Nitrogen assimilation in Actinomycetes

Kiran Babu Tiwari1,2*, Upendra Thapa Shrestha1 and Vishwanath Pd. Agrawal1
1Research Laboratory for Biotechnology and Biochemistry (RLABB), Maitidevi, Kathmandu, Nepal;
2Central Department of Microbiology, Tribhuvan University, Kirtipur, Nepal

*Corresponding Address
Kiran Babu Tiwari, Research Scientist, Research Laboratory for Biotechnology and Biochemistry, Maitidevi, Kathmandu, Nepal, email: gp120cdnashuffling@gmail.com, Ph.: 977-9841-37-47-38

ABSTRACT

Actinomycetes are aerobic, Gram-positive bacteria, which may form branching filaments or hyphae that may persist as a stable mycelium or may break up into rod-shaped or coccoid elements. Actinomycetes are widely distributed in terrestrial environment (Holt, 1989). For much of this time they were regarded as an exotic group of organisms with affinities to both bacteria and fungi. However, determination of their fine structure and chemical composition initiated in the 1950’s, confirmed their prokaryotic nature. The actinomycetes comprise a ubiquitous order of bacteria, which exhibit wide physiological and morphological diversity. Actinomycetes have long been a source of commercially useful enzymes and therapeutically useful bioactive molecules. Actinomycete biodiversity is vast frontier and potential goldmine for the biotechnology industry because it offers countless new genes and biochemical pathways to probe for enzymes, antibiotics and other useful molecules (Agrawal, 2003). Over past 60 years, products derived from microbial secondary metabolites have been used to meet medical, industrial and agricultural needs, e.g., antibiotics, anticancer drugs, antifungal compounds, immunosuppressive agents, enzyme inhibitors, anti parasitic agents, herbicides, insecticides and growth promoters (Busti et al., 2006). Hence, basic parameters for better growth and physiological characteristics of native micro-flora should be optimized.

METHODOLOGY

Isolation and identification of actinomycetes: Actinomycetes were isolated from soil samples in selective medium (Starch-Casein agar, pH 7.4) by serial dilution method and subsequently purified and identified by colonial characteristics and microscopy (Singh and Agrawal, 2002).

Effects of peptone concentration on actinomycetes growth: A series of peptone broths with or without glucose (10mM) were prepared and sterilized. The isolates were inoculated into the respective culture tubes, incubated at 28ºC and observed for growth characteristics in the broth subsequently on daily basis. Respective negative control tubes were also included.

Essential amino acids for actinomycetes: Twenty glucose broth tubes containing 10mM glucose and 0.3% Na2PO4 were prepared, and all but one aminoacid (1mM) were added in respective tubes. With 0.3% Na2PO4, other three tubes contained inorganic nitrogen (1mM KNO3), organic carbon (10mM glucose), and both (1mM KNO3 and 10mM glucose) respectively. The isolate was inoculated into the respective sterile broth, incubated at 28ºC and observed for growth characteristics in the broth on daily basis. A negative control tube was also included.

Carbohydrate (Glucose) utilization test for actinomycetes: Glucose broths containing 10mM glucose, 0.3% Na2PO4, 3mg% Phenol Red and 0.2% Peptone was prepared. A nitrogen control tube (0.2% Peptone) was also included. The isolates were inoculated into the respective sterile broth, incubated at 28ºC and observed for growth and coloration of the broth on daily basis.

RESULTS

Three representative actinomycete isolates (viz. Lob 1, 8 and 15) were assayed for various tests. Not only nitrogen source, actinomycetes can use peptone as carbon source too (Table 1). The optimum concentration of peptone for growth was from 0.2 – 0.5%, beyond which the growth was somewhat reduced. Addition of glucose as carbon source allows better growth, and upto 0.8% of peptone can be used along with glucose for growth assessment of actinomycetes (Table 2). Actinomycetes can use inorganic nitrogen (KNO3), however organic nitrogen enhanced growth (Table 3). At least five days of incubation is required for glucose utilization test for actinomycetes with conventional dark yellow color of Phenol red as indicator (Table 4).

DISCUSSIONS

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 (Holt, 1989). The present work explored importance and optimization of nitrogen substrates in basal culture media as a basic parameter for better growth and physiological characteristics of the organisms. The actinomycetes were isolated and purified from soil samples, of which only representative isolates were assayed for their physiological activities concerned with nitrogen assimilation.

Conventionally, peptone is used in various culture media as major nitrogen source for the organisms. As peptone is partial digest of protein complex, its carbon atom is liable to be used by organisms. Hence, actinomycetes can use peptone as carbon source when the culture medium lacks carbohydrates (e.g., sugar/s). Voelker and Altaba (2001) assayed role of various nitrogen sources (organic and inorganic) for growth and secondary metabolite production from a streptomycete. During balanced growth, either mineral or organic nitrogen sources were readily utilized. Glutamate and alanine were used as both nitrogen and carbon source, sparing the utilization of the primary carbon source, glucose. Without sugar, 0.2–0.5% peptone concentration is optimum for growth of actinomycetes. Higher peptone concentration disturbs C/N ratio and hence retards growth. With glucose, upto 0.8% of peptone can be used for growth; because, glucose, being an alternate to peptone carbon, increases the C/N ratio to the safe level.

Actinomycetes can fairly use inorganic nitrogen, which shows their wider biosynthetic capacities. It indicates that the organisms do not have any essential aminoacid as other prokaryotes. Growth in inorganic nitrogen, however, is pretty slow compared to organic nitrogen as found by Naeimpoor and Mavituna (2006). Glutamine synthetases (GS) are key enzymes of nitrogen metabolism. Most bacteria contain only one type of GS enzyme encoded by glnA. Streptomyces coelicolor, the model organism for Gram-positive streptomycetes, however is characterized by two functional GS (glnA, glnII) involved in nitrogen assimilation. The control of nitrogen assimilation and metabolism is mediated by transcriptional and post-translational regulation systems (Reuther and Wohlleben, 2007). Differences in growth patterns for structurally difficult amino acids and simpler ones are marked during the initial days of incubation (not shown in table).

Carbohydrate utilization properties are one of the important biochemical activities of microorganisms to identify and classify them (Dietz, 1988; Holt, 1989). Microorganisms reduce carbohydrates to organic acids and concomitant reduction pf pH of the culture medium is indicated by color change with appropriate indicator. As peptone is rich source of nitrogen (about 16% of molecular weight), microorganisms are forced to produce alkaline products in peptone broth without carbohydrate. However, with sugar as surplus carbon, actinomycetes produce more organic acids, which neutralize alkali from peptone and, further, lower pH of the culture medium. Actinomycetes are weak acid producers and, hence, the optimum peptone concentration for carbohydrate utilization test is 0.2% in the culture medium. At least five days of incubation is mandatory to interpret carbohydrate utilization test for actinomycetes. The interpretation should be based on color change of the culture medium, e.g., clear yellow color of phenol red.

Acknowledgement - The authors acknowledge the third batch (2004AD) students of Universal Science College, Maitidevi, Kathmandu, for laboratory help to accomplish this research work.

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