Rifamycin B Production Pattern In Nocardia Rsp-3 Strain And

Current Trends in Biotechnology and Pharmacy, Vol.2 (1) 173-181 (2008) ISSN:0973 - 8916

Rifamycin B Production Pattern in Nocardia RSP-3 Strain and Influence of Barbital on Antibiotic Production Y. Mahalaxmi, Ch. Subba Rao, G. Suvarnalaxmi, T. Satish, P. Sudhakar and R. S. Prakasham* Bioengineering and Environmental Centre, Indian Institute of Chemical Technology Hyderabad — 500 007 *For correspondence: [email protected]

Abstract Rifamycin B is a polyketide antibiotic from ansamycin family with a pronounced antimycobacterial activity and is extensively used in clinical treatment of tuberculosis and leprosy.˚ The present outbreak of AIDS-related mycobacterial infections further boosted the scientific research to improve this antibiotic production using novel microbial system.˚ In the present investigation, rifamycin B production pattern was studied under submerged fermentation using isolated Nocardia RSP-3 strain.˚ The strain was tested for its antibiotic productivity using complex medium consisting of soyabean meal, glucose, and calcium carbonate.˚ The analysis of growth vs antibiotic production pattern revealed that antibiotic production in this strain is started in exponential phase and maximum accumulation occurred in late stationery and death phases indicated mixed growth associated nature. Variation of carbon source supplementation improved the productivity from 800 — 1200 mg/liter with 10% (w/v) glucose addition. The effect of addition of barbital to the fermentation medium was studied in detail. A 100% improvement was found with barbital supplementation along with fermentation medium components.˚ However, its supplementation at different age cultures denoted further enhancement in rifamycin B productivity (approximately 120%) in this strain.˚Up on optimization, the antibiotic productivity was improved more than 8 times.

Key words Antibiotic, Bacteria, Barbital, Fermentation, Production, Rifamycin Introduction The rifamycins, commonly known by rifamycin B, belongs to ansamycin antibiotics family with pronounced anti-mycobacterial activity and occupy a major role in treatment of various mycobacterium infections caused by Mycobacterium tuberculum and Mycobacterium leprae (1, 2). These are extensively used in the clinical treatment of tuberculosis, leprosy and AIDS-related mycobacterial infections caused by Mycobacterium avium complex, an opportunistic pathogenic organism in AIDS (3). Formulations that are in use include rifampicin, rifabutin, rifamide etc., and these are obtained by chemical modification of native antibiotics rifamycin B and rifamycin SV (4). Amycolatopsis mediterranei is the only microorganism that has been in use for commercial production of Rifamycin B in the submerged cultivation and the volume ranges from 100 to 1000˚m3 (5) using complex fermentation medium. With the increasing demand of this antibiotic in terms of its utility spectrum in the current AIDS outbreak world forced the scientific community to look for novel antibiotic producers from exotic environment or increase the productivity with existing microbial system by modifying the cultivation procedures.

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Though different microbial strains have been isolated and characterized for production of rifamycin B wherein the yields ranging from 1.92 to 19.00 g/l, a few of them reached to industrial level. Industrial fermentation process are developed based on laboratory scale studies and typically use a multi-substrate economic complex medium especially locally available substrates (6). In ansamycin related antibiotics producing microbial strains, these secondary metabolites derived from molecular structures consisting of an aromatic nucleus (a naphthalenic or benzenic ring system) and a long handle-shaped aliphatic ansa bridge joining two opposite positions of the nucleus (7). Mejia et al., (4) working with mutant strain of Amycolatopsis mediterranei demonstrated that rifamycin B production could be improved with the supplementation of 5, 5diethylbarbituric acid commonly known as barbital in the culture medium and reported that this stimulation was mainly due to enhanced availability of oxygen for antibiotic synthesis indicating the influence of culture/fermentation conditions role in enhancement of this secondary metabolite synthesis. In the present investigation, an isolated microbial strain, Nocardia RSP-3, with rifamycin B production potential has been characterized for its growth and antibiotic production pattern in the presence and absence of barbital. Rifamycin B production in this microbial strain was dependent on the culture age, aeration levels and the production could be improved to more than 100% by optimizing fermentation conditions. Materials and methods Microorganism and Medium composition Isolated Nocardia RSP-3 strain was used in this study. Purified strain was grown and maintained in medium consisting (g/l) Dextrose —20, glycerol-20, yeast extract-5, beef extract-3, casein acid hydrolysate-3, peptone-2.5, malt extract-1. The pH of the medium was adjusted to 8.0 using 0.1 M NaOH or HCl solution. For growth studies,

