Abstract

Rifamycin B is a polyketide antibiotic fromansamycin family with a pronounced anti-mycobacterial activity and is extensively used inclinical treatment of tuberculosis and leprosy.˚The present outbreak of AIDS-relatedmycobacterial infections further boosted thescientific research to improve this antibioticproduction using novel microbial system.˚ In thepresent investigation, rifamycin B productionpattern was studied under submergedfermentation using isolated Nocardia RSP-3strain.˚ The strain was tested for its antibioticproductivity using complex medium consistingof soyabean meal, glucose, and calciumcarbonate.˚ The analysis of growth vs antibioticproduction pattern revealed that antibioticproduction in this strain is started in exponentialphase and maximum accumulation occurred inlate stationery and death phases indicated mixedgrowth associated nature. Variation of carbonsource supplementation improved theproductivity from 800 — 1200 mg/liter with 10%(w/v) glucose addition. The effect of addition ofbarbital to the fermentation medium was studiedin detail. A 100% improvement was found withbarbital supplementation along with fermentationmedium components.˚ However, its supplemen-tation at different age cultures denoted furtherenhancement in rifamycin B productivity(approximately 120%) in this strain.˚Up onoptimization, the antibiotic productivity wasimproved more than 8 times.

Rifamycin B Production Pattern in Nocardia RSP-3 Strain andInfluence 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: prakasam@iict.res.in

Key words

Antibiotic, Bacteria, Barbital, Fermentation,Production, Rifamycin

Introduction

The rifamycins, commonly known by rifamycinB, belongs to ansamycin antibiotics family withpronounced anti-mycobacterial activity andoccupy a major role in treatment of variousmycobacterium infections caused byMycobacterium tuberculum and Mycobacteriumleprae (1, 2). These are extensively used in theclinical treatment of tuberculosis, leprosy andAIDS-related mycobacterial infections caused byMycobacterium avium complex, an opportunisticpathogenic organism in AIDS (3). Formulationsthat are in use include rifampicin, rifabutin,rifamide etc., and these are obtained by chemicalmodification of native antibiotics rifamycin B andrifamycin SV (4).

Amycolatopsis mediterranei is the onlymicroorganism that has been in use forcommercial production of Rifamycin B in thesubmerged cultivation and the volume rangesfrom 100 to 1000˚m3 (5) using complexfermentation medium. With the increasingdemand of this antibiotic in terms of its utilityspectrum in the current AIDS outbreak worldforced the scientific community to look for novelantibiotic producers from exotic environment orincrease the productivity with existing microbialsystem by modifying the cultivation procedures.

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

Though different microbial strains have beenisolated and characterized for production ofrifamycin B wherein the yields ranging from 1.92to 19.00 g/l, a few of them reached to industriallevel. Industrial fermentation process aredeveloped based on laboratory scale studies andtypically use a multi-substrate economic complexmedium especially locally available substrates(6). In ansamycin related antibiotics producingmicrobial strains, these secondary metabolitesderived from molecular structures consisting ofan aromatic nucleus (a naphthalenic or benzenicring system) and a long handle-shaped aliphaticansa bridge joining two opposite positions of thenucleus (7). Mejia et al., (4) working with mutantstrain of Amycolatopsis mediterraneidemonstrated that rifamycin B production couldbe improved with the supplementation of 5, 5-diethylbarbituric acid commonly known asbarbital in the culture medium and reported thatthis stimulation was mainly due to enhancedavailability of oxygen for antibiotic synthesisindicating the influence of culture/fermentationconditions role in enhancement of this secondarymetabolite synthesis. In the present investigation,an isolated microbial strain, Nocardia RSP-3,with rifamycin B production potential has beencharacterized for its growth and antibioticproduction pattern in the presence and absenceof barbital. Rifamycin B production in thismicrobial strain was dependent on the culture age,aeration levels and the production could beimproved to more than 100% by optimizingfermentation conditions.

Materials and methods

Microorganism and Medium composition

Isolated Nocardia RSP-3 strain was used in thisstudy. Purified strain was grown and maintainedin medium consisting (g/l) Dextrose —20,glycerol-20, yeast extract-5, beef extract-3, caseinacid hydrolysate-3, peptone-2.5, malt extract-1.The pH of the medium was adjusted to 8.0 using0.1 M NaOH or HCl solution. For growth studies,

25ml of medium was taken in a 250 mlErlenmeyer flask, and incubated in a shakingincubator for 72 hrs at 250 rpm. Agar basedabove medium was used for development ofslants and stored at 4OC till further use.

