Biosynthesis of Bacitracin in Stirred Fermenter by Bacillus Licheniformis Using Defatted Oil Seed Cakes as Substrate

Bacitracin is being imported in India involving substantial amount of foreign exchange for its incorporation in poultry feed. The raw material for its production is readily available and cheap such as soybean meal, sunflower meal & wheat bran. Thus development of this technology in this country would result in saving a reasonable amount of foreign exchange by utilizing our resources. The present study is concerned with the biosynthesis of antibioticBacitracin by Bacillus licheniformison laboratory to scale up studies in StirredFermenter using defatted oil seed cakes of agricultural bye-products as startingmaterial for maximum production of the antibiotic Bacitracin. In stirred fermenter, antibiotic formation reached maximum (342 i.u. ml), 30 hours after inoculation at 37 C using different natural media such as defattedsoybean meal, glucose and metal ions.In solid-state fermentation, wheat bran, soybean meal, sunflower meal, rice hulls and their different combinationswere used. The antibiotic activity 48 hours after inoculation was 4540 i.u/g when only soybean was used.


Introduction and Literature Survey
Bacitracin is derived from cultures of Bacillussubtilis (Tracey).It is a white to pale buff, hygroscopic powder, odorless or having a slight odor.It is freely soluble in water; insoluble in acetone, chloroform, and ether.While soluble in alcohol, methanol, and glacial acetic acid, there is some insoluble residue.It is precipitated from its solutions and inactivated by many of the heavy metals.
The molecular formula is: C 66 H 103 N 17 O 16 S.Bacitracin is comprised of a polypeptide complex and Bacitracin A is the major component in this complex.The molecular weight of Bacitracin A is 1422.71.
Bacitracin consists of one or more of the antimicrobial polypeptides produced by certain strains of Bacillus licheniformis and Bacillus subtilis var Tracy and yields the Amino acids L-cysteine, D-glutamic acid, Lhistidine, D-phenylalanine, L-lysine, L-isoleucine, L-leucine, D-ornithine and DL-Aspartic acid on hydrolysis (BP 2002) and functions as an inhibitor of cell wall biosynthesis (Azevedo, 1993).Bacitracin of other micro-organism is an antibiotic as well as non-ribosomally produced by Bacillus licheniformis (Ohki, 2003).
Different types of Bacitracin like A, A1, B, C, D, E, F, F1, F2, F3 and G have been isolated.The most potent antibiotic is Bacitracin A, whereas Bacitracin B and C are less potent and the rest possess a very little antibacterial activity.This antibiotic is the most effective against Gram +ve and a few Gram -ve species of bacteria.It is almost exclusively used as a topical preparation in the treatment of infections (Brunner, 1965).
Bacillus licheniformis is a bacterium that is commonly found in soil and bird feathers.Birds that tend to stay on the ground more than the air (i.e.sparrows) and on the water (i.e.ducks) are common carriers of this bacterium; it is mostly found around the bird's chest area and back plumage.B. licheniformis is part of the subtilis group along with Bacillus subtilis and Bacillus pumilus.These bacteria are commonly known to cause food poisoning and food spoilage.B. licheniformis also is known for contaminating dairy products.Food borne outbreaks usually involve cases of cooked meats and vegetables, raw milk, and industrially produced baby food contaminated with B. licheniformis.This bacterium, although detrimental, can be modified to become useful.Researchers are trying to turn bird feathers into a nutritious livestock feed by fermenting non-digestible proteins on bird feathers with B. licheniformis.There is also research about the possibility that B. licheniformis causes changes in color in birds' feathers; this will provide information on the evolution of molting.Also, cultures of B. licheniformis are made to retain its protease, which is in turn used in laundry detergent.
Bacillus licheniformis, a Bacitracin producer, has an ABC transporter system which is hypothesized to pump out Bacitracin for self-protection (Podlesek, 1995).Bacitracin holds considerable importance.It is also widely used as supplement in poultry nutrition.Its addition to the feed increases feed efficiency and the incidence of infectious diseases are greatly reduced (Shalak, 1971;Smekal, et al., 1979).Zinc Bacitracin and Bacitracin methyl disalicylate (feed grade) are widely used for growth promotion.Addition of Bacitracin to the feed may affect the activity and synthesis of certain liver enzymes (Rybinska, 1977) and increase the level of proteases and amylases in the digestive tract of laying hens.

Gram Staining
Gram staining was carried out before Inoculum Preparation to ensure whether collected Bacillus licheniformisfrom N.C.L was pure.

Inoculum Preparation
The bacterial growth was aseptically scrapped from 48 hours old cultures lants and transferred to 50 ml sterilized basal medium (Table 1) in 250 ml conical flask and then shaken on rotary shaker at 150 rpm for 24 hours at 37 °C.The vegetative culture thus obtained was used for inoculation into fermentation media.4% v/v inoculum was used in this study.

Fermentation Media for Bacitracin Production
Fermentation media used for the production of Bacitracin by Bacilluslicheniformisis given in Table 2.

Fermentation Technique (Method)
10 g of the substrate was taken in 250 ml of conical flask.It was wetted by 10 ml of distilled water previously adjustedto pH 7 or phosphate buffer of pH 7 was used.Medium (Table 2) was autoclaved at 121 o C for 15 minutes; it was allowed to cool and then was inoculated with 1ml of seed culture.After inoculation, the flasks were shaken well and then incubated at 37 o C for 48 hours.At the end offermentation period, the fermented material was soaked in N/100 HCl for 1 hour and then centrifuged.The supernatant layer was assayed for calculation of antibiotic activity.
Rate of production of bacitracin by Bacillus licheniformis in wheat bran by solid-state fermentation is given in the following Table 3.