25ml of medium was taken in a 250 ml Erlenmeyer flask, and incubated in a shaking incubator for 72 hrs at 250 rpm. Agar based above medium was used for development of slants and stored at 4OC till further use. Production medium and fermentation conditions For antibiotic production, the following medium consisting (g/l) of soyabean meal-36, dextrose75, barbital-2, calcium carbonate-3, magnesium sulphate-0.02, distilled water-1000 ml (pH-8). Twenty-five ml medium was used in 250 ml flask and inoculated with 10% (v/v) inoculum and incubated on shaking incubator at 250 rpm and 280C. For studying the effect of pH on antibiotic production, medium pH was adjusted to predetermined pH before sterilization and used for fermentation. Effect of carbon sources and nitrogen sources Influence of different carbon and nitrogen sources on rifamycin B production during fermentation was studied by supplementing the selected nutrient to the fermentation medium before sterilization and culturing the microbe in the above growth conditions. Effect of barbital Barbital is used as inducer of rifamycin B and suppressor of other rifamycins. Its role on antibiotic production was studied by addition of separately sterilized barbital in sterilized fermentation medium containing cultures of age of 0hrs, 24hrs, 48hrs and 72hrs under sterilized conditions. The samples were collected for every 24 hours and analyzed for rifamycin production. Assay Rifamycin B in culture flasks was estimated colorimetrically according to the procedure of Pasqualucci et al., (8) 1970 by diluting the 0.1 ml of cell free fermentation broth with 5 ml with acetate buffer (pH - 4.63) and reading the

Rifamycin B Production patter in Nocardia RSP-3 absorbance at 425 nm against blank containing 0.1ml of fermentation broth diluted to 5ml with 0.1% Na N02 containing acetate buffer (pH 4.63). Rifamycin B was calculated using following formula Abs 425 * 50000 Rifamycin B (µg/ml) = 21.5 Isolation and identification of Rifamycin B The rifamycin was isolated according to Venkateshwarlu et al. (9). The cell free broth after fermentation was collected by centrifugation and the pH of the solution was adjusted to 2.0 using 2N HCl solution. The antibiotic was extracted using equal volumes of ethyl acetate. The organic fraction was separated and

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evaporated using rotary evaporator. The solid fraction was dissolved in methanol and used for analysis. Initially, the sample was checked for its purity using TLC using chloroform:methnol (4:1). The purified sample was further subjected for LC-MS and the spectrum was analyzed. Results and Discussion For the production of rifamycin, the isolated Nocardia RSP-3 strain was grown in fermentation medium for 9 days at 28OC and the cell free fermentation medium was subjected for extraction of antibiotic produced using ethyl acetate after adjusting the pH of the broth to 2.0 with 2N HCl (9). This ethyl acetate fraction was concentrated by vacuum evaporation and analyzed using LC-MS (Fig.1). The LC-MS data