Production medium and fermentationconditions

For antibiotic production, the following mediumconsisting (g/l) of soyabean meal-36, dextrose-75, barbital-2, calcium carbonate-3, magnesiumsulphate-0.02, distilled water-1000 ml (pH-8).Twenty-five ml medium was used in 250 ml flaskand inoculated with 10% (v/v) inoculum andincubated on shaking incubator at 250 rpm and280C. For studying the effect of pH on antibioticproduction, medium pH was adjusted topredetermined pH before sterilization and usedfor fermentation.

Effect of carbon sources and nitrogen sources

Influence of different carbon and nitrogen sourceson rifamycin B production during fermentationwas studied by supplementing the selectednutrient to the fermentation medium beforesterilization and culturing the microbe in theabove growth conditions.

Effect of barbital

Barbital is used as inducer of rifamycin B andsuppressor of other rifamycins. Its role onantibiotic production was studied by addition ofseparately sterilized barbital in sterilizedfermentation medium containing cultures of ageof 0hrs, 24hrs, 48hrs and 72hrs under sterilizedconditions. The samples were collected for every24 hours and analyzed for rifamycin production.

Assay

Rifamycin B in culture flasks was estimatedcolorimetrically according to the procedure ofPasqualucci et al., (8) 1970 by diluting the 0.1ml of cell free fermentation broth with 5 ml withacetate buffer (pH - 4.63) and reading the

174 Mahalaxmi et al

absorbance at 425 nm against blank containing0.1ml of fermentation broth diluted to 5ml with0.1% Na N0

2 containing acetate buffer (pH 4.63).

Rifamycin B was calculated using followingformula

Abs 425

* 50000Rifamycin B (µg/ml) =

21.5

Isolation and identification of Rifamycin B

The rifamycin was isolated according toVenkateshwarlu et al. (9). The cell free brothafter fermentation was collected by centrifugationand the pH of the solution was adjusted to 2.0using 2N HCl solution. The antibiotic wasextracted using equal volumes of ethyl acetate.The organic fraction was separated and

evaporated using rotary evaporator. The solidfraction was dissolved in methanol and used foranalysis. Initially, the sample was checked forits purity using TLC using chloroform:methnol(4:1). The purified sample was further subjectedfor LC-MS and the spectrum was analyzed.

Results and Discussion

For the production of rifamycin, the isolatedNocardia RSP-3 strain was grown infermentation medium for 9 days at 28OC and thecell free fermentation medium was subjected forextraction of antibiotic produced using ethylacetate after adjusting the pH of the broth to 2.0with 2N HCl (9). This ethyl acetate fraction wasconcentrated by vacuum evaporation andanalyzed using LC-MS (Fig.1). The LC-MS data

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

Rifamycin B Production patter in Nocardia RSP-3 175

products demands an optimal operation of theprocess (16, 17). In general, it was observed thatdown regulation of secondary metabolicbiosynthetic gene expression is essential for well-balanced primary metabolism (growth).Therefore, fermentation process supervision is ofparticular importance to ensure consistentoperation and thereby achieve high qualityproducts. Hydrogen ion concentration of mediumplays a vital role in metabolism of any organismand each organism has its optimum pH wheregrowth of selected microbial strain and its cellularmetabolism occurs in optimum levels comparedto other conditions (18). Hence, the influence ofmedium pH on the growth of the isolatedmicrobial strain has been investigated byinoculating the strain in the medium having pHrange of 3.0 to 9.0 and analyzing the growth andrifamycin production pattern regularly during thegrowth period. The growth data revealed thatthis microbial strain grows effectively in the pHrange of 5.0 to 9.0 with maximum growth at pH8.0 indicating its alkaline nature (Fig 2). Higheror lower media pH resulted in reduction ofgrowth. The antibiotic production pattern alsoshowed the similar influence, however, its

production observed in the medium having thepH of 6.0 to 9.0 with optimum at pH 8.0. Furtheranalysis of the antibiotic production patternrevealed that only growth of the strain wasnoticed in pH 4.0 medium not antibioticproduction. Such data reveal that the rifamycinB production in this isolated microbial strain isgrowth associated. Similar trend was noticed byVenkateshwarlu et al., (3) where maximumgrowth and antibiotic production was observedat pH 7.2.

