that must be supplied in large amounts
This is the brief representation of what is required for the L-arginine synthesis using Corynebacterium as the bacterial source or microorganism. Corynebacterium is a genus of bacteria that are Gram-positive and most are aerobic. As mentioned before, oxygen and ammonium sulphate is needed to produce L-arginine.
After L-arginine fermentation completed, the microorganisms are separated by centrifugal separation or membrane filtration. The supernatant or filtrate is charged to the resin column to separate organic acids or other amino acids.cation and anion types of resin are used to minimize the impurities in the L-arginine crystals.Active carbon powder is used for decoloration. After filtration through active carbon powder, the filtrate is concentrated to form the L-arginine crystals, which are separated by centrifugation. The crystals are dissolved in pure water and then ultrafiltered. The pure crystals are obtained by concentration and cooling. Because L-arginine dissolves well in hot water (Fig. 5), the concentrated solution must be cooled to 10°C to obtain L-arginine crystals. The crystals are dried with a dryer to minimize the moisture concentration at 0.5%. The obtained crystals (Fig. 6) are 98.5% pure.