Fig 1: LC-MS spectrum of antibiotic produced by isolated microbial strain

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indicated characteristic peaks at 756 (M. +H — 25%), 724 (OCH3 — 65%) and 697 (CH 3CO — 5%) which is similar to literature reported rifamycin B spectra (10). Hence, further studies were made for optimization of rifamycin B production with this microbial strain. Production of any biotechnological product whether it is of recombinant or classical fermentation economic feasibility plays an important role. Numerous applications are exampled in food, agrochemical and pharmaceutical Fig 2: Influence of medium pH on growth and rifamycin B industries (11- 15). Further, the production by isolated microbial strain cost-competitive nature of such products demands an optimal operation of the production observed in the medium having the process (16, 17). In general, it was observed that pH of 6.0 to 9.0 with optimum at pH 8.0. Further down regulation of secondary metabolic analysis of the antibiotic production pattern biosynthetic gene expression is essential for well- revealed that only growth of the strain was balanced primary metabolism (growth). noticed in pH 4.0 medium not antibiotic Therefore, fermentation process supervision is of production. Such data reveal that the rifamycin particular importance to ensure consistent B production in this isolated microbial strain is operation and thereby achieve high quality growth associated. Similar trend was noticed by products. Hydrogen ion concentration of medium Venkateshwarlu et al., (3) where maximum plays a vital role in metabolism of any organism growth and antibiotic production was observed and each organism has its optimum pH where at pH 7.2. growth of selected microbial strain and its cellular metabolism occurs in optimum levels compared The influence of incubation temperature was to other conditions (18). Hence, the influence of studied using fermentation by incubating the medium pH on the growth of the isolated fermentation medium in the selected O microbial strain has been investigated by temperatures (24 — 32 C) with an interval of 2OC. inoculating the strain in the medium having pH The results were presented in Fig 3. Maximum range of 3.0 to 9.0 and analyzing the growth and growth was noticed at 28OC and variation of this rifamycin production pattern regularly during the temperature resulted in reduction of biomass growth period. The growth data revealed that production at either sides. In fact, no growth was this microbial strain grows effectively in the pH noticed at 32 OC. Analysis of rifamycin B range of 5.0 to 9.0 with maximum growth at pH production during fermentation period revealed 8.0 indicating its alkaline nature (Fig 2). Higher that maximum antibiotic production noticed with or lower media pH resulted in reduction of 28OC incubated cultures. These results vary with growth. The antibiotic production pattern also the reported data on production of rifamycin by showed the similar influence, however, its Amycolatopsis mediterranei VA18 where

Rifamycin B Production patter in Nocardia RSP-3

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due to limitation in incubator shaker. Antibiotic production rate of the strain improved with increase in incubation rpm and it was noticed to be changing with rpm rates. Further it was noticed that the volume of the fermentation medium should be 10% of any given flask (25ml for 250ml flask).

Fig 3: Effect of incubation temperature on growth and rifamycin B production by isolated microbial strain

maximum antibiotic production observed with 32 OC grown culture (19). The influence of incubation temperature on microbial growth and product production was also reported in various strains (13, 20 & 21).

Since microbial productivity is directly proportional to biomass, the role of initial inoculum concentration on antibiotic production with this strain was studied. High yield of rifamycin B was obtained with 72 hours grown 10 % (v/v) inoculum level (Fig 5). It was interesting to note that increase of inoculum level up to 10% resulted in increased rifamycin B production and further increase resulted in reduction of antibiotic production. Such inoculum concentration dependent product production variations were reported in other microbial strains (18, 23, 24).

Rifamycin B production in Amycolatopsis mediterranei was associated with aeration levels (22). Hence, the influence of rpm on antibiotic production was investigated using this microbial strain. Antibiotic production was increased with increase in rpm during fermentation (Fig 4.) Maximum rifamycin B production was noticed at 250 rpm and further studies could not be made

Fig 5: Influence of inoculum concentration on rifamycin B production by isolated microbial strain

In general, antibiotic fermentation processes are cost intensive and the profitability is greatly dependent on the product yield per unit substrate consumed. In order to reduce cost, industrial processes use organic nitrogen substrates such as corn steep liquor and yeast extract. Thus, Fig 4: Effect of rpm on growth and rifamycin B although the stoichiometric analysis is the first production by isolated microbial strain logical step in process development, it is often

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difficult to achieve due to the ill-defined nature of the medium. In order to develop effective medium composition, the role of different carbon (Fig 6) and nitrogen (Fig 7) sources were

Fig 6: Effect of different carbon sources on rifamycin B production by isolated microbial strain

Fig 7: Effect of different nitrogen sources on rifamycin B production by isolated microbial strain

evaluated for their influence on growth and antibiotic production by this microbial strain. Of the various carbon sources that had been screened, glucose and galactose were found to be the best and lactose as carbon source indicated the least antibiotic production. These results are similar with the reported literature where glucose was found to be the best choice as carbon source for growth or production of any compound. In