Effect of Different Oil Seed Cake on the Production of Bacitracin
Bacitracin consists of a group of closely related peptides.Thus effect of different defatted oil seed cakes as a source ofamino acids, vitamins, minerals and sugars were investigated as in Table 4.
The production of Bacitracin on laboratory scale was carried out in 30-L Glass Stainless Steel Fermenter (B.E.Marubishi -MSJ -N2, Japan).It was connected with bioprocess operator-MSSD-1 and bioprocess controller-MEDIAC-93.The basal medium was sterilized inside the fermenter automatically.Temperature, pH, agitation and foaming were controlled automatically.The fermentation medium (Table 2) which gave the best results in shake flasks was used.The fermenter was run for 48 hours.The antibiotic activity during fermentation was determined from time to time.
Fermentation phenomenon can be occurred in simple condition.A rotary shaker is required.The fermentation medium would be a flask which is plugged by cotton.Note that transfer of inoculum (basal) medium to fermentation flask must be done in absolute sterilized environment.

Assay
The activity of the antibiotic Bacitracin present in the fermented material was determined by agar diffusion method (Table 5).
The pH of the medium was adjusted to 7.0 with 1N NaOH/HCl before the addition of agar.The medium was sterilized at 121 °C for 15 minutes.Approximately 20 ml of the medium was aseptically poured into the sterile Petri-plates and allowed to solidify.Then, 4 ml of melted assay medium which was previously inoculated with the pre-determined concentration of test organisms i.e.Staphylococcus aureus and Escherichia coli were spread uniformly over the first layer and were allowed to congeal.After setting the second layer, four holes 0.8 cm of diameter were made in the plates aseptically with stainless steel borer of uniform edge and size.
Two opposite holes were filled with working standard of 1:4 dilution (S1, S2) and the remaining two were filled with sample to be determined of 1:4 dilution (T1, T2) using micropipette.0.12 ml solution was poured in each digged hole.The plates were then very carefully placed in incubator for 24 hours at 37 °C.Clear zones of inhibition were developed both by standards and samples.Diameters of zones of inhibition were measured and compared with the known standard.
The potency of the sample was determined by the following formulae:

Units of Bacitracin
One unit of Bacitracin activity is the amount of antibiotic in 0.2 ml of culture supernatant broth that will cause a 1 mm inhibition zone outside the cylinder (Bernlohr, & Novelli, 1960).One unit of Bacitracin is equivalent to 26 μg of USP standard (Harvey, 1980).The USP Unit of Bacitracin is the Bacitracin activity exhibited by the weight of USP Bacitracin Reference Standard indicated on the label of the Standard.It has a potency of not less than 40 USP Units of Bacitracinmg-1 (Nichols, 2000).

Isolation of Bacitracin by Centrifugation
The fermented broth was centrifuged at 10,000 rpm for 15 minutes in a refrigerated centrifuge at 4 °C in order to remove cells and solid suspended particles.The clear supernatant solution was used for the isolation of antibiotic.

Partitioning
Physical separation may be used for isolation, purification, identification and determination of organic compounds.It is, of course, generally a two-phase process, and can be classified into solid-liquid, liquid-liquid, gas-liquid and gas-solid systems.An exception is electrophoresis, which takes place in a single phase.Applications of physical separation may cut across these categories-for example; phase titrations may result in separation of a solid or a liquid phase from the initial liquid phase.Similarly, some of the factors associated with physical separation have implications for figurative separation.

Results and Discussion
The production of antibiotic by solid state fermentation involves less consumption of energy compared to stirred fermenters where continuous aeration, agitation and control of foaming are necessary.
The rate of production was determined by using wheat bran as solid substrate.The antibiotic activity was determinedafter every 12 hours during the course of fermentation (Table 3).The antibiotic activity reached maximum (3287 i.u/g), 48 hours after inoculation.Further increases in fermentationperiod resulted in decline of bacitracin activity.It may be attributed to inhibition of "Bacitracin Synthetase" enzyme by bacitracin itself by feedback mechanism.4 shows that synthesis of bacitracin was maximum in of soybean meal (4540 i.u/g) while amount of bacitracin produced in sunflower meal was 1330 i.u/g.wheat bran also gave good antibiotic titre i.e. 3287 i.u/g but rice hulls only produced 562 i.u/g.The reason of low antibiotic production by rice hulls may be of its being poor source of carbon and nitrogen while soybean and wheat bran are ideal substrate providing all the nutrients required by Bacillus licheniformis.

Screening of Culture Media
The composition of the basal medium (Table 1) greatly influence theproduction of antibiotics.Replacing soybean meal with sunflower meal and/or wheat bran of same quantity (Table 2) in fermenting medium wasused for the screening purpose.The nutritional studies were carried out.
The antibiotic activity in the fermented broth was determined, 44-48 hours after inoculation with 4% v/v bacterial cell suspension obtained from theslant surface.Of the medium tested, soybean meal medium (Table 2) gave the best results of antibiotic titer.K 2 HPO 4 and KH 2 PO 4 were used as buffering agents, MnSO 4 .7H 2 O and MgSO 4 .4H 2 O as co-factors of enzymes while FeSO 4 .7H 2 O was used toassist the action of Manganese ion.Addition of citric acid leads to theformation of soluble coordinate complex with the metal ion thus makingthem available to the microorganism at adequate time (Haavik, 1976).
Organic and inorganic matter content is considered as an indicator of richresources of media for Nitrogen source (Varvel, 1994).The conditions like pH, temperature, aeration, different ratio of substrates as nitrogen sources and other parameters were optimized (Shabbir, 2001).

Table 1 .
Composition of basal medium