The influence of incubation temperature wasstudied using fermentation by incubating thefermentation medium in the selectedtemperatures (24 — 32OC) with an interval of 2OC.The results were presented in Fig 3. Maximumgrowth was noticed at 28OC and variation of thistemperature resulted in reduction of biomassproduction at either sides. In fact, no growth wasnoticed at 32OC. Analysis of rifamycin Bproduction during fermentation period revealedthat maximum antibiotic production noticed with28OC incubated cultures. These results vary withthe reported data on production of rifamycin byAmycolatopsis mediterranei VA18 where

indicated characteristic peaks at756 (M. +H — 25%), 724 (OCH

3 —

65%) and 697 (CH3CO — 5%)

which is similar to literaturereported rifamycin B spectra (10).Hence, further studies were madefor optimization of rifamycin Bproduction with this microbialstrain.

Production of any biotechnologicalproduct whether it is ofrecombinant or classicalfermentation economic feasibilityplays an important role. Numerousapplications are exampled in food,agrochemical and pharmaceuticalindustries (11- 15). Further, thecost-competitive nature of such

Fig 2: Influence of medium pH on growth and rifamycin Bproduction by isolated microbial strain

176 Mahalaxmi et al

maximum antibiotic production observed with32OC grown culture (19). The influence ofincubation temperature on microbial growth andproduct production was also reported in variousstrains (13, 20 & 21).

Rifamycin B production in Amycolatopsismediterranei was associated with aeration levels(22). Hence, the influence of rpm on antibioticproduction was investigated using this microbialstrain. Antibiotic production was increased withincrease in rpm during fermentation (Fig 4.)Maximum rifamycin B production was noticedat 250 rpm and further studies could not be made

due to limitation in incubator shaker. Antibioticproduction rate of the strain improved withincrease in incubation rpm and it was noticed tobe changing with rpm rates. Further it was noticedthat the volume of the fermentation mediumshould be 10% of any given flask (25ml for 250mlflask).

Since microbial productivity is directlyproportional to biomass, the role of initialinoculum concentration on antibiotic productionwith this strain was studied. High yield ofrifamycin B was obtained with 72 hours grown10 % (v/v) inoculum level (Fig 5). It wasinteresting to note that increase of inoculum levelup to 10% resulted in increased rifamycin Bproduction and further increase resulted inreduction of antibiotic production. Suchinoculum concentration dependent productproduction variations were reported in othermicrobial strains (18, 23, 24).

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

Fig 4: Effect of rpm on growth and rifamycin Bproduction by isolated microbial strain

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

In general, antibiotic fermentation processes arecost intensive and the profitability is greatlydependent on the product yield per unit substrateconsumed. In order to reduce cost, industrialprocesses use organic nitrogen substrates suchas corn steep liquor and yeast extract. Thus,although the stoichiometric analysis is the firstlogical step in process development, it is often

Rifamycin B Production patter in Nocardia RSP-3 177

evaluated for their influence on growth andantibiotic production by this microbial strain. Ofthe various carbon sources that had beenscreened, glucose and galactose were found tobe the best and lactose as carbon source indicatedthe least antibiotic production. These results aresimilar with the reported literature where glucosewas found to be the best choice as carbon sourcefor growth or production of any compound. In

this study, glucose being economically cheapcompared to galactose, glucose was selected asthe carbon source for further experiments.Glucose concentration dependent rifamycin Bproduction studies indicated that supplementationof 10% glucose into the medium resultedmaximum antibiotic production (1.2 g/l) andvariation of this carbon source concentration oneither sides showed reduced production (resultsnot shown). Among different nitrogen sourcesstudied, soyabean meal along with peptone (1.8g/l each) was found to be ideal for antibioticproduction (1.23 g/l) with this isolate.Approximately 60% improvement was noticedwith addition of these nitrogen sources incombination. Yeast extract supplementationshowed the least production of antibioticcompared to all other organic complex nitrogensources studied. In literature too, soyabean mealwas the most widely used nitrogen source in theproduction of rifamycin B and other metaboliteproducts (5, 18). In order to understand further,the impact of combination of nitrogen sourceson antibiotic production with this microbial strainwas studied by incorporating the combination ofstudied nitrogen sources in the fermentationmedium (insert of Fig 7). The results depictedthat rifamycin B production further improvedfrom 1.23 g/L to more than 2.0 g/L in soyabeanmeal and peptone (1.8g/L each) supplementedfermentation environments. However,combinations experiments with yeast extract andpeptone could not improve the antibioticproduction. In fact, antibiotic production wasreduced compared to other studied conditions.These results further suggested that soyabeanmeal in combination with peptone is effectiveantibiotic inducer in this microbial strain. Suchcombinatorial medium components influence hasbeen reported in production of extracellularenzyme in Bacillus sp. (18).