this study, glucose being economically cheap compared to galactose, glucose was selected as the carbon source for further experiments. Glucose concentration dependent rifamycin B production studies indicated that supplementation of 10% glucose into the medium resulted maximum antibiotic production (1.2 g/l) and variation of this carbon source concentration on either sides showed reduced production (results not shown). Among different nitrogen sources studied, soyabean meal along with peptone (1.8 g/l each) was found to be ideal for antibiotic production (1.23 g/l) with this isolate. Approximately 60% improvement was noticed with addition of these nitrogen sources in combination. Yeast extract supplementation showed the least production of antibiotic compared to all other organic complex nitrogen sources studied. In literature too, soyabean meal was the most widely used nitrogen source in the production of rifamycin B and other metabolite products (5, 18). In order to understand further, the impact of combination of nitrogen sources on antibiotic production with this microbial strain was studied by incorporating the combination of studied nitrogen sources in the fermentation medium (insert of Fig 7). The results depicted that rifamycin B production further improved from 1.23 g/L to more than 2.0 g/L in soyabean meal and peptone (1.8g/L each) supplemented fermentation environments. However, combinations experiments with yeast extract and peptone could not improve the antibiotic production. In fact, antibiotic production was reduced compared to other studied conditions. These results further suggested that soyabean meal in combination with peptone is effective antibiotic inducer in this microbial strain. Such combinatorial medium components influence has been reported in production of extracellular enzyme in Bacillus sp. (18). Barbital is known to induce rifamycin B production by suppressing the production of other rifamycin antibiotics (4). Therefore, impact of

Rifamycin B Production patter in Nocardia RSP-3 barbital addition on rifamycin B production was investigated. The data suggested that yield of rifamycin B influenced by the barbital addition to the medium (Fig 8). Higher antibiotic production was noticed in all barbital supplemented fermentation conditions compared with control. This enhanced production was associated with the fermentation time indicating the barbital role on altering the strains metabolism and improving the rifamycin B production. Barbital addition improved antibiotic production more than 100% compared to control (without barbital in the medium) (Fig 8). This improvement was also associated with the growth of the organism. This could be evidenced based on the observation that the values of antibiotic productivity were noticed to be 15.27 in control and 23.08 mg/h/L barbital supplemented

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indicated that higher and prolonged antibiotic production period. This is evidenced based on the observation that maximum rifamycin production was noticed at 144 and 168 hours of incubation in control and barbital supplemented conditions, respectively (Fig.8) with a productivity improvement of 51%. This data revealed that barbital has impact on cellular metabolism associated antibiotic production in two angles, i.e., in improving the productivity and enhancing the production period. Barbital has been reported as toxic to the microbial growth (25). Hence, the impact of barbital addition at different cell growth periods was investigated. The data depicted that a slight improvement in rifamycin B production was noticed in variation of barbital addition at different fermentation incubation times. A 10% of antibiotic production improvement was noticed when barbital supplemented to the fermentation medium after 24 hours of incubation compared to 0.0 hour. Whereas, supplementation of barbital in subsequent fermentation times reduced the rifamycin B production, however, the production was higher compared to control fermentations (without barbital) (Fig 8). Concentration dependent barbital influential studies revealed that 0.2% was effective for rifamycin production by this isolated strain compared to other studied range of Fig 8: Effect of barbital on rifamycin B production by isolated 0.1 to 0.4% (w/v) (results not shown) microbial strain and maximum production of 4.4 g/l was observed under optimized fermentations indicating that barbital associated conditions. rifamycin B production improvement is associated with the altered metabolism of the microbial strain. In literature too, it was reported Conclusions that barbital negatively regulate the electron transport system and increases the availability of The potential of isolated microbial strain, Nocardia RSP-3, on rifamycin B production was oxygen to other cellular activities (25). investigated under different growth and medium Further analysis of the rifamycin production trend conditions. The growth and antibiotic production in fermentations with and without barbital occurred simultaneously in this isolated strain.

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Maximum growth was noticed at pH 8.0, at 28OC with glucose and galactose as carbon source. Among all nitrogen sources tested, soyabean meal showed maximum rifamycin B production compared to other nitrogen sources tested. Barbital improved the antibiotic production in this strain was noticed with supplementation of barbital (0.2%) to the 24 hours grown cultures compared to other tested conditions. Under optimized environment rifamycin B production was improved from 0.8 to 4.4 g/l with this microbial isolate indicating a productivity improvement from 3.63 to 26.18 mg/h/L after optimization.

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