Nitrogen Assimilation in Actinomycetes

Published by: Nepalese Society for Microbiology 

Original Article Nepalese Journal of Microbiology (NJM), 2003, Vol 2, No 1, 2011, pp 61-65)

Nitrogen Assimilation in Actinomycetes

Kiran B Tiwari^1,2* and Upendra Thapa Shrestha^1,2

1Research Laboratory for Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal

2Kantipur College of Medical Science, Sitapaila, Kathmandu, Nepal

Abstract

As actinomycetes are one of the diverse groups of soil bacteria possessing commercially useful enzymes and therapeutically useful bioactive molecules, biochemical characterization of the individual isolates is of utmost importance to understand their basic physiology. The work explored importance and optimization of nitrogen substrates in basal culture media. Besides nitrogen source, actinomycetes can use peptone as carbon source too. However, the growth will be retarded at a concentration above 0.5% of peptone. The organisms can use inorganic nitrogen but growth is pretty slow. Actinomycetes have no essential amino acid; however, organic nitrogen enhances the growth. Actinomycetes are weak acid producers. With 10mM glucose, 0.2% peptone is the optimum concentration for glucose (carbohydrate) utilization test. Hence, carbohydrate utilization test should be done in 0.2% peptone and appreciable lowering of pH should be expected at least with five days of incubation at 28ºC.

Keywords: Actinomycetes, aminoacid, nitrogen assimilation, growth optimization

Mass Spectroscopy

MASS SPECTROSCOPY

- Branch of Spectroscopy

- Analytical technique that gives information concerning the molecular structure the of organic and inorganic compounds

- It can determine molecular weight of as high as 4000

- Qualitative analytical tool to characterize different organic substances

- The quantitative analysis of mixtures (gases or liquids and sometimes solids)

- Based on the simple principle

- Yet it is a very complex and very expensive instrument

- Commonly used because of its high speed and reliability

PRINCIPLE

The compounds under investigation are bombarded with a beam of electrons which produce an ionic molecules or ionic fragments of the original species then they are separated on the basis of the difference in their masses. Suppose ionization is as follows;

M + e^- ® M⁺ + 2e^-

Where:

M⁺ = an ionized molecule

e^- = an electron

The ions are then accelerated in an electric field at voltage V. Now the energy given to each particle is eV and this is equal to the kinetic energy of the ions.

½ mn² = e V

Or n = Ö 2e V/m

Where:

n = velocity of the particle of mass, m

e = charge on an electron

m = mass of the particle

V = accelerating voltage

All the particles possess the same energy, eV. Also, all particles have the same kinetic energy, ½ mn². As the value of m varies from particle to particle, the velocity of particle in motion also changes to meet the potential energy. E.g. For a particle with mass, m₁ and n₁

½ m₁n₁²= eV

Similarly, for particles of mass m₂, m₃, m₄,………………..m_n the velocities are n₁, n₂, n₃, …………….n_n. Now their kinetic energies are:

½ m₂n₂²= eV ½ m₃n₃²= eV ½ m₄n₄²= eV………………½ m_nn_n²= eV

From above, we have;

½ m₂n₂²= ½ m₃n₃²= ½ m₄n₄²= ………………½ m_nn_n²= eV

After the charged particles have been accelerated in an applied voltage, they enter into the magnetic field, H. This field attracts the particles and move in a circular around it. The attractive force due to magnetic field is Hen, where the balancing centrifugal force on the particle is mn²/r. When the particle starts moving uniformly around the circular path the two forces become equal. i.e.

mn²/r = H.e.n

r = mn / H.e

Where, r is the radius of circular path of the particle in the motion.

From above equations;

r = [m / H.e] Ö 2eV/m

On squaring both sides;

r² = [m² / H².e²] 2eV/m

\ m/e = [H² . r²]/ 2V

The radius of circular path of the particles depends on the accelerating voltage- V, the magnetic field-H and the ratio of mass upon charge-m/e. As e, V and H are constants; the radius of ionized molecule depends on the mass only which is actually the main basis of separation of particles.

INSTRUMENTATION

pH Value, indicators, charge relations

pH Value, indicators, charge relations

pH Value:

The pH value is the negative logarithm to the base 10 of the hydrogen ion concentration.

Derivation:

In pure water, the concentration of hydrogen ions [H₃O⁺] is equal to the concentration of hydroxyl ions [OH^–], viz

[H₃O⁺] = [OH^-] = 10^-7 mol/L

At this equilibrium, the water has a neutral reaction. In practice, the minus sign is dropped, 10^-7 = pH 7.

According to the above equation, if the hydrogen ion concentration in an aqueous system is increased, the hydroxyl ion concentration becomes less, and the system has an acid reaction.

If the hydroxyl ion concentration is increased, the hydrogen ion concentration decreases accordingly, and the solution has an alkaline reaction.

pH = 1 ←	pH = 7	→ pH = 14
acid	neutral	alkaline

Determination of pH value:

1. Potentiometric (electrometric)

With a hydrogen electrode or glass electrode (measuring range pH 0 – pH 14)

2. Colorimetric

With indicators or indicator papers.

Common indicators

Indicator	Effective pH range	Color change	Concentration of solution
Thymol Blue	1.2 – 2.8	red – yellow	0.1% in 20% alcohol
Dimethyl Yellow	2.9 – 4.0	red – yellow	0.1% in 90% alcohol
Bromophenol Blue	3.0 – 4.6	yellow – blue	0.1% in 20% alcohol
Congo Red	3.0 – 5.0	blue – red	1% in water
Methyl Orange	3.2 – 4.4	red – yellow	0.1% in water
Bromocresol Green	3.8 – 5.4	yellow – blue	0.1% in 50% alcohol
Methyl Red	4.8 – 6.0	red – yellow	0.2 % in 90% alcohol
Litmus	5.0 – 8.0	red – blue	0.3 % in 90% alcohol
Bromocresol Purple	5.2 – 6.8	yellow – purple red	0.04 % in 90% alcohol
Bromothymol Blue	6.0 – 7.6	yellow – blue	0.1% in 20% alcohol
Neutral Red	6.8 – 8.0	red – yellowish orange	0.1 in 70% alcohol
Phenol Red	6.6 – 8.0	yellow – red	0.02 % in 90% alcohol
o-Cresol Red	7.0 – 8.8	yellow – purple red	0.1% in 20% alcohol
Tropaeoline 000	7.6 – 8.9	yellowish green – pink	1% in water
Phenolphthalein	8.2 –10.0	colorless – pink	1.0 % in 50% alcohol
Thymol Blue	8.0 – 9.6	yellow – blue	0.1% in 20% alcohol
Thymolphthalein	9.4 –10.6	colorless – blue	0.1% in 90% alcohol
Alizarin Yellow R	10.1 –12.0	yellow – red	0.1% in 50% alcohol
Tropaeoline O	11.3 –13.0	yellow – orange red	0.1% in water

Example: pH colour range of bromocresol green

Color: Yellow = pH 3.5 and lower

Yellow-green = pH 4.0

Green = pH 4.5

Blue-green = pH 5.0

Blue = pH 5.5 and higher