Barbital is known to induce rifamycin Bproduction by suppressing the production of otherrifamycin antibiotics (4). Therefore, impact of

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

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

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

178 Mahalaxmi et al

barbital addition on rifamycin B production wasinvestigated. The data suggested that yield ofrifamycin B influenced by the barbital additionto the medium (Fig 8). Higher antibioticproduction was noticed in all barbitalsupplemented fermentation conditions comparedwith control. This enhanced production wasassociated with the fermentation time indicatingthe barbital role on altering the strains metabolismand improving the rifamycin B production.Barbital addition improved antibiotic productionmore than 100% compared to control (withoutbarbital in the medium) (Fig 8). Thisimprovement was also associated with the growthof the organism. This could be evidenced basedon the observation that the values of antibioticproductivity were noticed to be 15.27 in controland 23.08 mg/h/L barbital supplemented

indicated that higher and prolonged antibioticproduction period. This is evidenced based onthe observation that maximum rifamycinproduction was noticed at 144 and 168 hours ofincubation in control and barbital supplementedconditions, respectively (Fig.8) with aproductivity improvement of 51%. This datarevealed that barbital has impact on cellularmetabolism associated antibiotic production intwo angles, i.e., in improving the productivity andenhancing the production period. Barbital hasbeen reported as toxic to the microbial growth(25). Hence, the impact of barbital addition atdifferent cell growth periods was investigated.The data depicted that a slight improvement inrifamycin B production was noticed in variationof barbital addition at different fermentationincubation times. A 10% of antibiotic production

Fig 8: Effect of barbital on rifamycin B production by isolatedmicrobial strain

improvement was noticed whenbarbital supplemented to thefermentation medium after 24 hoursof incubation compared to 0.0 hour.Whereas, supplementation ofbarbital in subsequent fermentationtimes reduced the rifamycin Bproduction, however, the productionwas higher compared to controlfermentations (without barbital) (Fig8). Concentration dependent barbitalinfluential studies revealed that 0.2%was effective for rifamycinproduction by this isolated straincompared to other studied range of0.1 to 0.4% (w/v) (results not shown)and maximum production of 4.4 g/lwas observed under optimizedconditions.

fermentations indicating that barbital associatedrifamycin B production improvement isassociated with the altered metabolism of themicrobial strain. In literature too, it was reportedthat barbital negatively regulate the electrontransport system and increases the availability ofoxygen to other cellular activities (25).

Further analysis of the rifamycin production trendin fermentations with and without barbital

Conclusions

The potential of isolated microbial strain,Nocardia RSP-3, on rifamycin B production wasinvestigated under different growth and mediumconditions. The growth and antibiotic productionoccurred simultaneously in this isolated strain.

Rifamycin B Production patter in Nocardia RSP-3 179

Maximum growth was noticed at pH 8.0, at 28OCwith glucose and galactose as carbon source.Among all nitrogen sources tested, soyabeanmeal showed maximum rifamycin B productioncompared to other nitrogen sources tested.Barbital improved the antibiotic production inthis strain was noticed with supplementation ofbarbital (0.2%) to the 24 hours grown culturescompared to other tested conditions. Underoptimized environment rifamycin B productionwas improved from 0.8 to 4.4 g/l with thismicrobial isolate indicating a productivityimprovement from 3.63 to 26.18 mg/h/L afteroptimization.

Acknowledgements

Authors of this paper Y Mahalaxmi, Ch SubbaRao and T Satish thankful to CSIR (JRF), CSIR(SRF) and APNL for financial support in the formof fellowship, respectively.

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